A Comparative Analysis of Listeria Monocytogenes Plasmids: Presence, Contribution to Stress and Conservation

Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations

2020

A comparative analysis of Listeria monocytogenes plasmids: Presence, contribution to stress and conservation

Annabel Naditz Iowa State University

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Recommended Citation Naditz, Annabel, "A comparative analysis of Listeria monocytogenes plasmids: Presence, contribution to stress and conservation" (2020). Graduate Theses and Dissertations. 17859. https://lib.dr.iastate.edu/etd/17859

This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. A comparative analysis of Listeria monocytogenes plasmids:

Presence, contribution to stress and conservation

Annabel Naditz

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Microbiology

Program of Study Committee: Stephan Schmitz-Esser, Major Professor Gwyn Beattie James Dickson Larry Halverson Melha Mellata

The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred.

Iowa State University

Ames, Iowa

2020

DEDICATION

I would like to dedicate this dissertation to my son, Aidan, whose patience and kindness

during my graduate career has been unfathomable. Thank you for making life a little

easier and rolling with it when it was a little harder. I would also like to dedicate this to my parents and my sister, whose never-ending support kept me going, especially during

the roughest times. Thanks for the food, the Kleenex, the chocolates, and grandson-

sitting.

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TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... v

ABSTRACT ...... vi

CHAPTER 1. GENERAL INTRODUCTION ...... 1 Background ...... 1 Conservation of Plasmids in L. monocytogenes Strains ...... 6 Plasmids and Survival of L. monocytogenes in Food and FPEs ...... 9 Genetic Content of L. monocytogenes Plasmids in Relation to Stress Survival ...... 11 References ...... 14

CHAPTER 2. A LARGE-SCALE SEQUENCING-BASED SURVEY OF PLASMIDS IN LISTERIA MONOCYTOGENES ...... 23 Abstract ...... 23 Introduction ...... 24 Materials and Methods ...... 27 Strain Selection ...... 27 Dataset Generation, BLAST Analyses and Plasmid Identification ...... 35 Gene Content Analysis and Gene Conservation ...... 37 Results and Discussion ...... 38 Overview of the Full Dataset ...... 38 Summary of the Generated Dataset ...... 42 Presence of Plasmids ...... 43 Size Range of Plasmids ...... 47 Overall Conservation of Plasmids ...... 49 Presence and Conservation of Stress-Response Plasmid Genes Across STs ..... 51 Conclusion ...... 59 References ...... 61

CHAPTER 3. PLASMIDS CONTRIBUTE TO FOOD PROCESSING ENVIRONMENT- ASSOCIATED STRESS SURVIVAL IN LISTERIA MONOCYTOGENES ST121, ST8, AND ST5 STRAINS ...... 70 Abstract ...... 70 Introduction ...... 71 Materials and Methods ...... 74 Bacterial Strains, Growth Conditions, Plasmid Curing and Plasmid Screening .... 74 Stress Survival Assays ...... 75 Sequence Analysis ...... 77 Statistical Analysis ...... 78 Results ...... 78 Gene Content of the Analyzed Plasmids ...... 78 Survival of Wildtype Strains Compared to Plasmid-Cured Strains Without Stress Conditions ...... 87

Survival of Wildtype Strains Compared to Plasmid-Cured Strains Exposed to Stress Conditions ...... 88 Discussion ...... 92 Conclusion ...... 97 Acknowledgements ...... 97 References ...... 97

CHAPTER 4. TRANSCRIPTOME SEQUENCING OF LISTERIA MONOCYTOGENES EXPOSED TO OXIDATIVE AND ACID STRESS REVEALS HIGH EXPRESSION OF CHROMOSOMAL AND PLASMID NON-CODING RNAS ...... 105 Abstract ...... 105 Introduction ...... 106 Materials and Methods ...... 109 Bacterial Strains Used in this Study ...... 109 Experimental Stress Conditions ...... 109 RNA Extraction, Library Preparation, and Sequencing ...... 110 Sequence Analysis ...... 112 BLAST Methods ...... 112 Results and Discussion ...... 113 Chromosomal Gene Expression ...... 113 Chromosomal Gene Expression Changes in Response to Hydrogen Peroxide . 122 Chromosomal Gene Expression Changes in Response to Lactic Acid Treatment ...... 124 σB Genes ...... 129 Plasmid Gene Expression ...... 135 Plasmid Gene Expression Changes in Response to Hydrogen Peroxide Treatment ...... 136 Plasmid Gene Expression Changes in Response to Lactic Acid Treatment ...... 137 Conclusion ...... 141 Availability of Sequencing Data ...... 142 Author Contributions ...... 142 Acknowledgements ...... 142 Conflicts of Interests ...... 143 References ...... 143

CHAPTER 5. GENERAL CONCLUSION ...... 153 Conclusion ...... 153 References ...... 160

APPENDIX. SUPPLEMENTARY FIGURES ...... 183

ACKNOWLEDGMENTS

I would like to thank my committee chair, Stephan Schmitz-Esser, and my committee members, Gwyn Beattie, James Dickson, Larry Halverson, and Melha

Mellata, for their guidance and support throughout the course of this research. My thanks to Cassi Wattenburger, Chase Mayers, and Jennifer Chang for their assistance in editing and proofreading. In addition, I would also like to thank my friends, colleagues, the department faculty and staff for making my time at Iowa State University a wonderful experience. In particular, I would like to thank my family, without whom I would not have been able to accomplish any of this. I am forever grateful for your love and support.

ABSTRACT

Listeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis and is commonly isolated from food products and production facilities. Some of these isolates are known to contain plasmids, though previous plasmid studies were conducted with a limited data set of 14 sequenced strains. To determine the presence of plasmids in a variety of L. monocytogenes strains and from various sequence types

(ST), a large-scale analysis of 1924 L. monocytogenes genome sequences was conducted to determine the presence of plasmids in different STs, years, countries, and isolation sources. On average, 53% of all L. monocytogenes strains, and up to 92% of the strains within a ST (i.e. ST121), analyzed contained a plasmid. These plasmids ranged in size between 3 kb to 140 kb, with the majority being around 58 kb (± 1kb) and the other around 77 kb (± 1 kb). Plasmid conservation was a minimum of 91.9% nucleotide identity across all plasmids among all STs and the average similarity was

92.3% similarity. Examination of six previously identified plasmid-encoded stress-related genes indicated the genes present in L. monocytogenes plasmids are highly conserved, but their presence in plasmids is highly variable. Analysis of the dataset also identified a novel mercury-resistance operon containing seven genes involved in increasing tolerance in the presence of mercury.

Understanding that plasmids often play a role in stress survival, it was necessary to determine the contribution of L. monocytogenes plasmids to stress tolerance when under such conditions. For this aim, we studied three isogenic pairs of L. monocytogenes ST5, ST8, and ST121 strains, isolated from dairy foods and production facilities, and that either contained or were cured of plasmids harboring putative stress

vii response genes. Wildtype (wt) and plasmid-cured (pc) strains were exposed to sublethal concentrations of oxidative stress (0.01% H 2O2), salinity (15% wt/vol NaCl, pH

5), acidity (1% v/v lactic acid, pH 3.4), heat stress, or disinfectant (benzalkonium chloride, BC). When exposed to H 2O2, NaCl, or lactic acid, CFU counts for all wt strains were significantly higher (P < 0.05) than those of the pc strains. Our data revealed that

L. monocytogenes ST5, ST8, and ST121 plasmids confer increased tolerance against elevated temperature, salinity, acidic environments, oxidative stress and disinfectants.

Further examination of the phenotypic and functional characteristics of plasmids was required to better understand the mechanisms used by L. monocytogenes plasmids to confer tolerance to stress conditions. Therefore, a transcriptomics survey of plasmid- carrying L. monocytogenes strains 6179 and R479a was conducted under acidic and oxidative stress conditions, using the acidic and oxidative conditions of functional characterization, with an exposure time of 30 minutes at 20°C. The data showed no significant gene expression in the oxidative stress conditions for the R479a strain, and a single differentially expressed (DE) gene in the 6179 strain, a protein of unknown function.

For the lactic acid conditions, pLM6179 had 35 DE genes, 16 of which were upregulated, including clpL , a gene predicted to be involved in general stress response.

For pLMR479a, the most upregulated DE gene under lactic acid stress was a putative zinc riboswitch, found upstream of a zinc-transporting ATPase. Seventy percent of the transcripts of pLMR479a during lactic acid stress were mapped to this putative zinc riboswitch, indicating heavy metal riboswitches and their potentially corresponding transporters may be important for L. monocytogenes stress response.

CHAPTER 1. GENERAL INTRODUCTION

Background

Food and food product contamination from bacterial pathogens is a serious concern to the food industry and a threat to human health. The transmission of foodborne pathogens to consumers via contaminated food products can result in a variety of diseases, and the pathogens responsible for these diseases are just as diverse, including bacteria such as Salmonella , Escherichia coli , Campylobacter and Listeria monocytogenes . These illnesses range from mild symptoms such as nausea or vomiting, to severe symptoms, where some foodborne illnesses can be fatal (Mead et al., 1999; Kaur et al., 2007; Scallan et al., 2011). Surveillance and epidemiological studies are routinely conducted to monitor these food-borne pathogens and to identify contaminated products, issuing recalls to prevent contaminated products from further endangering the public.

The genus Listeria is currently comprised of 20 species, with new species added in the last few years (Orsi and Wiedmann, 2016; Leclercq et al., 2019). The genus

Listeria is further divided into two major groups: Listeria sensu stricto and Listeria sensu lato . Listeria sensu stricto includes the species L. monocytogenes, L. innocua, and L. ivanovii. All other Listeria species belong to Listeria sensu lato. Among the genus

Listeria, only L. monocytogenes and L. ivanovii are considered to be pathogenic, and as infectious cases due to L. ivanovii are uncommon, L. monocytogenes represents the primary concern to global human health.

L. monocytogenes is a facultative intracellular pathogen and is responsible for listeriosis, a rare but severe disease in humans with a high mortality rate. It is typically

acquired through the consumption of contaminated food products (Deng et al., 2016;

Allard et al., 2018). The disease primarily affects newborns, pregnant women, older adults, and immunocompromised individuals. Listeriosis can also manifest varying disease symptoms depending upon the host. For example, in older adults and those with weakened immune systems, the patient will often experience septicemia or meningitis, whereas pregnant women may experience fevers and non-specific symptoms, followed by fetal loss, bacteremia or meningitis in newborns (Pouillot et al.,

2012). In contrast, healthy individuals may experience acute febrile gastroenteritis or have no clinical presentations. Although listeriosis cases are relatively rare, with approximately 0.26 cases per 100,000 individuals and 1600 cases annually in the U.S., listeriosis is characterized by a high mortality rate ranging from 15 to 30%, resulting in

260 deaths occurring every year in the U.S. (Allerberger and Wagner, 2010; Scallan et al., 2011). As previously mentioned, continued monitoring of food quality results in product recalls of L. monocytogenes contaminated food products, with the aim to prevent potential listeriosis outbreaks. However, even with increased observations, outbreaks still occur, with high health and economic costs. The 2011 cantaloupe listeriosis outbreak was the largest listeriosis outbreak in the U.S., affecting 28 states, with 147 cases and 33 deaths (McCollum et al., 2013). The overall annual costs of L. monocytogenes in the US is estimated to be between $2 and $22 billion (Ivanek et al.,

2004; Batz et al., 2012; Scharff, 2012). The economic consequences of contaminated food product recalls due to L. monocytogenes contamination is considered to be between $1.2 and $2.4 billion USD annually (Ivanek et al., 2004) and in 2008, a single

outbreak of 57 listeriosis cases in Canada was estimated to have a cost of $242 million

CAD (Thomas et al., 2015).

L. monocytogenes is of particular research interest, as it has been isolated often from ready-to-eat (RTE) foods and can survive in food and food production environments (FPEs) (Allerberger and Wagner, 2010; Ferreira et al., 2014). RTE foods are particularly high-risk, because of the lack of a heating step, which is especially important as heat is an effective and efficient method for inactivation of L. monocytogenes . Recently, listeriosis outbreaks have been traced to food products that were thought to be of moderate or low risk by current risk assessments, including stone fruits, caramel apples, cantaloupe and celery (Gaul et al., 2013; McCollum et al., 2013;

Chen et al., 2016; Angelo et al., 2017; Zhu et al., 2017).

L. monocytogenes strains are routinely exposed to a wide array of environmental stresses during the food production process, including high salinity, acidity, alkalinity, high or low temperatures, and low nutrients, which can influence their survivability

(Leistner and Gorris, 1995; Gandhi and Chikindas, 2007; Larsen et al., 2014; Schirmer et al., 2014). To reduce contamination with food-borne pathogens, the food production and processing industries utilize a variety of methods including strict hygiene plans, cleaning agents, disinfectants, and food additives to prevent bacterial growth. Thus, L. monocytogenes also undergoes additional challenges due to these routine FPE cleaning procedures, as these disinfectants and detergents often include heavy metals, toxic compounds or reactive oxygen species, all of which can be detrimental to bacterial growth and survival (Barker et al., 2003; Ratani et al., 2012). Food-specific stress conditions, such as high levels of salinity or acidity due to compounds such as acetic

acid, lactic acid, sodium chloride or other fermentation products, may also expose L. monocytogenes to additional stress (Gahan et al., 1996; O'Driscoll et al., 1996; Davies et al., 1997; Luna-Guzman and Barrett, 2000). Moreover, L. monocytogenes is well- known for colonizing specific areas in FPEs, such as hard-to-clean surfaces and drainage ducts, where L. monocytogenes can form biofilms which allow L. monocytogenes to survive, adapt and grow, making it difficult to eliminate these bacteria from FPEs (Ferreira et al., 2014).

Due to L. monocytogenes’ ability to colonize inaccessible areas in FPEs, the pathogen can be highly persistent in food production facilities. Here, we define persistence as genetically indistinguishable or highly similar strains of L. monocytogenes that have been repeatedly isolated from the same FPE over long periods of time. Additionally, genetically highly similar strains may be found in the same period of time, but in geographically unrelated facilities, indicating that persistence of L. monocytogenes may not be specific to individual food production facilities (Autio et al.,

2002; Carpentier and Cerf, 2011; Ferreira et al., 2014).

Plasmids are mobile genetic elements which can provide survival or fitness benefits to their host strains. Plasmids can be lost due to the maintenance costs to the host and are typically retained only if they provide an advantage for survival. Prior to the availability of genome sequencing, studies examined the presence of plasmids in L. monocytogenes using a variety of methods, such as alkaline plasmid extraction, pulse field gel electrophoresis (PFGE) or multilocus enzyme electrophoresis (MEE) (Perez-

Diaz et al., 1982; Baloga and Harlander, 1991; Lebrun et al., 1992; Harvey and Gilmour,

2001). A number of these studies of L. monocytogenes isolates collected from various

food production facilities over several years found that the strains had retained their plasmids, indicating there is a benefit for the retention of plasmids that outweigh maintenance costs (Harvey and Gilmour, 2001; Kuenne et al., 2010; Ferreira et al.,

2014; Martin et al., 2014; Knudsen et al., 2017). Moreover, food- and FPE-isolated L. monocytogenes strains were found to carry plasmids at a higher percentage than sporadic or clinical isolates (Lebrun et al., 1992; McLauchlin et al., 1997; Harvey and

Gilmour, 2001).

Interestingly, knowledge about Listeria plasmids is surprisingly low. Previously, detected plasmids were considered rare and were found to be similar in size (Perez-

Diaz et al., 1982; Lebrun et al., 1992; McLauchlin et al., 1997). Therefore, profiling and replication studies were considered superfluous. In contrast to other bacteria such as

Enterobacteriaceae , the knowledge about plasmid replication in Listeria is very limited.

Listeria utilizes similar minimal replicons belonging to the pAMbeta1 family of theta- replication plasmids for plasmid replication. This family of plasmids is closely related to plasmids found in Firmicutes genera Bacillus, Streptococcus, and Enterococcus

(Weaver et al., 1993; Wilcks et al., 1999; Francia et al., 2004; Tinsley et al., 2004; Van der Auwera et al., 2005; Francia et al., 2007; Kuenne et al., 2010). The replicon consists of three genes that include the gene for replication ( repA ) and partition ( repB/repC ) as well as genes encoding DNA polymerase IV, and the origin of replication (Weaver et al.,

1993; Wilcks et al., 1999; Francia et al., 2004; Tinsley et al., 2004; Van der Auwera et al., 2005; Francia et al., 2007; Kuenne et al., 2010). It should also be noted that incompatibility types of Listeria plasmids have not yet been described.

Listeriosis outbreaks are well-documented and monitored and whole-genome sequencing approaches are now regularly used to identify the outbreak-associated strains and provide in-depth analysis on the spread of the disease (Deng et al., 2016;

Allard et al., 2018). However, while knowledge on bacterial strains and listeriosis epidemiology is increasing, the molecular mechanisms for survival of L. monocytogenes in food and FPEs are still not fully understood, even as new genome sequences become more readily available. Molecular mechanisms for L. monocytogenes survival in food processing plants due to chromosomally encoded mechanisms is well- characterized, whereas our understanding of the role of plasmids in L. monocytogenes stress survival is still highly limited. This lack in knowledge is remarkable, as plasmids are known to be pivotal in providing advantages to their hosts, allowing for adaptations to altering environments (Shintani et al., 2015). This leads to the primary research questions of this dissertation:

1. What is the distribution and genetic content of plasmids in different L.

monocytogenes strains?

2. Do plasmids contribute to survival of L. monocytogenes in food and FPEs?

3. If so, which plasmid genes are responsible for this contribution?

Conservation of Plasmids in L. monocytogenes Strains

There are few data available on the distribution of plasmids in L. monocytogenes strains, as sequence analysis of plasmids in L. monocytogenes has been limited to few sequence types (STs). Examination of the genetic content of various L. monocytogenes plasmids has shown that plasmids have a modular structure and plasmids within

sequence types can be highly conserved, in some cases virtually identical, yet these L. monocytogenes plasmids may have been isolated from different countries and years(Kuenne et al., 2010).

Previous research has shown L. monocytogenes ST121 plasmids are highly conserved and essentially identical, with greater than 99.9% nucleotide similarity

(Schmitz-Esser et al., 2015b; Rychli et al., 2017). Given the large size (62-kb) of these plasmids, as well as the costs for plasmid maintenance and replication, it is likely that there is a high evolutionary pressure to retain the plasmids. Studies on plasmids in other L. monocytogenes STs have also shown a high degree of conservation in some

STs. Examination of L. monocytogenes ST204 plasmids found plasmids in 86.6% of the isolates studied, and yet, though the ST204 plasmid size was diverse, the sequences were highly conserved (Fox et al., 2016). Furthermore, L. monocytogenes ST5 plasmids were found in 55% of the isolates studied under stress conditions (Hingston et al.,

2017b). On the other hand, research on ST8 plasmids has shown higher levels of variability (Fagerlund et al., 2016), indicating the level of conservation may differ depending upon the ST.

Research has shown that plasmids across L. monocytogenes STs are highly modular with long segments of conserved sequences, often containing genes that are virtually identical (Kuenne et al., 2010). For example, we conducted an examination of three L. monocytogenes plasmids from different sequence types, a ST5 plasmid

(pLM4KSM), a ST8 plasmid (pLMR479a), and a ST121 plasmid (pLM6179), all of which derive from different countries, sources and years (Schmitz-Esser et al., 2015a;

Schmitz-Esser et al., 2015b; Muhterem-Uyar et al., 2018; Naditz et al., 2019). We

identified 22 genes shared among all three plasmids, with variation in genetic content clearly denoting these as three different plasmids (Chapter 3).

Moreover, independent studies on individual L. monocytogenes ST plasmids have identified homologous genes on plasmids from different STs. One study conducted by Kuenne et al. has been the most comprehensive study to date on the presence of plasmids across the genus Listeria (Kuenne et al., 2010). Phylogenetic analysis of L. monocytogenes replication protein RepA amino acid sequences revealed that the plasmids clustered into two different subtypes, termed group 1 and group 2 plasmids (Kuenne et al., 2010). Additionally, the authors found that the gene content among L. monocytogenes plasmids mirrored the replicon-clustering, with highly similar clusters of sequences for plasmids within the same plasmid group but from differing

STs. However, this study was limited to 14 strains, and therefore, although informative, is not comprehensive due to the exclusion of some L. monocytogenes STs.

The identification of highly similar genes on different L. monocytogenes ST plasmids supports the concept of gene modularity and shared sequence similarity between L. monocytogenes plasmids from many STs. Consequently, in light of the previous studies, we hypothesize the answer to “ What is the distribution and genetic content of plasmids in different L. monocytogenes strains? ” would be that there is a larger distribution of plasmids than currently known in various L. monocytogenes STs and the genetic content of plasmids will show a high degree of similarity among plasmids of a comparable subtype.

Plasmids and Survival of L. monocytogenes in Food and FPEs

Previous studies have found that L. monocytogenes is capable of survival and growth in a variety of both natural and man-made habitats, including (but not limited to) soil, marine and fresh water, silage, vegetation, sewage, food processing plants, and domestic and wild animals (Sauders et al., 2012; Ferreira et al., 2014). L. monocytogenes is also able to adapt to varying stress conditions found in these habitats, such as large temperature ranges, salinity concentrations, low pH, low water availability and a multitude of sanitizers (Sauders et al., 2012; Ferreira et al., 2014).

Persistence of L. monocytogenes has been observed in a variety of STs and facilities, and therefore is a challenge for food safety in a variety of food production areas

(Carpentier and Cerf, 2011; Ferreira et al., 2014; Larsen et al., 2014). Persistent L. monocytogenes is also believed to be the primary source of contamination in post- processing (Buchanan et al., 2017). It is therefore necessary to further elucidate the processes used to aid in persistence to increase food safety and cultivate mechanisms to control L. monocytogenes in the future.

Earlier studies analyzed the genomes of persistent L. monocytogenes strains and found the presence of strain-specific putative candidate genes for persistence, but these studies also indicated there was no common molecular or genetic mechanism found in all or most strains that could contribute to persistence (Holch et al., 2013;

Fagerlund et al., 2016; Fox et al., 2016; Ortiz et al., 2016; Knudsen et al., 2017). Multi- locus sequence typing (MLST) is widely used as the primary method for classifying and characterizing L. monocytogenes strains from clinical samples, food, or FPE.

Furthermore, whole-genome sequencing-based typing is now routinely used for

epidemiology and surveillance of food-borne pathogens and provides even higher resolution for differentiating strains based on single nucleotide polymorphisms (SNPs).

In-depth studies on L. monocytogenes epidemiology indicate that particular STs are predominantly found in food products, whereas other STs are more commonly found in human clinical samples and still others are isolated more frequently in FPEs (Carpentier and Cerf, 2011; Maury et al., 2016; Buchanan et al., 2017; Moura et al., 2017).

L. monocytogenes strains are typically sorted among of MLST sequence types or clonal complexes (CC), which are clusters of related MLST sequence types. Of these classifcations, STs 5, 8, 9 and 121 can be abundant in food and FPEs (Fagerlund et al.,

2016; Hingston et al., 2017b; Moura et al., 2017; Rychli et al., 2017). These L. monocytogenes STs have increased in abundance since the year 2000, with up to 90% of ST5, ST8, ST121, and ST204 strains harboring plasmids (Chenal-Francisque et al.,

2011; Martin et al., 2014; Fagerlund et al., 2016; Fox et al., 2016; Henri et al., 2016;

Maury et al., 2016; Moura et al., 2017; Rychli et al., 2017; Bergholz et al., 2018;

Muhterem-Uyar et al., 2018). Plasmid sizes ranged from 14 to 106 kbp and the plasmid- carrying frequency among these strains ranged from 28 to 90% (Kolstad et al., 1992;

Lebrun et al., 1992; McLauchlin et al., 1997; Harvey and Gilmour, 2001; Kuenne et al.,

2010; Schmitz-Esser et al., 2015b; Fagerlund et al., 2016; Hingston et al., 2017b; Rychli et al., 2017). A recent genome sequencing study conducted by Hingston et al. revealed the presence of plasmids in 55% of a diverse set of 166 L. monocytogenes isolates, with the majority of those plasmid-carrying strains belonging to the same ST, furthering the implication of the presence of L. monocytogenes plasmids that are ST-specific

(Hingston et al., 2017b).

L. monocytogenes strains are often exposed to variable stress conditions, where plasmids may aid in growth and survival (Cruz et al., 2014; Martin et al., 2014;

Fagerlund et al., 2016; Henri et al., 2016; Maury et al., 2016; Knudsen et al., 2017;

Maury et al., 2019). Even if the stress survival mechanisms of L. monocytogenes in general have been well-characterized, particularly those involving chromosomally encoded genes (Soni et al., 2011), the mechanism contributing to stress survival found on plasmids is not as understood. Due to these previous studies, and prior research conducted in our lab, we respond to the question “ Do plasmids contribute to survival of

L. monocytogenes in food and FPEs?” with the hypothesis that plasmids confer an advantage to survival when L. monocytogenes strains are exposed to stress conditions found in food and FPEs.

Genetic Content of L. monocytogenes Plasmids in Relation to Stress Survival

L. monocytogenes responds to food and FPE environmental stress conditions by activating various chromosomally-encoded mechanisms that confer stress tolerance

(Dutta et al., 2013; Müller et al., 2013; Harter et al., 2017; Bucur et al., 2018; Hingston et al., 2019b). These mechanisms may include antimicrobial resistance, disinfectant resistance, and transfer of genetic material among different bacteria from varied sources and origins. Recent studies on L. monocytogenes plasmids have identified novel genes that may contribute to survival of L. monocytogenes in food and FPEs, but these analyses were conducted on a sequence level or during genome sequencing projects. Some studies showed that L. monocytogenes plasmids confer increased tolerance to disinfectants, heavy metals, heat, or antibiotic resistance (Lebrun et al.,

1994a; b; Elhanafi et al., 2010; Li et al., 2016; Kremer et al., 2017; Pöntinen et al.,

2017). The plasmids carry a variety of genes, including putative multidrug resistance efflux pumps, as well as genes shown to be involved in resistance or stress responses, such as the transposon Tn 5422 , conferring cadmium resistance (Lebrun et al., 1994a),

NADH peroxidases (Hingston et al., 2019b) possibly involved in oxidative stress responses, or Clp-like proteases (Pöntinen et al., 2017) thought to be involved in general stress response, as well as increased heat tolerances. All of these genes may provide a benefit to L. monocytogenes for growth under suboptimal conditions in food and FPEs (Kuenne et al., 2010).

Besides functional characterization of selected genes of interest, multiple studies analyzing gene expression patterns have implicated the expression of chromosomal genes responsible for L. monocytogenes responses to temperature, oxidative, disinfectant, pH, and osmotic stresses (Oliver et al., 2009; Soni et al., 2011; Tessema et al., 2012; Casey et al., 2014; Tang et al., 2015a; Hingston et al., 2017b). However, to date, L. monocytogenes transcriptomic analyses have almost exclusively been conducted on L. monocytogenes isolates devoid of plasmids. Gene expression analysis studies can be useful to identify genes that might be involved in a certain phenotype of interest due to changes in gene expression patterns. Previous research from our lab has shown that L. monocytogenes plasmids pLM6179 and pLMR479a contribute to survival when L. monocytogenes strains were exposed to heat, acidic, salt and oxidative stress (CHAPTER 3)(Naditz et al., 2019).

With the exceptions of our research and a recently conducted study (Pöntinen et al., 2017), little data are available on the functional characteristics or the contributions of

L. monocytogenes plasmids to stress response, besides studies on antibiotic and disinfectant resistance. Furthermore, the study conducted by Hingston et al. provided indirect evidence of L. monocytogenes plasmid contribution to acid tolerance, based on a correlation of plasmid presence with increased stress tolerance. However, the authors did not examine plasmid-cured strains nor did they identify the responsible plasmid genes (Hingston et al., 2017b). Another recent study showed increased heat tolerance conferred by a ClpL protein encoded on pLM58, a 58-kbp L. monocytogenes ST9 strain plasmid (Pöntinen et al., 2017).

The 4.3-kb plasmid pLMST6, which confers increased tolerance towards the disinfectant benzalkonium chloride, is associated with more severe meningitis listeriosis cases (Kremer et al., 2017). The 77-kb plasmid pLM5578 was identified in the L. monocytogenes strain responsible for the 2008 Canadian listeriosis outbreak caused by

RTE meat, yet the function of the plasmid is currently unknown (Gilmour et al., 2010).

More recently, a lineage IV strain FSL J1-208, potentially responsible for an animal listeriosis outbreak, contained a novel 80 kb plasmid termed pLMIV (den Bakker et al.,

2012). A unique observation was that the plasmid annotation did not reveal genes involved in resistance to antibiotics or heavy metals (den Bakker et al., 2012). Instead, some of the genes on pLMIV encode members of a family of virulence-associated proteins, known as the internalin family (surface proteins used for the invasion of mammalian cells), indicating that pLMIV may be a putative virulence plasmid. Recently, it was demonstrated that a 21-kb region of pLMIV includes three putative internalins integrated into the chromosome of an Austrian listeriosis outbreak strain (Rychli et al.,

2014) contributes to virulence (den Bakker et al., 2012). These studies indicate a need

to better understand the role plasmids play, not only in food production and FPE- associated L. monocytogenes strains, but also those found in clinical L. monocytogenes strains.

Thus, transcriptomic analysis may provide insights into which L. monocytogenes plasmid genes might be involved in stress resistance. It may also identify novel candidate genes that permit L. monocytogenes persistence in food and FPEs. Thus, in response to “ Which plasmid genes are responsible for this contribution?” , we hypothesize transcriptome sequencing will reveal an upregulation in L. monocytogenes plasmid genes that aid in stress responses and will also identify potentially novel genes that might also be involved in persistence in the presence of environmental stresses.

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CHAPTER 2. A LARGE-SCALE SEQUENCING-BASED SURVEY OF PLASMIDS IN LISTERIA MONOCYTOGENES

Modified from a manuscript to be submitted for publication in Frontiers in Microbiology

Annabel L. Naditz a,b , Justin Anast a,b , Stephan Schmitz-Esser a,b

aDepartment of Animal Science, Iowa State University, Ames, IA, 50011 USA; bInterdepartmental Microbiology Graduate Program, Iowa State University, Ames, IA, 50011 USA

Abstract

Listeria monocytogenes causes the disease listeriosis and is responsible for a high economic cost due to food recalls, patient health career, and production losses. L. monocytogenes is known to contain plasmids, however, prior studies analyzing plasmids in Listeria consisted of a limited data set of 14 sequence strains. To ascertain the presence of plasmids among L. monocytogenes strains, an in-depth dataset was gathered, consisting of 1924 genomes from 14 sequence types (STs) collected over 60 years, from 32 countries, and 3 primary source types (i.e. food, clinical, environment).

Analysis of the dataset identified an average of 53% of all L. monocytogenes strains in the dataset contained a plasmid. The plasmid sizes ranged from 3 kb to 140 kb, but the majority of these fell within two plasmid subtypes. One subtype consisted of plasmids around 58 kb (± 1kb) and the other subtype was approximately 77 kb (± 1 kb). The genetic content in the plasmids were highly conserved and similar across all STs, with a minimum of 91.9% nucleotide identity.

Six previously identified stress-related genes were selected to assess the conservation of stress-related genes among L. monocytogenes plasmids. The data indicated the selected genes present in L. monocytogenes plasmids exhibited high

sequence conservation, but their presence in plasmids is highly variable depending upon the ST of the plasmid. Additionally, we also identified a novel set of seven genes that comprise part of a mercury-resistance operon that we hypothesize may increase mercury tolerance and has not previously been identified in L. monocytogenes . We provide the largest plasmid survey of L. monocytogenes to date, with a comprehensive examination on the distribution of plasmids among L. monocytogenes .

Introduction

The food-borne pathogen Listeria monocytogenes is of high concern in food production facilities since it is responsible for the disease listeriosis, which can be fatal, particularly for infants, pregnant women and immunocompromised individuals (Mead et al., 1999; Kaur et al., 2007; Scallan et al., 2011). L. monocytogenes can be isolated from food and ready-to-eat (RTE) foods, which typically are high in salinity or acidity and are often refrigerated. Many listeriosis outbreaks have been linked to L. monocytogenes contamination of RTE foods such as deli meat, cheeses, fruits or vegetables. L. monocytogenes can also be present and persist for long time periods in food production environments (FPEs) despite that FPEs regularly undergo harsh sterilization and cleaning techniques.

Extrachromosomal mobile genetic elements, such as plasmids, are often utilized by bacteria to enhance survival under stressful conditions. Plasmids can be transferred within and between bacterial species, lost, or kept if they provide an advantage to survival. Plasmids carry a variety of genes that may be beneficial for the microorganisms, such as genes contributing to stress, disinfectant and antibiotic

resistance. Previous studies have shown that plasmids can be found in many L. monocytogenes strains, with frequencies of carrying plasmids reaching up to 90% of the analyzed strains, but that the percentage of plasmid-carrying strains varies depending on the sequence type (ST) (Kuenne et al., 2010; Schmitz-Esser et al., 2015b; Fagerlund et al., 2016; Fox et al., 2016; Hingston et al., 2017b; Rychli et al., 2017; Bergholz et al.,

2018; Muhterem-Uyar et al., 2018). Prior L. monocytogenes studies have also indicated that within certain STs, such as ST121, L. monocytogenes strains can be found carrying virtually identical plasmids, yet these strains may have been isolated decades apart, or originate from different countries and different years (Schmitz-Esser et al., 2015b;

Rychli et al., 2017).

Whereas most L. monocytogenes plasmid research has focused primarily on antibiotic and disinfectant resistance, some recent studies indicate that some plasmids may confer an advantage to survival the stress encountered in FPE and RTE foods.

These advantages include increased tolerance to acidic stress, oxidative, heat, and cold stress (Hingston et al., 2017b; Pöntinen et al., 2017; Naditz et al., 2019). Some plasmids confer increased tolerance to disinfectants, such as quaternary ammonium compounds, and heavy metals, allowing L. monocytogenes to tolerate higher levels of cleaners and detergents when these compounds are used at sublethal concentrations

(Lebrun et al., 1994b; a; Elhanafi et al., 2010; Li et al., 2016; Kremer et al., 2017;

Pöntinen et al., 2017). Sublethal concentrations of disinfectants are often due to dosage failures or dilution of the disinfectants. Still other plasmids provide adaptability in the presence of detrimental conditions during the food process, such as high salinity, high

to low pH levels, lactic acid and fermentation compounds (Naditz et al., 2019), all of which could be present on the food products or during the food processing routine.

Previous research has suggested that L. monocytogenes isolates of certain STs might carry plasmids at a higher percentage than other STs, and that plasmid-carrying strains may be more abundant than previously thought (Schmitz-Esser et al., 2015b;

Rychli et al., 2017; Hingston et al., 2019b). However, these preceding studies into the presence of plasmids in the genus Listeria were limited, as only a comparatively low number of genomes of the ST of interest was available for analysis. Moreover, those studies have been limited to specific STs or stress conditions. For example, Kuenne et al. conducted a plasmid survey that examined 14 plasmids (Kuenne et al., 2010). This study revealed important genetic features of Listeria plasmids and is - until now – the only general overview on Listeria plasmids. In 2016, Fox et al. studied L. monocytogenes ST204 isolates and found plasmids in 86.6% of the strains (Fox et al.,

2016). A study conducted on 70 ST121 L. monocytogenes genomes observed 81.4% of the strains harbored almost identical plasmids (Rychli et al., 2017). Studies conducted in 2017 by Hingston et al. found that 55% of 166 isolates from various L. monocytogenes STs contained a plasmid (Hingston et al., 2017b). Today, thousands of

L. monocytogenes genomes are available, with metadata information available for many of those genomes.

The aim of this study was to perform a plasmid survey in Listeria monocytogenes with a much larger dataset than in previous studies (Kuenne et al., 2010; Hingston et al., 2017b). Our dataset consists of 1924 L. monocytogenes genomes comprising 14 different and highly abundant sequence types that were isolated from 32 countries and

multiple isolation origins (i.e. food, clinical, environment). The survey was conducted with a focus on the presence/absence of plasmids, sequence conservation between and within STs, as well as the gene content of these plasmids focusing on genes possibly important for survival in food and FPE.

Materials and Methods

Strain Selection

We performed an in-depth literature search on studies describing Listeria monocytogenes genomes focusing on assembled genomes (to reduce the bioinformatic burden associated with assembling hundreds or thousands of genomes) and with published metadata available for each of the genomes. Based on these criteria, 33 peer-reviewed research articles (Table 1) were selected for this study (as of September

2019). If the Multi-Locus Sequence Type (MLST) were unknown, genome sequences were submitted to the Center for Genomic Epidemiology (CGE) server (Thomsen et al.,

2016). Additionally, selection criteria for genomes included focusing on strains representing the most abundant L. monocytogenes STs found in food and FPE, based upon large scale studies conducted previously (Maury et al., 2016; Moura et al., 2017;

Bergholz et al., 2018; Painset et al., 2019). Strains were selected without a priori knowledge of plasmid harborage to reduce bias towards selecting for plasmid-carrying strains. Furthermore, our dataset included all genomes from the selected STs from each study. To provide a sufficiently high coverage for each L. monocytogenes STs included in this study, the minimum number of assembled genomes per ST was set to 50. The dataset also included both closed and draft genomes.

Table 1. Studies used for selection of L. monocytogenes isolates included in this study. All isolates selected were assembled genomes with metadata available and without a-priori knowledge of plasmid harborage. F: Food, C: Clinical, E: Environmental, U: Unknown.

Reference Title Country/countries Isolate year Isolation range source Allam et al. Whole Genome Sequences of Listeria monocytogenes Sequence South Africa 2017-2018 FCE (2018) Type 6 Isolates Associated with a Large Foodborne Outbreak in South Africa, 2017-2018 Bergholz et Determination of Evolutionary Relationships of Outbreak-Associated USA, Switzerland, Italy, 1966-2012 FCE al. (2018) Listeria monocytogenes Strains of Serotypes 1/2a and 1/2b by Canada, Finland, Whole-Genome Sequencing Denmark Burall et al. A clade of Listeria monocytogenes serotype 4b variant strains linked USA, Australia, Canada 2010-2015 FCE (2017) to recent listeriosis outbreaks associated with produce from a defined geographic region in the US Chen et al. Comparative Genomics Reveals the Diversity in Restriction- USA 1989-2011 FCE (2017) Modification Systems and DNA Methylation Sites in Listeria monocytogenes Chiara et al. Draft Genome Sequences of Six Listeria monocytogenes Strains Italy 2012 F (2014) Isolated from Dairy Products from a Processing Plant in Southern Italy 34 Fagerlund et Genome Analysis of Listeria monocytogenes Sequence Type 8 Norway, Denmark, China, 1994-2014 FE al. (2016) Strains Persisting in Salmon and Poultry Processing Environments Italy, Canada, and Comparison with Related Strains Switzerland Fox et al. Comparative Genomics of the Listeria monocytogenes ST204 Australia, Ireland 2000-2015 FCE (2016) Subgroup Halbedel et Whole-Genome Sequencing of Recent Listeria monocytogenes Germany 2007-2017 C al. (2018) Isolates from Germany Reveals Population Structure and Disease Clusters Hilliard et al. Genomic Characterization of Listeria monocytogenes Isolates Ireland 2013-2015 CE (2018) Associated with Clinical Listeriosis and the Food Production Environment in Ireland Hingston et Genotypes Associated with Listeria monocytogenes Isolates Canada, Switzerland 1990-2013 FCE al. (2017b) Displaying Impaired or Enhanced Tolerances to Cold, Salt, Acid or Desiccation Stress Horlbog et al. Whole-Genome Sequences of Six Listeria monocytogenes Strains Switzerland 2011-2014 F (2018) Isolated from Food

Table 1. Continued

Reference Title Country/countries Isolate year Isolation range source

Hyden et al. Whole genome sequence-based serogrouping of Listeria Austria, France, 1958-2015 FCE (2016) monocytogenes isolates Denmark, USA, Germany, Canada, UK, New Zealand Kim et al. Genetic diversity and virulence profiles of Listeria monocytogenes USA 2002-2014 FE (2018) recovered from bulk tank milk, milk filters, and milking equipment from dairies in the United States (2002-2014) Kwong et al. Prospective Whole-Genome Sequencing Enhances National Australia 1995-2015 FCE (2016) Surveillance of Listeria monocytogenes Lomonaco et The evolution and epidemiology of Listeria monocytogenes in Italy 2002-2015 FCE al. (2015) Europe and the United States Lopez- Whole-Genome Sequencing of Seven Listeria monocytogenes Spain 2004 E Alonso et al. Strains from Different Stages of a Poultry Meat Production Chain (2019) Maury et al. Uncovering Listeria monocytogenes hypervirulence by harnessing France, Italy, USA 1997-2012 FCE 35 (2016) its biodiversity Morganti et Processing-Dependent and Clonal Contamination Patterns of Italy 2011-2014 E al. (2016) Listeria monocytogenes in the Cured Ham Food Chain Revealed by Genetic Analysis Moura et al. Whole genome-based population biology and epidemiological Canada, Denmark, 1960-2015 FCE (2017) surveillance of Listeria monocytogenes Finland, France, Italy, Switzerland, UK, USA Muhterem- New Aspects on Listeria monocytogenes ST5-ECVI Predominance Austria 2010-2012 E Uyar et al. in a Heavily Contaminated Cheese Processing Environment (2018) Nowak et al. Persistent Listeria monocytogenes strains isolated from mussel New Zealand 1992-2011 FE (2017) production facilities form more biofilm but are not linked to specific genetic markers Nwaiwu et al. Draft Genome Sequences of Listeria monocytogenes , Isolated from Nigeria 2017 F (2017) Fresh Leaf Vegetables in Owerri City, Nigeria Orsini et al. Whole-Genome Sequences of Two Listeria monocytogenes Serovar Italy 2015-2016 C (2018) 1/2a Strains Responsible for a Severe Listeriosis Outbreak in Central Italy

Table 1 Continued Reference Title Country/countries Isolate year Isolation range source Ortiz et al. The Connection between Persistent, Disinfectant-Resistant Listeria Spain 2008, 2010 E (2016) monocytogenes Strains from Two Geographically Separate Iberian Pork Processing Plants: Evidence from Comparative Genome Analysis Pasquali et Listeria monocytogenes Sequence Types 121 and 14 Repeatedly China, France, 2004-2017 FCE al. (2018) Isolated Within One Year of Sampling in a Rabbit Meat Processing Germany, Italy, Poland, Plant: Persistence and Ecophysiology Spain, UK, USA Pirone- Genes significantly associated with lineage II food isolates of Listeria Chile, Germany, UK, 1975-2015 FCE Davies et al. monocytogenes USA (2018) Reimer et al. Shared genome analyses of notable listeriosis outbreaks, Canada 1981-2011 FCE (2019) highlighting the critical importance of epidemiological evidence, input datasets and interpretation criteria Rychli et al. Comparative Genomics of Human and Non-Human Listeria Australia, Austria, China, 1968-2015 FCE (2017) monocytogenes sequence type 121 strains Denmark, Egypt, France, Germany, Ireland, Italy, Rep. of 36 Moldavia, Romania, Russia, Spain, Switzerland, Turkey, USA Schjorring et Cross-border outbreak of listeriosis caused by cold-smoked salmon, Denmark, France 2015-2017 F al. (2017) revealed by integrated surveillance and whole genome sequencing (WGS), Denmark and France, 2015 to 2017 Stasiewicz et Whole-Genome Sequencing Allows for Improved Identification of USA 2001-2011 E al. (2015) Persistent Listeria monocytogenes in Food-Associated Environments Tasara et al. Genome Sequences of Listeria monocytogenes Strains Responsible Switzerland 2005, 2011 C (2016) for Cheese- and Cooked Ham Product-Associated Swiss Listeriosis Outbreaks in 2005 and 2011 Toledo et al. Genomic Diversity of Listeria monocytogenes Isolated from Clinical Chile 2008-2011 FCE (2018) and Non-Clinical Samples in Chile Zhang et al. Evolution and Diversity of Listeria monocytogenes from Clinical and China 2004-2012 FC (2016) Food Samples in Shanghai, China

Dataset Generation, BLAST Analyses and Plasmid Identification

Datasets for all included STs were created as Microsoft Excel sheets, listing strain, assembly accession number, source, location and year. Once sorted, the FASTA files for each of the strains were downloaded using their WGS (Whole Genome

Shotgun) or accession numbers from NCBI. A Basic Local Alignment Search Tool

(BLAST) database containing all contigs was created for each ST (Altschul et al., 1990).

Nucleotide BLAST (BLASTn) searches were conducted against the created database using selected reference plasmid sequences as the query and required a minimum of

90% nucleotide identity. The selected reference plasmid used as query against all STs was the plasmid from strain N1-011a, a ST3 environmental isolate from the United

States, collected in 2013. This reference plasmid was selected as it is the largest known

Listeria plasmid with a size of 148,959 bp and contained homologs of many plasmid- specific genes identified in plasmids among a variety of STs. For STs with described and sequenced plasmids, BLASTn searches were also performed with these as additional reference queries for further confirmation (Table 2).

Table 2. Selected L. monocytogenes plasmids used as queries for BLASTn analysis for determination of plasmid presence. ST4 and ST155 did not have a known reference query. Source types are F: food, C: clinical, E: Environmental, U: unknown.

ST Reference Plasmid Plasmid Country of Source GenBank or Reference strain size origin / year WGS (bp) accession number 1 LM-C-273 pLM-C-273 48,140 Canada C / KX467250- Liang et 2009 KX467259 al. (2016) 2 PIR00544 p1-3PIR00544 28,550 Switzerland U / CP025562- Portmann 2018 CP025564 et al. (2018) 3 N1-011a* pLMN1-011a 148,959 US E / CP006611 Chen et al. 2013 (2007) 5 4KSM pLM4KSM 90,542 Austria E / JYOJ01 Muhterem- 2011 Uyar et al. (2018)

Table 2 continued

ST Reference Plasmid Plasmid Country of Source GenBank or Reference strain size origin / year WGS (bp) accession number 6 ST6 pLMST6 4,268 Netherlands C / LT732640 Kremer et 2017 al. (2017) H7858 pLM80 82,248 US F / AADR01 Nelson et 1998 al. (2004) 7 2015TE24968 pl2015TE24968 57,530 Italy C / NZ_CP015985 Orsini et al. 2015 (2018) LM-F-131 pLM-F-131 81,666 Canada E / NZ_CM009923 Fortuna et 2011 al. (2018) 08 R479a* pLMR479a 86,652 Denmark F / HG813248 Schmitz- 1996 Esser et al. (2015a) 9 AT3E pLM58 58,523 Finland F / NZ_CP023753 Lunden et 1995 al. (2008) 14 LM1 pLM33 32,307 Spain F / GU244485 Margolles 1992 and Reyes- Gavilan (1998) 120 08-5578 pLM5578 77,054 Canada F / NC_013767 Gilmour et 2008 al. (2010) 121 6179 pLM6179 62,206 Ireland F / HG813250 Schmitz- 2000 Esser et al. (2015b) 204 2882 pLM2882 91,396 Australia F / LXQP01 Fox et al. 2000 (2016) * denotes plasmids utilized as queries for BLASTn searches against other STs for plasmid identification.

Once BLASTn results were obtained as tab-separated files and imported into

Microsoft Excel, the results were manually reviewed for each ST and filtered for

BLASTn comparison matches equal to or greater than 2000 bp alignment length with minimum nucleotide sequence identity of 90%. Using the filtered results, subsequent files were created to include only the strain name, accession number, contig number, contig alignment length and total alignment length. Those identified contigs were then considered as potential plasmids.

In a next step, to provide additional evidence that the potential contigs identified using BLASTn were plasmids, selected representative genomes (based on the BLASTn

results) for each ST were uploaded into the PATRIC database for annotation (Wattam et al., 2017). Plasmid replication protein RepA amino acid sequences from two different sequence types (GenBank accession number ZP_00231652.1 for RepA pLM80_ Listeria monocytogenes H7858, AOA49245.1 for pLM-C-273 Listeria monocytogenes ) representing a group 1 (pLM33) and group 2 plasmid (pLM80) (Kuenne et al., 2010) were compared against the representative genomes in PATRIC using BLASTp for confirmation of plasmid presence. RepA BLASTp results of > 98% amino acid identity and > 98% query coverage were considered as positive for identification of plasmid contigs. Additionally, for each ST, representative strains for each plasmid subtype were also annotated using Patric and only contigs with no known chromosomal genes (to eliminate potential chromosomal plasmid-gene insertion) were considered as putative plasmid contigs after manual review of the annotations and the BLASTp results.

Gene Content Analysis and Gene Conservation

Plasmid gene content was analyzed for conservation by reviewing the PATRIC annotations for the BLASTn and BLASTp-identified candidate plasmid contigs for the presence of known plasmid-encoded proteins. Presence of pertinent plasmid genes on the contigs were recorded for gene content analysis. Alignments of plasmids were done with MAUVE (Darling et al., 2010).

Results and Discussion

Overview of the Full Dataset

In total, from the 33 published datasets (Table 1), 1924 assembled genomes were included in this dataset representing 14 abundant L. monocytogenes STs. The ST with the lowest number of strains was ST204 (Figure 1). The STs with the highest number of strains were ST9, ST5, ST121, and ST1, which is consistent with previous reports indicating that these L. monocytogenes STs are among the most abundant L. monocytogenes STs (Maury et al., 2016; Moura et al., 2017; Bergholz et al., 2018;

Painset et al., 2019).

350

300

250

200

150

100

NumberofGenomes 50

0 1 2 3 4 5 6 7 8 9 14 120121155204 Sequence Types

Figure 1. Number of L. monocytogenes genomes per sequence type included in this study.

Isolates used in this study originated from 32 countries and from all 6 inhabited continents. Of the strains collected 42% (n=810/1924) selected were collected in the

United States, the most prevalent country of origin (Figure 2). This was followed by Italy at 26% of the strains (n=502) and Canada with 10% of the strains (n=201). Seven countries were represented with only a single isolate: Argentina, (ST1), Brazil (ST204),

Ecuador (ST6), Egypt (ST204), Finland (ST9) and Mexico (ST1). A low number of strains in our dataset from those countries may be due to lower reporting, a decrease in incidences, or differences in L. monocytogenes surveillance procedures resulting in fewer isolates subjected to whole genome sequencing.

Austria China Germany UK New Ireland Norway 1% 1% 1% 1% Zealand 1% 1% 1% Netherlands South Africa 2% 1% France Denmark 2% 1%

Chile 2%

<10 isolates 2%

Australia 4% United States 42% Switzerland 4% Canada 10%

Italy 26%

Figure 2. Country of origin of L. monocytogenes strains included on this study. Countries with less than 10 isolates per country are shown together (“<10 isolates”).

Isolates were obtained between 1958 to 2018 and to facilitate representation, isolates were grouped in sets of 5 years (Figure 3). Prior to 1990, there are no years with more than six isolates collected. The number of strains included were fairly consistent between 1986 to 2000, with an average of 43 strains per 5-year period.

There was a marked increase in sequenced isolates after that for each sequential 5- year set. Highest strain numbers originated from 2011-2015 (n= 791). It should be noted that the dataset from 2016-2018 contains only three years and has 365 isolates. The overall trend shows a higher number of included strains over time. However, this increase in sequence availability is most likely due to the advent of next generation sequencing technologies in 2006, resulting in the reduction of sequencing costs and the subsequent implementation of whole genome sequencing for routine L. monocytogenes surveillance in a number of countries.

800

700

600

500

400

300

200 NumberofGenomes 100

Year of Isolation

Figure 3. Year of origin of the L. monocytogenes genomes included in this study, grouped in five- year increments.

All 1924 isolates included were sorted according to source of isolation: clinical, environmental, food, or unknown (Figure 4). Clinical isolates included human and animal listeriosis isolates. Food-related strains were 37% (n=707) of the total dataset, whereas 34% (n=655) of the strains were environmental strains, including strains from the environment and food production environments, and 28% (n=542) of the strains were clinical isolates. 1% (n=20) of the strains were from an unknown source, as the metadata was not available. Thus, the strains used for this survey provide a roughly equal representation of strains from the three main sources of L. monocytogenes .

Unknown 1%

Clinical 28% Food 37%

Environment 34%

Figure 4. Isolates sorted by source of origin. Clinical sources included human or animal clinical samples, whereas food sources were strictly isolated from food products. Environmental sources included, but were not limited to, production facilities, environmental swabs, and equipment.

Summary of the Generated Dataset

For the first time, we present a large and representative dataset consisting of

1924 genomes from 33 peer-reviewed journal articles, that includes isolates from 14

STs, 32 countries and 60 years. Thus, this is by far the largest dataset used for a plasmid survey in L. monocytogenes. It contains representative isolates from all three characterized isolation sources, and high numbers of strains for each ST (at least 52) for examination of plasmid presence. Previous molecular studies examining the presence of plasmids were limited in scope, focusing either on specific sequence types or specific conditions. One of the largest conducted by Moura et al. (2017) used whole- genome-based surveillance to investigate L. monocytogenes population diversity. That data set contained 1696 isolates, on par with the size of our dataset. However, the study by Moura et al. (2017) primarily focused upon evolutionary aspects, virulence and stress resistance chromosomal genomic features of clinical and food isolates.

Moreover, the genetic content of plasmids was not examined, nor was there examination for plasmid presence.

Research conducted by Hingston et al. (2017b) examined 166 L. monocytogenes genome sequences primarily for classification based upon food-related stress conditions from primarily food and FPE isolates, with only six isolates representing clinical samples, from two countries. In comparison, our present study contains more than 10 times the number of genomes and provides an equal representation of isolates from food, clinical, and environmental sources.

Previous plasmid surveys were primarily focused on strains associated with outbreaks using clinical isolates, leaving a gap in knowledge on putative plasmid genes

and their molecular function in isolates from different sources. Moreover, despite the fact that up to 90% of the strains were reported to contain plasmids, the genetic content of those plasmids remained largely unexamined.

It should be noted however that there remain some limitations in the scope of this dataset. Requirements for nucleotide BLASTn comparisons are inherently more rigid, requiring a match for highly similar sequences, thereby leaving the potential open for more distantly related plasmids to be missed in the BLASTn analysis. This potential issue was mitigated using plasmid reference sequences from different STs, with the intention of finding plasmids within one ST that may carry a shared, and maybe partial, relatedness to plasmids from different ST (Table 2). Previous reports have shown that

L. monocytogenes plasmids are highly conserved (with more than 90% nucleotide identity) and have a modular structure (Kuenne et al., 2010; Fox et al., 2016; Hingston et al., 2017b; Rychli et al., 2017). Thus, BLASTn should reliably identify plasmid contigs in L. monocytogenes . Additionally, BLASTp searches for the plasmid replication protein

RepA provides an additional approach to identify plasmid contigs since homologs with lower similarity can be detected, permitting the identification of subgroups of a putative plasmid family.

Presence of Plasmids

Overall, out of the 1924 isolates, 53% were identified as harboring potential plasmids using the plasmid pLMN1-011a as well as ST-specific plasmids mentioned in

Table 2 as reference queries (Table 3). The highest percentage of putative plasmids was found in ST121 with 92% of the 287 strains carrying possible plasmids. This is

similar to what has been previously reported for ST121, as a prior study found 81.4% of analyzed ST121 isolates carried a plasmid (Rychli et al., 2017). ST5, ST3, ST8, and

ST204 also showed high abundances of potential plasmids in 85%, 78%, 75%, and

69% of the strains, respectively. The high abundance of potential plasmids in ST8 is supported by preceding research that showed 6 out of 6 strains contained a plasmid identical to the reference plasmid pLMR479a. However, those results were based on a much smaller dataset, as the research was primarily focused on comparison with other

ST8 strains (Fagerlund et al., 2016). The results obtained here for ST204 are similar to what has been indicated by Fox et al., who found that 86.7% of the ST204 isolates studied carried plasmids (Fox et al., 2016). However, the study by Fox et al. only examined 15 strains, whereas our dataset for ST204 contains a greater volume of sequences and is based on 52 ST204 isolates. In L. monocytogenes STs 14, 155, 120,

6, and 9 between 35 and 50% of the strains contained plasmids. A low (<15%) abundance of plasmids was found in the ST1 and ST2 strains in this study.

Interestingly, plasmids were not identified in ST4.

Table 3. Total number and percentage of potential plasmids identified for each L. monocytogenes ST. L. monocytogenes Number of total Number of strains with Percentage of putative sequence type strains per ST potential plasmids per ST plasmids per ST 121 286 264 92% 5 287 243 85% 9 323 159 49% 6 147 66 45% 8 67 50 75% 3 59 46 78% 7 108 44 41% 204 52 36 69% 155 74 29 39% 120 62 25 40% 14 72 25 35% 2 124 18 15% 1 209 13 6% 4 54 0 0% Total 1924 1018 53%

The 1018 identified putative plasmids were also arranged according to source of isolation. In contrast to equal representation of L. monocytogenes strains among the three main sources, the source of origins for the strains containing putative plasmids are more unevenly distributed. Of the putative plasmids, 48% (n=487) of the plasmids originated from environmental strains. Plasmids in food-related strains account for 39%

(n=391) putative plasmids and plasmids from clinical strains make up 13% (n=136), with the remaining 1% (n=7) of plasmids originating in a strain from an unknown source.

Unknown 1% Clinical 13%

Environment 48%

Food 39%

Figure 5. Putative plasmids sorted by source of origin. Clinical sources included human or animal clinical samples, whereas food sources were strictly isolated from food products. Environmental sources included, but were not limited to, production facilities, environmental swabs, and equipment.

In a few sequence types, two RepA proteins were found in the same strain but on different contigs, each matching to a different group RepA protein. This was found for example in strain P61-18, a ST5 strain, FSL R8-5805, a ST6 strain, and Lmo33H08, a

ST9 strain. This may indicate the possibility of two plasmids present in these strains,

which has been reported previously in other L. monocytogenes strains (Lebrun et al.,

1992; Hingston et al., 2017b), although the presence of two plasmids has not been functionally confirmed and appears to be an uncommon occurrence. Similarly, also the results obtained here indicate that – if present at all – the presence of multiple plasmids seems to be rare in L. monocytogenes . Therefore, further research into the possibility of multiple plasmids within L. monocytogenes strains is necessary.

One limitation of this method of plasmid determination remains that the analyses are conducted in silico only. All analysis conducted has been sequence-based, and the actual strains are not available in the laboratory Thus, we cannot and have not been able to verify experimentally that these strains do in fact contain plasmids. Therefore, these plasmids should be considered as putative plasmids. Moreover, most of the genomes included in this analysis are draft genomes and have not been closed, often resulting in chromosomal and plasmid assemblies spread across multiple contigs. This results in difficulties with plasmid identification in strains with multiple contigs identified using BLASTn and Patric annotation: are these contigs truly part of the plasmid, or are these segments of a plasmid that have been incorporated into the chromosome?

Incorporation of plasmid genes and parts of plasmids into the chromosome has been documented previously in L. monocytogenes (Rychli et al., 2014), particularly for genes that provide a consistent benefit to the host. Nevertheless, in spite of the limitations mentioned above, the combination of multiple BLAST searches, annotation and analysis of representative plasmid subtypes and the previously reported high conservation and modular structure of L. monocytogenes plasmids, suggests that the results obtained reliably identified putative plasmid contigs.

Size Range of Plasmids

Overall, the predicted plasmid sizes based on the sizes of putative plasmid contigs ranged from approximately 3 kb (ST6) to 176 kb (ST3). Within different STs, plasmid size range trends were apparent. Two commonly identified plasmid sizes were found in different STs: ST1, ST3, ST5, ST6, ST7, ST9, and ST121 had plasmids of approximately 56 kb (± 1kb) (Table 4). In comparison, ST2, ST5, ST6, and ST120 had plasmids of approximately 77 kb (± 1 kb). Based on these different plasmid sizes and differences between the plasmids found in BLAST searches, these different plasmids within a ST can be considered plasmid subtypes. This agrees with the findings of

Kuenne et al., where plasmids in L. monocytogenes differentiated by the RepA replication protein clustered into two group sizes, one ranging between 32-57 kb and the other ranging between 77-83 kb (Kuenne et al., 2010). All STs had a minimum of two different plasmid general sizes, while some STs had a wider variety in plasmid sizes, resulting in plasmid sizes that were not considered a member of an ST plasmid subtype. The plasmid sizes in ST9 and ST5 were particularly variable, with 10 and 12 different plasmid size subtypes, respectively. These results are similar to those found in previous studies that stated the plasmids in L. monocytogenes variable in sizes were still highly modular and contain highly conserved sequences – called modules - with conservation in some cases as high as 99.7% average nucleotide identity (Schmitz-

Esser et al., 2015b; Fox et al., 2016; Rychli et al., 2017).

Table 4. Plasmid size ranges for different L. monocytogenes STs. Plasmid subtypes shown were identified as plasmids with that size in at least 2 strains per size subtype. Sizes are approximate, within ± 2 kb. Outliers (based on plasmid size) were present in all plasmid types and listed as “Other”.

SEQUENCE TYPE Plasmid Size ST1 ST2 ST3 ST5 ST6 ST7 ST8 ST9 ST14 ST120 ST121 ST155 ST204 4 kb 7 18 kb 12 25-27 kb 12 37 28-32 kb 6 20 3 37-40 kb 6 2 8 43-45 kb 3 2 3 48-50 kb 6 9 14 19 53 kb 40 55-57 kb 2 15 9 7 10 20 15 58-60 kb 10 170 61-63 kb 14 45 64-66 kb 4 4 6

70 kb 2 2 2 48 75 kb 2 77-78 kb 3 4 5 17 80-82 kb 8 18 20 6 3 86-88 kb 35 3 12 89-92 kb 12 18 6 11 19 97-100 kb 10 6 3 >105 kb 17 3 Other 5 9 7 89 10 8 12 33 2 2 34 6 14 Total 13 18 46 243 66 44 50 159 25 25 264 29 36

Sequences of representative plasmids of the most common plasmid subtypes were analyzed using Mauve alignments to determine the relatedness and conservation of the plasmids. It has been shown that most plasmids within a ST can be separated into at least two subtypes based upon nearly identical genetic content, but across subtypes the plasmids have a higher degree of divergence (Kuenne et al., 2010). The same general division into subtypes is seen here in the Mauve alignments for multiple

STs, indicated by the modularity of L. monocytogenes plasmid genes for plasmids in the same subtype and the same ST (Figure 6). Some STs show a high degree of similarity within their plasmid subtypes, as e.g. in the ST121 plasmids. Conversely, there is a high degree of variability across plasmid subtypes, as seen e.g. in ST5 and ST9 plasmids.

However, the presence of such conserved regions within plasmid subtypes provides strong evidence for the modularity of Listeria plasmids. This is in line with what has been previously described for L. monocytogenes plasmids, irrespective of STs (Kuenne et al., 2010; Fagerlund et al., 2016; Hingston et al., 2017b; Rychli et al., 2017).

Overall Conservation of Plasmids

The minimum nucleotide identity among the plasmids among all strains was

91.9% nucleotide identity. Thirteen out of the fourteen STs had as high as 100% nucleotide identity for some plasmids. The average similarity among all strains in all STs was 92.3% nucleotide identity. ST3 and ST155 plasmids were the most conserved, with a range of 99.8 to 100% and 99.9 to 100% nucleotide identity range, respectively.

Conversely, ST7 plasmids were the most diverse, with a percent nucleotide identity range of 91.9% to 100% (Table 5).

A B

C D 50

Figure 6. Mauve alignments of representative plasmid subtypes within L. monocytogenes STs. A: ST1 plasmids, B: ST5 plasmids, C: ST9 plasmids, D: ST121 plasmids. Homologous regions are shown in the same color. The height of the similarity profile within each block corresponds to the average level of conservation in that region of the plasmids. Mauve alignments were conducted for the remaining STs as well and are shown in the appendix.

Table 5. Percent nucleotide identity ranges of plasmid-carrying strains based on ST.

ST % nucleotide identity range Average % nucleotide identity 1 95.7 - 100 99.6 2 95.8 - 100 99.1 3 99.8 - 100 99.9 5 98.8 - 100 99.9 6 95.7 - 100 99.3 7 91.9 - 100 98.8 8 92.8 - 100 98.4 9 95.4 - 100 99.7 14 96.4 - 100 99.8 120 95.7 - 99.9 99.3 121 95.8 - 100 98.8 155 99.6 - 100 99.9 204 93.8 - 100 99.7

Data from various studies correspond with those from this study, that plasmids within a ST can be highly similar and, in some cases, nearly identical (Kuenne et al.,

2010; Fagerlund et al., 2016; Hingston et al., 2017b; Rychli et al., 2017). Moreover, while some variable regions among plasmids were found, the high nucleotide identities indicate that L. monocytogenes plasmids exhibit a modularity with highly conserved regions shared between some L. monocytogenes plasmids, irrespective of ST, although plasmids may still be sorted into two basic plasmid groups based on their replication protein A phylogeny.

Presence and Conservation of Stress-Response Plasmid Genes Across STs

For most of the L. monocytogenes plasmid genes, putative function based on sequence similarity cannot be deduced. Analysis of the potential plasmid gene annotations identified a selection of genes often located on L. monocytogenes plasmids which have been found or suggested to be involved in various stress response mechanisms. Due to this commonality, we selected six loci of particular interest

because of their role in stress survival (Table 6). These loci of interest were encode a multicopper oxidase (MCO) (Sitthisak et al., 2005), cadmium efflux proteins (Lebrun et al., 1994b), BcrABC proteins for quaternary ammonium compound resistance (Dutta et al., 2013), multidrug resistance proteins EbrAB, heat shock protein ClpL (Pöntinen et al., 2017), and an NADH peroxidase (Npx) (Hingston et al., 2019b), as well as a locus that serves as a novel riboswitch identified during transcriptome sequencing (Chapter

4). While only some of these genes of interest have been functionally characterized, based on annotation, they are predicted to play a role in stress survival and may confer increased survival in a variety of stress conditions, including oxidative, acidic, heat, cold, and osmotic stress (Kuenne et al., 2010; Hingston et al., 2017b; Naditz et al., 2019).

Table 6. Selected plasmid-encoded genes of interest involved in stress response.

Plasmid stress response genes Proposed/demonstrated function Multicopper oxidase (MCO) Copper homeostasis; mediates oxidative stress tolerance in Staphylococcus aureus Cadmium resistance transposon Tn 5422 Cadmium detoxification and resistance BcrABC Quaternary ammonia compound (benzalkonium chloride) resistance EbrAB Multidrug resistance efflux pump ClpL Heat stress response in L. monocytogenes plasmids; general stress response NADH peroxidase (Npx) Reduces hydrogen peroxide, mitigating oxidative stress Zinc riboswitch Intergenic region linked to heavy metal binding

Selected reference sequences identified in other L. monocytogenes plasmids were used for all BLAST analyses for the plasmid gene analyses. Furthermore, BLASTn results of these loci were analyzed for nucleotide identity for all the genes, to evaluate if these were highly similar or identical. For all selected stress response genes, the nucleotide identity was 99% or greater, showing that the genes used as queries and the genes identified on the putative plasmids are highly conserved or essentially identical.

Multicopper oxidases (MCOs) are known to confer copper homeostasis but also contribute to increased oxidase activity in the presence of other substrates, as well as possibly play a role in iron transport (Askwith et al., 1994; Huston et al., 2002).

Moreover, the MCO identified on L. monocytogenes plasmids share 95% amino acid identity with a homolog of the Staphylococcus aureus strain ATCC12600, which is involved in both copper resistance and oxidative stress response (Sitthisak et al., 2005).

It should, however, be noted that the Listeria MCO homologs are shorter than those of

S. aureus. Additionally, an identical homolog of a putative MCO was upregulated when exposed to mild (pH 5) acidic stress (Hingston et al., 2019a) and under a more severe

(pH 3.4) acidic stress in our transcriptome analyses (Chapter 4), indicating MCOs may have a more diverse function in stress response than copper homeostasis and oxidative stress response. In this dataset, the presence of a MCO gene is variable, with an average of 40% of all putative plasmids carrying this gene, with a range from 0%

(ST155) to 96% (ST8 and ST120) (Table 7).

Cadmium detoxification and the role of cadmium ATPases in this process have been studied in depth in L. monocytogenes in the studies on the transposon Tn 5422

(Lebrun et al., 1994a). Furthermore, cadmium ATPases are often found within transposons in both plasmids and chromosomes in L. monocytogenes (Lebrun et al.,

1994b). In a prior study, the transfer of the L. monocytogenes cadAC plasmid genes to a Bacillus subtilis cadmium-sensitive strain, they induced cadmium resistance (Lebrun et al., 1994b). Here, the cadmium-resistance genes cadAC found on plasmid-encoded transposon Tn 5422 were used as the reference query. cadAC genes were found in an average of 40% of the plasmids. All of the ST3 and ST120 plasmids contained the

cadmium ATPases encoded by cadA , whereas none of the ST155 plasmids contained the cadmium ATPases. ST121 and ST8 plasmids also had a high percentage of plasmids carrying the cadmium ATPases, with 98% and 96%, respectively. These results confirm previous research where plasmid-carrying cadmium resistance was functionally confirmed in 95.3% of the L. monocytogenes plasmids studied (Lebrun et al., 1992; Lebrun et al., 1994b; a; McLauchlin et al., 1997; Harvey and Gilmour, 2001). It also corresponds with studies conducted by Kuenne et al. (2010), which found 95% of the L. monocytogenes strains studied were resistant to cadmium; all L. monocytogenes plasmids analyzed were carrying the cadAC genes, and the L. monocytogenes cadAC plasmid genes were similar to those found in S. aureus.

Disinfectants such as quaternary ammonium compounds (QAC) are often used in food production or retail facilities, or household products (Merianos, 1991; McDonnell and Russell, 1999). Therefore, disinfectant resistance and tolerance to QACs can be beneficial for L. monocytogenes strains that are present in food production facilities

(Mereghetti et al., 2000; Kovacevic et al., 2016; Martinez-Suarez et al., 2016; Moretro et al., 2017). Research has shown the presence plasmid-mediated benzalkonium chloride resistance genes on plasmids is probable, particularly the cassette bcrABC , which located on a plasmid-encoded transposon (Romanova et al., 2006; Elhanafi et al.,

2010). The bcrABC cassette encodes two transporters ( bcrBC ) and a regulator ( bcrA ) conferring increased benzalkonium chloride tolerance (Elhanafi et al., 2010; Dutta et al.,

2013). In this dataset, bcrABC was found on average in 44% of the plasmids. All ST155 plasmid contained the bcrABC cassette, and ST14 and ST204 also showed a high percentage of plasmids carrying the bcrABC cassette (96% and 92%, respectively). In

contrast, ST8 and ST120 plasmids did not contain bcrABC and only 1% of the ST121 plasmids harbored bcrABC . Previous studies found that the bcrABC cassette is highly conserved, with variability primarily occurring in the flanking transposase regions.

Moreover, the bcrABC cassettes were either found on highly similar plasmid types or were encoded in the chromosome, adjacent to novel segments (Dutta et al., 2013). The presence of bcrABC on putative plasmid contigs was verified using the BLASTn results described above used to identify plasmid contigs.

ClpL, a heat shock protein (HSP), found in the L. monocytogenes plasmid pLM58, has been demonstrated to confer increased heat stress tolerance (Pöntinen et al., 2017). Additionally, other ClpL proteins have shown to be involved in other stress response mechanisms, including acid stress response, detergent and penicillin resistance, and long-term survival (Kajfasz et al., 2009; Tran et al., 2011b; a). A ClpL homolog found in Lactobacillus shared 67% amino acid identity with the pLM58 ClpL, and its expression was upregulated during acid stress (Wall et al., 2007). These data suggest that the L. monocytogenes plasmid-encoded ClpL proteins may also be involved in responses to other stresses. In line with this, the clpL gene in L. monocytogenes ST121 plasmid pLM6179 was significantly upregulated in response to lactic acid stress (pH3.4) (Chapter 3). All L. monocytogenes ST3 strain plasmids contain the clpL gene, as do 98% of the ST121 strains. Conversely, ST8 and ST155 plasmids do not carry the clpL gene. It should be noted that genes encoding other HSPs distantly related to ClpL have been identified on the Patric annotations for some plasmids; these include genes encoding ClpB- and ClpE-like proteins. However, these other clp genes

on L. monocytogenes plasmids have not been characterized functionally and have not been analyzed here in more detail.

L. monocytogenes strains can be exposed to oxidative stress, when exposed to of certain cleaners and disinfectants that contain quaternary ammonium compounds, such as benzalkonium chloride, bleach, or hydrogen peroxide. Moreover, exposure to sublethal concentrations of hydrogen peroxide appears to mitigate oxidative stress and induce oxidative stress adaptation, aiding in increased survival when exposed to subsequent potentially lethal concentrations (De Abrew Abeysundara et al., 2016).

Thus, plasmids carrying proteins that potentially aid in oxidative stress response, such as the putative NADH peroxidases (Npx), might be advantageous to the host during oxidative stress. Interestingly, all strains had at least a small percentage of plasmids carrying the npx gene, with an average of 61% of the plasmids in the dataset harboring npx . The npx gene was found in the highest percentage among ST8 and ST120 plasmids, with 96%, as well as in 93% of ST155 plasmids. Only 2% of ST121 plasmids encode the Npx protein.

Transcriptomic research in our lab examining the gene expression of L. monocytogenes strain R479a exposed to lactic acid stress identified a putative zinc riboswitch (Chapter 4). The majority of the reads mapped to this intergenic putative zinc riboswitch and expression of this noncoding region were significantly upregulated during acidic stress. The riboswitch shared 56% nucleotide identity to the RFAM family

RF02683, a family recently described as nickel-cobalt riboswitches involved in transition metal resistance. Firmicutes , such as Clostridiales and Erysipelotrichaceae utilize

transition metal riboswitches that bind to ions such as zinc, cobalt, and nickel, for detection and response to toxic concentrations of heavy metals (Furukawa et al., 2015).

However, it should be noted that the function of this putative L. monocytogenes plasmid-encoded riboswitch still unknown. Using the putative riboswitch identified in

R479a as a query sequence, we determined that nearly 60% of the plasmids in this dataset contain this putative riboswitch. Thus, overall, this putative novel riboswitch is highly abundant in L. monocytogenes plasmids. It was detected in 96% of ST120 plasmids and 93% of ST155 plasmids. Additionally, a high percentage of other ST plasmids also harbored the zinc riboswitch, including 88% of the ST8 plasmids (the ST of the query strain) and 86% of ST204 plasmids. Conversely, ST121 plasmids had by far the lowest presence with 3% of the ST121 plasmids carrying this putative riboswitch.

Interestingly, the functionally characterized homologs of these riboswitches in

Firmicutes are encoded on the chromosome (Furukawa et al., 2015). Notably, these riboswitches are found upstream of putative heavy metal transporters that correspond with the heavy metals used by the riboswitches for binding. Also, whereas the functionally characterized riboswitches and the L. monocytogenes homologs are highly similar, their adjacent transporters show no similarity, indicating these transporters are highly specified in relation to the corresponding riboswitches (Chapter 4).

Table 7. Percentage of selected plasmid-encoded stress response genes, by ST. All plasmid- encoded genes share more than 99% nucleotide identity.

ST mco cadA/C bcrABC clpL npx zinc riboswitch 1 77% 77% 8% 77% 38% 38% 2 8% 31% 50% 17% 39% 39% 3 62% 100% 4% 100% 43% 41% 5 21% 39% 56% 32% 64% 64% 6 3% 3% 78% 3% 46% 44% 7 43% 43% 57% 23% 45% 45% 8 96% 96% 0% 0% 96% 88% 9 33% 87% 25% 69% 64% 60% 14 10% 10% 96% 8% 80% 80% 120 96% 100% 0% 76% 96% 96% 121 2% 98% 1% 98% 2% 3% 155 0% 0% 100% 0% 93% 93% 204 16% 14% 92% 14% 86% 86% Average 36% 40% 44% 40% 61% 60%

In addition to identifying known genes demonstrated or suggested to be involved in stress response, our dataset can also be used to identify novel genetic features on L. monocytogenes plasmids. A putative mercury resistance locus was detected in the plasmid of L. monocytogenes ST6 strain FDA00012105 by manual review of the Patric annotations (Figure 7). This locus probably represents an operon encoding the following proteins: MerR1 - a mercuric resistance operon regulatory protein, MerE - a mercury transporter identified in other Firmicutes, MerT - a mercuric transport protein, MerP - a mercuric transport protein periplasmic component, MerA - a mercury (II) reductase,

MerR2- a mercury-family transcriptional regulator, and MerB - an organomercurial lyase. Tolerance of L. monocytogenes strains to mercury has been described in only one study (Lebrun et al., 1992). But, until now, no data have been published regarding

merR1 merE merT merP merA merR2 merB

Figure 7. Probable genes of a novel putative mercuric resistance operon in L. monocytogenes plasmid of strain FDA00012105. Genes were located on a single contig, in order. Genes are drawn to scale.

the molecular mechanisms of mercury resistance in L. monocytogenes . A function of this locus in mercury resistance will need to be verified in future studies.

The size of the dataset, with 1924 strains comprising of 1018 plasmid-carrying strains, precluded identifying novel plasmid genes via manual review of the annotation of plasmid contigs. The results nevertheless revealed the both known and novel genes using this dataset. However, further research and identification of novel genes requires analysis of the annotations as well as a more in-depth protein analysis, which is not feasible for all plasmids within a large dataset of this size. Automated annotations can be inaccurate and require manual verification of genes of interest. The use of representative strains for each ST could have missed novel genes in other strains. For analysis of known target genes of interest speculated to be present on L. monocytogenes plasmids, this is a large and comprehensive data set which can be utilized using both BLASTn and BLASTp analyses allowing for high throughput searches.

Conclusion

Here, we present an in-depth comparative analysis of the presence of plasmids across a variety of STs, countries, years, and isolation sources based on 1924 L. monocytogenes genomes. Only assembled genomes from peer-reviewed articles were included to capture the metadata and the gene content of plasmids, it was decided to include

We determined that an average of 53% of the 1924 L. monocytogenes strains in this dataset harbored plasmids, ranging from 0% to 92% of the isolates, depending on

ST. Some STs such as ST5 and ST121 have a higher percentage of strains that harbor plasmids, whereas ST4 did not have any strains that carry plasmids. This indicates that some L. monocytogenes STs are more likely to carry plasmids than other STs.

Stress genes identified in previous studies are conserved across STs, but were not uniformly or consistently found in all STs and plasmids. Classification of plasmids into subtypes was apparent, with very high genetic conservation within the subtypes, and more diverse genetic arrangements across subtypes. Some STs exhibited greater variation in genetic content, as seen e.g. in ST9. Our results confirm a high degree of modularity of L. monocytogenes plasmids.

All plasmids had greater than 91.9% nucleotide identity, indicating a high degree of similarity. Two STs, ST3 and ST155, had nearly 100% nucleotide identity, indicating plasmids identified in this STs are almost identical. This may possibly be due to strong selective pressures of the particular environments isolates experienced.

As well as variation in plasmid harborage in L. monocytogenes STs, there also appears to be a high variety among the genes that are present on those plasmids. This is not necessarily surprising, as different strains may derive from unique niches and face differing environmental conditions. We also identify a novel putative mercury resistance operon on L. monocytogenes ST6 strain FDA00012105 plasmid, indicating the usefulness of this dataset for detection of both putative plasmid genes and novel genetic elements.

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CHAPTER 3. PLASMIDS CONTRIBUTE TO FOOD PROCESSING ENVIRONMENT- ASSOCIATED STRESS SURVIVAL IN LISTERIA MONOCYTOGENES ST121, ST8, AND ST5 STRAINS

Modified from a manuscript published in International Journal of Food Microbiology

Annabel L. Naditz ab , Monika Dzieciol c, Martin Wagner c,d , Stephan Schmitz-Esser a,b

aDepartment of Animal Science, Iowa State University, Ames, IA, 50011 USA; bInterdepartmental Microbiology Graduate Program, Iowa State University, Ames, IA, 50011 USA; cInstitute for Milk Hygiene, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austia; dAustrian Competence Center for Feed and Food Quality, Safety and Innovation (FFoQSI), Technopark C, 3430 Tulln, Austria

Abstract

Listeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis and is commonly isolated from food and food production facilities. Many L. monocytogenes strains contain plasmids, though the contributions of plasmids to survival in food production environments is unknown. Three L. monocytogenes ST5,

ST8, and ST121 strains containing plasmids, which harbor putative stress response genes, were cured of their plasmids. Wildtype (WT) and plasmid-cured strains were exposed to disinfectant, oxidative, heat, acid, or salt stress. After stress exposure, cells were plated for colony forming unit (CFU) counts to determine survivors. L. monocytogenes WT strains exposed to 0.01% (vol/vol) H 2O2, 1% (vol/vol) lactic acid, and 15% (wt/vol) NaCl, pH 5 showed significantly higher counts of survivors compared to the plasmid-cured strains. The number of survivors for the ST5 WT strain exposed to

10 µg/mL benzalkonium chloride (BC) was significantly higher than in the plasmid-cured strain. The ST8 and ST5 strains were exposed to elevated temperature (50° and 55°C

respectively); only the ST5 WT strain had significantly higher numbers of survivors than the plasmid-cured strains. Our data revealed that L. monocytogenes ST5, ST8, and

ST121 plasmids contribute to tolerance against elevated temperature, salinity, acidic environments, oxidative stress and disinfectants.

Introduction

In food production facilities, food-borne pathogens are of high concern and can – when consumed – cause a variety of food-borne illnesses, ranging from relatively mild symptoms such as nausea, vomiting, or diarrhea to severe symptoms; some foodborne illnesses can be life-threatening (Mead et al., 1999; Kaur et al., 2007; Scallan et al.,

2011). Food-borne pathogens are constantly exposed to environmental stresses during food processing, including high or low temperatures, high salinity, acidity/alkalinity and low nutrient availability, all of which temper their ability to survive (Leistner and Gorris,

1995; Gandhi and Chikindas, 2007; Larsen et al., 2014; Schirmer et al., 2014).

Furthermore, the routine cleaning and disinfection procedures in food processing environments (FPEs) provide additional challenges for microorganisms such as detergents and disinfectants resulting in toxic conditions for bacterial growth and survival (Barker et al., 2003; Ratani et al., 2012). Food-borne pathogens are also exposed to food-specific stress conditions such as varying levels of salinity or acidity due to an assortment of compounds, such as acetic acid, lactic acid, sodium chloride, calcium chloride, or other fermentation products (Gahan et al., 1996; O'Driscoll et al.,

1996; Davies et al., 1997; Luna-Guzman and Barrett, 2000). Among food-borne pathogens, L. monocytogenes is of particular concern due to its presence in ready-to-

eat food, its capability to survive in food and FPEs, and the high mortality of listeriosis

(Allerberger and Wagner, 2010). L. monocytogenes is known to colonize niche areas such as drainage and hard-to-clean surfaces, which allow the bacteria to survive or even proliferate and thus, making it difficult to completely eradicate from FPEs (Ferreira et al., 2014).

Persistence, as used in this article, is defined as the repeated occurrence of genetically indistinguishable L. monocytogenes strains in the same food production facility over a long period of time (Carpentier and Cerf, 2011; Ferreira et al., 2014).

Furthermore, identical strains may be found in the same time period but in unrelated facilities, indicating that persistence of L. monocytogenes is not necessarily plant specific (Autio et al., 2002).

The L. monocytogenes strain classification via multi-locus sequence types (ST) is widely used for characterization of strains from clinical samples, food or FPEs. In-depth epidemiological studies have identified STs that are found predominantly in human clinical samples, while other STs have been shown to be particularly abundant in food and FPEs (Maury et al., 2016). The L. monocytogenes sequence types ST121, ST5 and

ST8, can be highly abundant in food and FPE, and therefore exposed to variable stress conditions (Cruz et al., 2014; Martin et al., 2014; Fagerlund et al., 2016; Henri et al.,

2016; Maury et al., 2016; Knudsen et al., 2017). L. monocytogenes strains isolated from food and FPEs are often found to contain plasmids. Plasmid frequencies ranged from

28% to 81% and plasmids sizes ranged from 14 to 106 kbp (Kolstad et al., 1992; Lebrun et al., 1992; McLauchlin et al., 1997; Harvey and Gilmour, 2001; Kuenne et al., 2010;

Schmitz-Esser et al., 2015b; Fagerlund et al., 2016; Hingston et al., 2017b; Rychli et al.,

2017). Some Listeria plasmids can be identical and can be recovered from strains over multiple years and from different geographic locations, indicating a high level of conservation of some Listeria plasmids (Kuenne et al., 2010; Schmitz-Esser et al.,

2015b; Fox et al., 2016; Rychli et al., 2017; Muhterem-Uyar et al., 2018). However, a lower level of conservation of plasmids was found e.g. in ST8 strains (Fagerlund et al.,

2016). Additionally, L. monocytogenes plasmids exhibit a modular genetic structure

(Canchaya et al., 2010; Kuenne et al., 2010), but the function of most of these plasmid genes has not yet been identified. Some functional data on the effect of plasmids on stress survival has been described recently, such as their contribution to heat stress survival, heavy metal, antibiotics, or benzalkonium chloride tolerance (Lebrun et al.,

1994a; Elhanafi et al., 2010; Li et al., 2016; Kremer et al., 2017; Pöntinen et al., 2017).

In general, plasmids can be easily lost as they impose a maintenance cost and are only retained if they confer an evolutionary or selective advantage for survival. L. monocytogenes strains isolated from food production facilities over several years were found to retain their plasmids, indication of a benefit for the retention of these plasmids that compensates for their maintenance cost (Kuenne et al., 2010; Ferreira et al., 2014;

Martin et al., 2014; Knudsen et al., 2017).

We hypothesize that these plasmids confer an advantage to survival when L. monocytogenes strains are exposed to stress conditions found in food and FPEs. To examine this, we used three sets of isogenic L. monocytogenes strains belonging to

ST5, ST8 and ST121, each consisting of a pair of plasmid-harboring wildtype and plasmid-cured strains to determine the effect of plasmids on stress survival. The plasmids were carefully analyzed with regard to their gene contents and retained after

exposure to a variety of stress conditions. We reveal a higher stress tolerance among strains containing plasmids, thereby showing that plasmids confer advantages for survival in stress environments that L. monocytogenes is exposed to in food and FPEs.

Materials and Methods

Bacterial Strains, Growth Conditions, Plasmid Curing and Plasmid Screening

Plasmids were cured from three L. monocytogene s ST121, ST8, and ST5 strains

(Table 1). These strains have been identified as persistent in food and FPEs previously, see Table 1, and have been used as models for studying various aspects of survival of

L. monocytogenes in a number of studies (Müller et al., 2013; Casey et al., 2014; Müller et al., 2014; Fagerlund et al., 2016; Fox et al., 2016; Harter et al., 2017; Rychli et al.,

2017; Muhterem-Uyar et al., 2018).

Table 1. Listeria monocytogenes wildtype strains harboring plasmids used in this study.

Strain Sequence Country Isolation Plasmid Plasmid GenBank Reference Type of origin source, size accession year (kbp) number 6179 ST121 Ireland Cheese, pLM6179 62.2 HG813250 Schmitz- 2000 Esser et al. (2015b) R479a ST8 Denmark Smoked pLMR479a 86.6 HG813248 Schmitz- Salmon, Esser et al. 1996 (2015a) 4KSM ST5 Austria Food p4KSM 90.5* JYOJ01000032.1*, Muhterem- production Uyar et al. environment, JYOJ01000033.1* (2018) 2011 * p4KSM has not been closed and consists of 2 contigs.

For plasmid curing, strains were grown overnight at 37°C in tryptic soy broth and yeast extract (TSB+y) in a shaking incubator at 100 rpm. 100 µL of the overnight culture was pipetted into 10 mL of TSB+y that contained sub inhibitory concentrations (0.2 and

0.3 µg/mL) of novobiocin (Oxoid). The cultures inoculated with novobiocin were then

exposed to increased heat, 40°C overnight, with shaking at 125 rpm. Exposure to both elevated temperature and the novobiocin should result in curing of the plasmid. Diluted cultures of 10 -1 to 10 -4 were then streaked on tryptic soy agar (TSA, BD) or ALOA plates

(Chromogenic Listeria Agar, Oxoid) and incubated overnight at 37°C. Single colonies were screened for the presence of plasmids with PCR using primers targeting a 600 bp region of the plasmid replication protein gene rep A (Forward: 5’ –

CGCCGTTTTTGATCACTGTA-3; Reverse: 5’-AGCAAGTACCAATCGGAAGG-3’; T A:

62°C). Primers were designed using Primer3 (Untergasser et al., 2012). In a PCR final volume of 50 µL, the following concentrations were used: 10 µM of each primer, 50 mM

MgCl 2, 10 mM dNTP mix, buffer, 1.5 U Platinum Taq polymerase (Life Technologies), and DEPC-treated water. PCR cycle conditions were as follows: initial denaturation for 3 minutes at 94°C, 35 cycles of denaturation at 94°C for 30 seconds, annealing at 62°C for 30 seconds and elongation at 72°C for 30 seconds, final elongation at 72°C for 4 minutes. Genomic DNA was isolated from all three sets of isogenic L. monocytogenes strain pairs for use as positive controls. Negative controls with no template and positive controls were used in all PCR reactions. All PCR products were confirmed with agarose gel electrophoresis. After confirmation of curing, wildtype and cured strains were routinely grown overnight on TSA or in TSB at 20°C.

Stress Survival Assays

For all stress survival assays, each strain was inoculated into 5 mL of TSB

(Fisher Chemical) and grown overnight at 20°C with shaking at 200 rpm. Overnight cultures were adjusted to a starting inoculum of an optical density (OD) at 600nm of 0.2

in TSB. 100 µL of starting inoculum was used to inoculate 5 mL of TSB and exposed to the stress conditions as described below. The exposure time for all stress conditions was two hours. Experimental cultures were conducted in triplicate for each experiment and placed in a shaking incubator at 200 rpm at 20°C for 2 hours. A temperature of

20°C was chosen to provide conditions similar room temperature. Tenfold serial dilutions of the WT and plasmid-cured cultures after stress exposure were plated on

TSA plates with duplicates for each concentration to determine the difference in CFUs between the WT and plasmid-cured strains. Determination of CFUs was conducted after

24 hours of incubation of the plates at 37°C. Log 10 reduction values were calculated based on the obtained CFU counts using the following formula: Log 10 reduction = mean log 10 (CFUs WT) – mean log 10 (CFUs plasmid-cured).

Experimental culture media and strains varied depending on the stress conditions tested: The wildtype strains 6179, R479a, and 4KSM, and their plasmid- cured derivatives were subjected to an oxidative stress, acidic stress, and salt stress survival test. For the oxidative stress test, a final concentration of 0.01% (vol/vol) H 2O2 was applied. For acidic stress survival, TSB, with a final concentration of lactic acid of

1% (vol/vol), pH 3.4 was used. To test for combined salt and mild acidic stress survival, experimental cultures were inoculated into TSB, with 15% (wt/vol) NaCl, adjusted to a pH of 5 with hydrochloric acid. For each strain (6179, R479a, 4KSM; for both wildtype and plasmid-cured strains), experiments with each of the three different stress conditions (15% NaCl, pH 5; 0.01% H 2O2; 1% lactic acid, pH 3.4) were conducted in triplicate.

Testing for benzalkonium chloride survival was conducted with the WT and the plasmid-cured strain of 4KSM only, as only 4KSM contains the bcrABC cassette, which confers increased tolerance to BC, on the plasmid p4KSM, whereas 6179 contains

Tn 6188 , a chromosomally encoded BC resistance marker (Müller et al., 2013). The

R479a WT strain is naturally sensitive to BC (Müller et al., 2013) and thus was not tested for BC tolerance here. For the BC stress test, each strain was inoculated into

TSB with a final concentration of 10 µg/mL BC.

The ST5 and ST8 strains were also analyzed in a heat stress assay. As pLM6179 contains an identical copy of the clpL gene found in pLM58, recently shown to be responsible for heat stress tolerance (Pöntinen et al., 2017), we did not test heat stress with 6179. Maximum growth temperatures were determined prior to survival experiments, indicating a maximum growth temperature of 50°C for R479a, and 55°C for 4KSM. Initial overnight setup and OD measurements for the heat stress assay were identical to the other stress assays. Cultures were inoculated into TSB prewarmed to

50° or 55°C and placed in a shaking incubator at 50° or 55°C for 2 hours. All serial dilutions, plating, and CFU counts were conducted identically to the previous stress assays.

Sequence Analysis

Sequence analysis including annotation and comparative BLAST searches of the plasmids was performed in PATRIC (Wattam et al., 2017). The average nucleotide identity between the plasmids was determined using the Jspecies webserver (Richter et al., 2016). Phylogenetic analyses of plasmid replication initiation protein RepA amino

acid sequences was performed with MEGA7 using maximum likelihood based phylogenetic inference and the JTT amino acid substitution model with 1000x bootstrapping (Kumar et al., 2016).

Statistical Analysis

Statistical analysis was conducted on Microsoft Office Excel 2013. Differences in

CFU counts between WT and plasmid-cured strains were tested using student paired two-tailed t-test. Graphing was conducted in JMP Pro 14, with standard error used for calculation of error bars.

Results

Gene Content of the Analyzed Plasmids

To determine the overall relatedness of the plasmids, we analyzed the average nucleotide identity (ANI) between the three plasmids. pLM6179 and pLMR479a shared a higher ANI than they did with the ST5 plasmid p4KSM. Whereas the ANI values were above 95%, the overall overlap between the plasmids was lower than 17%, except for pLM6179 and pLMR479a, with more than 52% coverage (Table S1, Fig. S1). Similarly, pLM6179 and pLMR479a showed higher average amino acid identity between shared plasmid proteins compared to p4KSM (Table S2). pLM6179 and pLMR479a shared 46 proteins. P4KSM and pLM6179 shared 26 proteins, whereas p4KSM and pLMR479a shared 40 proteins. 22 proteins where shared among all three plasmids (Fig. S2).

We analyzed the gene content of the three plasmids from the ST121, ST5, and

ST8 strains to determine putative plasmid genes that may contribute to stress survival

(Table 2). Many of the plasmid genes cannot be assigned with a putative function, but for some, a possible function can be deduced. Furthermore, there were some genes that we identified on all three plasmids. All three strains used for our experiments contain the transposon Tn 5422 on their plasmids, which is involved in cadmium tolerance (Lebrun et al., 1994a). However, while the pLM6179 and pLMR479a Tn 5422 cadA and cadC genes share 100% amino acid identity, the p4KSM Tn5422 shows 69% and 55% amino acid identity with the pLM6197 and pLMR479a homologs, respectively.

All three plasmids contain a RepA plasmid replication protein. We found that the pLM6179 RepA shared 99% amino acid identity with the RepA protein on p4KSM. The pLMR479a RepA protein shared 97% amino acid identity with the p4KSM and pLM6179

RepA proteins. Phylogenetic analyses of RepA amino acid sequences revealed that all three plasmids belonged to group 2 Listeria plasmids established by (Kuenne et al.,

2010) (Fig. S3).

The plasmid of ST121 L. monocytogenes strain 6179 contains a ClpL protein, a member of the HSP100 subgroup of heatshock proteins. The clpL gene found on pLM6179 is identical to the clpL gene found in the plasmid pLM58, which has been shown to be responsible for increased heat stress survival in L. monocytogenes

(Pöntinen et al., 2017).

The L. monocytogenes ST8 strain R479a plasmid genes possibly involved in stress survival include a putative multicopper oxidase and copper transporter, which have been shown in Staphylococcus aureus to be involved in copper homeostasis and oxidative stress response (Sitthisak et al., 2005; Kosman, 2010; Ladomersky and Petris,

2015). The Staphylococcus aureus and R479a proteins share 95% and 100% amino

Table 2. Presence and conservation of plasmid encoded candidate genes involved in stress survival.

Plasmid Tn 5422 Tn 5422 BcrABC ClpL ClpB NADH Triphenylmethane Multicopper GbuC (CadA) (CadC) Peroxidase reductase Oxidase

pLM6179 + + - + - - - - - *(100%) *(100%)

pLMR479a + + - - - + - + +

*(100%) *(100%) *(100%) *(100%)

p4KSM + + + - + + + - +

*(69%) *(55%) *(100%) *(100%)

* (amino acid identity) 86

acid identity with the oxidase and copper transporter, respectively. Two putative heavy metal transporting ATPase genes that may be involved in resistance to heavy/transition metals are present on pLMR479a (Lewinson et al., 2009). pLMR479a also contains a putative NADH peroxidase, Npx, which has been shown to act as a hydrogen peroxide reducer in Lactobacillus casei , thus decreasing oxidative stress (Gibson et al., 2000; La

Carbona et al., 2007; Serata et al., 2012). Additionally, a putative glycine betaine transport binding protein, GbuC, is present on pLMR479a, which may reduce the effect of osmotic stress (Ko and Smith, 1999; Angelidis and Smith, 2003).

One notable locus on the L. monocytogenes ST5 strain 4KSM plasmid is the bcrABC cassette that conveys increased tolerance to quaternary ammonium compounds and was first characterized on pLM80 (Elhanafi et al., 2010). The p4KSM

BcrABC copy shares 100% amino acid identity with BcrABC from pLM80. The 4KSM plasmid also contains a putative ClpB protein, a distant homolog to the ClpL heatshock protein found in pLM6179 and pLM58. The p4KSM Clp protein belongs to the ClpA/B family (InterPro domain: IPR001270). Similar to pLMR479a, a putative heavy metal transporting ATPase, a NADH peroxidase Npx, as well as a GbuC protein are found on p4KSM. P4KSM also contains an identical homolog of the triphenylmethane reductase characterized from the L. monocytogenes plasmid pLM80 which has been shown to be involved in crystal violet detoxification (Dutta et al., 2014).

Survival of Wildtype Strains Compared to Plasmid-Cured Strains Without Stress Conditions

Our study is based upon the premise that plasmids aid in survival under stress conditions found in food and food production facilities. To more accurately reflect

temperatures found in such conditions, our studies were conducted at 20°C and not at

37°C. To ensure that the plasmid curing had no detrimental effect on growth under regular growth conditions without stress, we analyzed the growth difference of the WT and plasmid-cured derivatives of the L. monocytogenes 6179, R479a, and 4KSM strains at 20°C with no stressors added. While the CFUs were numerically lower for the plasmid-cured strains, we found no significant effect upon growth differences due to the temperature or the removal of the plasmid from the WT strains (Fig. S4).

Survival of Wildtype Strains Compared to Plasmid-Cured Strains Exposed to Stress Conditions

In this study, we analyzed and compared the stress survival between L. monocytogenes WT and plasmid-cured strains when exposed to environmental stress typically found in food and food production facilities. In order to observe the effect of plasmids on oxidative stress survival, we compared the stress survival between the L. monocytogenes WT strains 6179, R479a, and 4KSM and their plasmid-cured derivatives when exposed to sublethal concentrations of H 2O2 for two hours. All three of the WT strains tested were significantly more tolerant to oxidative stress in comparison to their plasmid-cured derivatives (P < 0.001) (Fig. 1 A). Curing of the plasmids resulted in 0.06 (pLM6179), 0.2 (p4KSM) and 0.42 (pLMR479a) log 10 reduction of the survivors.

As bacteria are often exposed to various acidic stress in food and food production facilities, L. monocytogenes WT and plasmid-cured strains were exposed to a pH of 3.4, adjusted with lactic acid. Our results show that all three WT strains tested were also significantly more tolerant to acidic stress in comparison to their plasmid-

cured derivatives (P < 0.001) (Fig. 1 B). Plasmid-cured strains exhibited a 0.08

(pLM6179), 0.18 (p4KSM) and 0.26 (pLMR479a) log 10 reduction of the survivors.

Figure 1. Survival of wildtype (WT) and plasmid-cured L. monocytogenes strains under different stress conditions at 20°C, displayed as log 10 CFU/ml values and log 10 reduction values based on CFU/ml. To display log 10 reduction values, the CFU/ml values for the wildtype strains were set as zero. Error bars show standard deviation among three replicate data sets. Stress conditions were: A) 0.01% H2O2; B) 1% lactic acid, pH 3.4; C) 15% NaCl, pH 5. Stress exposure time was two hours for all conditions. P-values are as follows: *: P ≤ 0.05, ***: P ≤ 0.001.

Acidity and high salinity are frequent co-stressors observed e.g. in meat and dairy production facilities and fermented foods. Therefore, we compared the stress survival response between the L. monocytogenes strains 6179, R479a, and 4KSM, both

WT and the plasmid-cured derivatives, when exposed to high salinity and low pH (pH

5). We found L. monocytogenes WT strains 6179 and 4KSM to be more tolerant in comparison to the plasmid-cured strains (P < 0.05). A much stronger contribution of plasmids to the same stress conditions was found for R479a (P < 0.001) (Fig. 1 C).

Absence of the plasmids resulted in 0.06 (pLM6179), 0.13 (p4KSM) and 0.18

(pLMR479a) log 10 reduction of the survivors.

We analyzed and compared the stress survival between the 4KSM WT and plasmid-cured strains, when exposed to sublethal concentrations of BC, a quaternary ammonium compound often found in industrial disinfectants. We anticipated an effect on stress survival for the 4KSM WT strain over the plasmid-cured derivative as p4KSM encodes a bcrABC cassette that provides increased tolerance to BC (Elhanafi et al.,

2010). This assumption was confirmed as, after two-hour stress exposure period, the L. monocytogenes 4KSM WT strain proved to be significantly more tolerant to BC in comparison to the plasmid-cured strain (P < 0.001) when exposed to sublethal concentrations of BC (Fig. 2) resulting in a 0.17 log 10 reduction.

Figure 2. Survival of wildtype (WT) and plasmid-cured L. monocytogenes ST5 strain 4KSM exposed to 10 µg/mL of benzalkonium chloride for two hours displayed as log 10 CFU/ml values and log 10 reduction values based on CFU/ml. To display log 10 reduction values, the CFU/ml values for the wildtype strain were set as zero. Error bars show standard deviation among three replicate data sets. P-values are as follows: ***: P ≤ 0.001.

In addition to cold temperatures, L. monocytogenes can be exposed to heat stress during food production in FPEs. Due to the presence of a Clp-like protein on the p4KSM, we anticipated an increased tolerance to heat stress in the WT strain over the plasmid-cured. We found that the 4KSM WT strain was significantly more tolerant to heat stress when exposed to 55°C for two hours in comparison to the plasmid-cured strain (P <0.001) (Fig. 3). Since we did not identify a putative protein that may mediate heat stress response in the R479a plasmid we hypothesized no effect for this isolate.

Consistently, the R479a WT strain showed no significant difference in tolerance to heat stress when compared to the plasmid-cured strain when exposed to 50°C for two hours

(P = 0.74) (Fig. 3).

Figure 3. Survival of wildtype (WT) and plasmid-cured L. monocytogenes ST5 strain 4KSM exposed to heat stress at 55°C for 2 hours, and WT and plasmid-cured ST8 strain R479a to heat stress at 50°C for 2 hours. Values are displayed as log 10 CFU/ml and log 10 reduction values based on CFU/ml. To display log 10 reduction values, the CFU/ml values for the wildtype strain were set as zero. Error bars show standard deviation among three data sets. P-values as follows: ***: P ≤ 0.001, NS: No significant difference between the WT and plasmid-cured strains.

Discussion

Listeria monocytogenes is a major contributor to global foodborne illnesses and foodborne deaths and is capable of long term survival in food and FPEs and its ability to adapt to stresses contributes to the persistent nature of the pathogen. Moreover, it has been noted that while the L. monocytogenes core genome is highly stable, there are a variety of mobile genetic elements interspersed (den Bakker et al., 2010; Kuenne et al.,

2013), and some of them contribute to stress response and environmental adaptation

(Orsi et al., 2008; Ryan et al., 2010a; Verghese et al., 2011; Dutta et al., 2013; Müller et al., 2013; Harter et al., 2017). However, little data is available on the functional characteristics of L. monocytogenes plasmids in regard to stress survival mechanisms in food and FPEs. Therefore, we examined the differences between WT and plasmid- cured strains exposed to stress conditions that may be found in such conditions.

Experimental data on Listeria plasmids has almost exclusively been focused primarily on antibiotic, heavy metal and disinfectant resistance (Lebrun et al., 1994a; Elhanafi et al., 2010; Li et al., 2016; Kremer et al., 2017; Pöntinen et al., 2017). A recent study provided preliminary indirect evidence for a contribution of L. monocytogenes plasmids to acid tolerance but did not analyze this in more detail using e.g. plasmid-cured strains and/or identification of the responsible plasmid genes (Hingston et al., 2017b). Here, we perform a broad analysis comparing three distinct plasmids from strains from three different L. monocytogenes STs under various stress conditions.

Our results show that plasmids in the analyzed L. monocytogenes ST5, ST8, and

ST121 strains confer increased tolerance under stress conditions relevant for food and

FPE. More specifically, we show that p4KSM provides increased tolerance to heat stress at 55°C. Recently, pLM58 from an ST9 L. monocytogenes strain has been described to confer heat stress tolerance at 55°C through a ClpL protein encoded on pLM58 (Pöntinen et al., 2017). The ClpL from pLM58 is identical to the ClpL found on pLM6179, strongly suggesting that the ClpL in pLM6179 does also provide increased heat stress tolerance. P4KSM does not encode a ClpL protein, but a ClpB-like protein, which is only distantly related to the pLM58 ClpL (29% amino acid identity, 38% coverage). The p4KSM ClpB-like protein is also significantly shorter than the pLM58

ClpL: 704 amino acid residues compared to 372 amino acid residues for the ClpB-like protein in p4KSM. Highly similar or identical proteins are found in numerous Listeria other Firmicutes genomes, all with highly similar predicted lengths, thus suggesting that the 4KSM Clp protein is most likely not a truncated pseudogene. Clp proteins have been described to be involved in various stress response reactions (Chastanet et al.,

2004; Tao and Biswas, 2013); a contribution of the p4KSM Clp protein to heat stress response might be conceivable. No significant differences in heat stress tolerance at

50°C were found for the R479a WT and plasmid-cured strains. Thus, pLMR479a does not seem to provide heat stress tolerance. In line with this, and in contrast to p4KSM and pLM6179, based on sequence analysis, no proteins were identified on pLMR479a that may confer heat stress tolerance.

p4KSM also confers increased tolerance to BC which is most likely encoded by the bcrABC cassette present on p4KSM. The p4KSM BcrABC proteins share 100% amino acid identity with BcrABC from pLM80 which have been demonstrated to provide increased tolerance to BC (Elhanafi et al., 2010). The efflux transporter proteins BcrBC from p4KSM also share 53% and 55% amino acid identity with the EmrC protein on pLMST6 which also confers BC tolerance (Kremer et al., 2017). In this context, it is interesting to note that p4KSM derives from a strain that was isolated from a food production facility with a long-term L. monocytogenes contamination. The ST5 strains

(including 4KSM) persisted in this plant for several years and became the dominant ST in this facility, in spite of massive usage of various disinfectants including BC-based disinfectants. Interestingly, none of the initially abundant L. monocytogenes strains in this plant harbored a plasmid and after three years, the two remaining abundant STs

(ST5 and ST204) from this facility harbored identical plasmids. Thus, plasmids have been suggested to be important for the persistence of ST5 strains in this FPE based on sequence analyses (Muhterem-Uyar et al., 2018). Here, we provide experimental evidence for the contribution of plasmids in 4KSM, an ST5 strain from this FPE, to survival under stress conditions.

All three plasmids analyzed here significantly increased the tolerance of the strains to 0.01% hydrogen peroxide, 1% lactic acid, pH 3.4 and 15% sodium chloride, pH 5 (Fig. 1). For hydrogen peroxide, p4KSM and pLMR479a encode candidate genes which might be responsible for mediating the oxidative stress response. P4KSM and pLMR479a encode an identical putative NADH peroxidase, both show 45% amino acid identity to homologs in Enterococcus faecalis which are responsible for oxidative stress response (La Carbona et al., 2007). Other candidate genes possibly involved in oxidative stress response on pLMR479a include a putative multicopper oxidase, a Dps family protein, and YbbML homologs which have been shown to be involved in oxidative stress response in other bacteria (Olsen et al., 2005; Sitthisak et al., 2005; Tu et al.,

2012; Nicolaou et al., 2013).

All three plasmids contributed significantly to tolerance to 1% lactic acid, pH 3.4.

Similar to the results from oxidative stress response, pLMR479a and p4KSM showed a higher contribution to stress response compared to pLM6179. Based on the annotation and predicted functions of the pLM6179 proteins, no candidate gene which might mediate acid stress response could be identified.

All three plasmids were also involved in response to mild acidic stress combined with high osmotic stress (15% sodium chloride, pH 5). While pLM6719 and p4kSM contributed to a lesser, still significant, degree to stress response, the contribution of pLMR479a was much higher. R479a was isolated from smoked salmon and persisted in this fish processing facility for several years (Fonnesbech Vogel et al., 2001). Thus, the higher salt tolerance conferred by pLMR479a might reflect an adaptation to high salt concentrations commonly found in fish processing and smoking. Based on the predicted

functions of the pLM6179 proteins, no candidate genes which might be responsible in mild acid stress and high osmotic stress response could be identified. The plasmids p4KSM and pLMR479a contain identical putative GbuC proteins which are glycine/betaine binding proteins of the L. monocytogenes GbuABC transporter and have been shown to be involved in osmotic stress response (Angelidis and Smith, 2003).

However, p4KSM and pLMR479a contain only a GbuC homolog, no GbuA or GbuB homologs are found on the plasmids. The benefit of the additional GbuC homologs may thus be in conjunction with other chromosomally encoded osmotic stress response proteins such as the chromosomally encoded GbuABC proteins, which are present in the R479a and the 4KSM chromosomes (data not shown).

As shown for the first time on phenotypic and genomic level, plasmids pLM6179, pLMR479a and p4KSM have a clear survival-triggering function in the L. monocytogenes isolates to stresses that can be encountered in many FPEs. For all stress conditions analyzed, the highlighted proteins potentially involved in mediating stress response are candidates only. It is possible that other plasmid proteins are responsible for conferring the observed stress responses; these might include many of the uncharacterized plasmids proteins for which we cannot assign a putative function based on sequence annotation and similarity. Further functional characterization such as gene expression data, expression of proteins in other hosts, or deletion mutants of candidate genes will be required to identify which plasmid proteins are involved in response to certain stress conditions.

Conclusion

Taken together, our study provides evidence that the analyzed plasmids provide increased tolerance against different stress conditions including disinfectants, heat, high salt concentrations combined with mild acid stress, and acid stress. Our results thus broaden our knowledge about the function of L. monocytogenes plasmids in stress response and show that L. monocytogenes plasmids can aid in the response to different stress conditions. Based on the reported high conservation of some, or large parts of, L. monocytogenes plasmids, the results of our study could thus potentially also apply to other L. monocytogenes plasmids.

Acknowledgements

We thank Nicola Pacher and Myrte van der Heijden for help with curing of the plasmids and Beatrix Stessl for providing the 4KSM strain. We would also like to thank

Bienvenido Cortes, Allison Brost and Debarpan Dhar for their assistance in running

PCRs and collecting and collating data for the heat stress experiments. SSE and ALN were supported by the USDA National Institute of Food and Agriculture Hatch project no. 1011114. Martin Wagner’s work was partly financed by the Austrian Competence

Center for Feed and Food Quality, Safety and Innovation (FFoQSI).

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CHAPTER 4. TRANSCRIPTOME SEQUENCING OF LISTERIA MONOCYTOGENES EXPOSED TO OXIDATIVE AND ACID STRESS REVEALS HIGH EXPRESSION OF CHROMOSOMAL AND PLASMID NON-CODING RNAS

Modified from a manuscript published in Frontiers in Microbiology

Bienvenido W. Cortes a,b# , Annabel L. Naditz a,b# , Justin M. Anast a,b , Stephan-Schmitz-

Esser a,b

aInterdepartmental Microbiology Graduate Program, Iowa State University, Ames, IA, 50011 USA; bDepartment of Animal Science, Iowa State University, Ames, IA, USA

#B. Cortes contributed to the chromosomal portions and A. Naditz contributed to the

plasmid portions of the manuscript

Abstract

Listeria (L.) monocytogenes is a well-characterized pathogen that represents a major threat to food safety. In this study, we examine the chromosomal and plasmid transcriptomes of two different L. monocytogenes strains, 6179 [belonging to sequence type (ST) 121] and R479a (ST8), in response to 30 min exposure to oxidative (0.01% hydrogen peroxide) and acid (1% lactic acid, pH 3.4) stress. The exposure to oxidative stress resulted in 102 and 9 differentially expressed (DE) genes in the chromosomal transcriptomes of 6179 and R479a, respectively. In contrast, 2280 and 2151 DE genes were observed in the respective chromosomal transcriptomes of 6179 and R479a in response to lactic acid stress. During lactic acid stress, we observed upregulation of numerous genes known to be involved in the L. monocytogenes stress response, including multiple members of the σB regulon, many of which have not been functionally characterized. Among these genes, homologs of lmo2230 were highly upregulated in

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both strains. Most notably, the σ B-dependent non-coding RNA Rli47 was by far the most highly expressed gene in both 6179 and R479a, accounting for an average of 28 and

38% of all mapped reads in the respective chromosomal transcriptomes.

In response to oxidative stress, one DE gene was identified in the 6179 plasmid transcriptome, and no DE genes were observed in the transcriptome of the R479a plasmid. However, lactic acid exposure resulted in upregulation of the stress response gene clpL, among others, on the 6179 plasmid. In R479a, a number of uncharacterized plasmid genes were upregulated, indicating a potential role in stress response.

Furthermore, an average of 65% of all mapped transcriptome reads for the R479a plasmid following acid stress were mapped to an intergenic region bearing similarity to riboswitches involved in transition metal resistance.

The results of this study support the conclusion that members of the σB regulon, particularly lmo2230 and the non-coding RNA Rli47, play an integral role in the response of L. monocytogenes to acid stress. Furthermore, we report the first global transcriptome sequencing analysis of L. monocytogenes plasmid gene expression and identify a putative, plasmid-encoded riboswitch with potential involvement in response to acid exposure.

Introduction

Food-borne pathogens present a major concern to public health, causing diseases ranging from gastroenteritis to meningitis. In some cases, these infections are severe enough to result in hospitalization or death. Therefore, preventing food-borne pathogens from contaminating foods and food production environments (FPEs) is

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essential. To mitigate contamination, the food production and processing industries utilize a variety of methods including strict hygiene plans, cleaning agents, disinfectants, and food additives to prevent bacterial growth. As a result, food-borne pathogens are routinely exposed to a variety of stress conditions such as high salinity and extremes in both pH and temperature. Cleaning routines and disinfecting agents can expose foodborne pathogens to heavy metals or reactive oxygen species, and the presence of preservatives such as salts and organic acids, produced by fermentation or added intentionally, present additional environmental stressors for microorganisms.

Despite these efforts, the food-borne pathogen Listeria monocytogenes readily persists in FPEs (Carpentier and Cerf, 2011; Ferreira et al., 2014). L. monocytogenes is the causative agent of listeriosis, a disease that has the potential to cause severe conditions such as meningitis, sepsis, and spontaneous abortions in susceptible individuals. Although listeriosis has a low morbidity rate, the high rates of hospitalization and mortality associated with this disease are of significant concern (Allerberger and

Wagner, 2010; Scallan et al., 2011). Furthermore, the economic consequences of product recalls caused by L. monocytogenes contamination have been estimated to be

1.2–2.4 billion United States dollars annually (Ivanek et al., 2004), and in 2008, a single outbreak of 57 cases of listeriosis in Canada was estimated to have cost a total of 242 million Canadian dollars (Thomas et al., 2015). L. monocytogenes is also concerning because it is considerably more resilient to the numerous stress conditions used in

FPEs to mitigate contamination than are many other food-borne pathogens. As a result,

L. monocytogenes is able to colonize niche environments in FPEs such as drainage and hard-to-clean surfaces, and therefore the complete eradication of L. monocytogenes

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from a FPE is a difficult task (Ferreira et al., 2014). In addition to persistence in FPEs themselves, the tolerance of L. monocytogenes to food preservatives provides the organism with a competitive advantage when growing in certain types of foods, particularly in “ready-to-eat” foods that are highly processed and have a long shelf life.

Upon exposure to environmental stress conditions found in food and FPEs, L. monocytogenes responds by activating a wide variety of mechanisms that confer stress tolerance (Dutta et al., 2013; Müller et al., 2013; Harter et al., 2017; Bucur et al., 2018;

Hingston et al., 2019a). In addition to functional characterization, multiple gene expression analyses have implicated many genes in the response of L. monocytogenes to temperature, oxidative, disinfectant, pH, and osmotic stresses (Oliver et al., 2009;

Soni et al., 2011; Tessema et al., 2012; Casey et al., 2014; Tang et al., 2015b; Hingston et al., 2017b). Until now, transcriptomic research on L. monocytogenes has been conducted almost exclusively on strains that lack plasmids. Previous research from our group has demonstrated that the plasmids of L. monocytogenes strains 6179 and

R479a contribute to survival under heat, acidic, salt, and oxidative stress (Naditz et al.,

2019). Therefore, transcriptomic analysis may provide valuable insight into the effect of plasmids on stress tolerance and may also identify novel candidate chromosomal features permitting L. monocytogenes persistence in food and FPEs. Thus, this study conducted transcriptome sequencing of the plasmid-carrying strains 6179 and R479a to analyze their chromosomal and plasmid gene expression patterns after short-term exposure to oxidative and acid stress.

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Materials and Methods

Bacterial Strains Used in this Study

L. monocytogenes 6179 is a persistent strain that belongs to ST121 and was isolated from Irish cheese in 2000 (Fox et al., 2011a; Fox et al., 2011b). It possesses a

3.01 Mbp genome and a 62.2 kbp plasmid, designated pLM6179 (Schmitz-Esser et al.,

2015b). Notably, the stress survival characteristics of L. monocytogenes 6179 have been extensively characterized (Müller et al., 2013; Casey et al., 2014; Müller et al.,

2014; Makariti et al., 2015; Rychli et al., 2016; Harter et al., 2017; Naditz et al., 2019). L. monocytogenes strain R479a is an ST8 strain isolated from smoked salmon in Denmark in 1996 where it persisted for more than two years in the same FPE (Fonnesbech Vogel et al., 2001; Schmitz-Esser et al., 2015a). L. monocytogenes R479a possesses a 2.94

Mbp genome and a 86.6 kbp plasmid, pLMR479a. Similarly to 6179 , L. monocytogenes

R479a has been subject to multiple stress survival analyses (Müller et al., 2013;

Fagerlund et al., 2016; Rychli et al., 2016; Naditz et al., 2019).

Experimental Stress Conditions

Recently, we demonstrated that plasmids pLM6179 and pLMR479a contribute to the survival of L. monocytogenes strains 6179 and R479a during acidic (1% vol/vol lactic acid, pH 3.4) and oxidative stress (0.01% vol/vol hydrogen peroxide) (Naditz et al.,

2019). To elucidate plasmid genes with potential involvement in stress response, we sequenced the transcriptomes of 6179 and R479a under the same stress conditions as applied in Naditz et al. (2019). Overnight cultures of 6179 and R479a were made by inoculating a loop of colonies into 25 mL of tryptic soy broth (TSB; Becton, Dickinson

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and Company) and incubating at 20 °C with 200 rpm shaking for 22 hours. 7.5 mL of

6179 culture and 15 mL of R479a culture were harvested and spun down at 4696 x g at

20 °C for 10 minutes before pouring off the supernatants, and the 6179 and R479a pellets were then resuspended in 5 mL and 7.5 mL sterile 1x PBS, respectively. OD600 values were measured using a spectrophotometer (SmartSpec 3000, Bio-Rad

Laboratories), and the cultures were adjusted to an OD600 of 3.5 ± 0.2. 0.5 mL of adjusted overnight culture was then inoculated into 4.5 mL TSB with either a final concentration of 0.01% (vol/vol) hydrogen peroxide or 1% (vol/vol) lactic acid (pH 3.4).

These tubes were then incubated at 20 °C with 200 rpm shaking. To reduce the chance of significant RNA degradation, stress exposure was conducted for 30 minutes rather than 2 hours as previously conducted (Naditz et al., 2019). Controls for both strains were created in a similar manner by inoculating 0.5 mL of adjusted overnight culture to

4.5 mL of TSB without the addition of hydrogen peroxide or lactic acid. Each condition was conducted in biologically independent triplicates. For the triplicates, three tubes for each condition were combined to ensure sufficiently high RNA concentrations for transcriptome sequencing.

RNA Extraction, Library Preparation, and Sequencing

Following stress exposure for 30 minutes, the three tubes for each condition were combined and centrifuged at 4696 x g at 20 °C for 3 minutes. Supernatants were poured off, and the pellets were each resuspended in 600 µL of Invitrogen Purelink RNA

Mini Kit lysis buffer containing 1% β-mercaptoethanol. The protocol for the Invitrogen

Purelink RNA Mini Kit was then followed for RNA extraction, and chemical lysis was

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complemented with mechanical lysis using a bead-beater (Lysing Matrix E, MP

Biomedicals; Bead Mill 24 Homogenizer, Fisher Scientific). 1 µL Superase RNase inhibitor (Invitrogen) was added to each sample, and any remaining DNA was removed using the Turbo DNA-Free kit (Invitrogen) following the instructions of the manufacturer.

A PCR targeting the prfA gene using the primers Lip1 (5’ – GAT ACA GAA ACA

TCG GTT GGC – 3’) and Lip2 (5’ – GTG TAA TCT TGA TGC CAT CAG G – 3’)

(Rossmanith et al., 2006) yielded no amplified DNA, confirming the absence of L. monocytogenes DNA in the extracted RNA samples. PCR was conducted using the

Platinum Taq DNA Polymerase system (Invitrogen) according to manufactures specifications. PCR cycle conditions were as follows: initial denaturation at 94 °C (4 min), 35 cycles of denaturation at 94 °C (30 sec), annealing at 64 °C (30 sec), elongation at 72 °C (30 sec), and final elongation at 72 °C (5 min). PCR products were then confirmed with agarose gel electrophoresis.

The RNA integrity of all samples was then measured using an RNA 6000 Nano chip via an Agilent 2100 Bioanalyzer (Prokaryote Total RNA Nano assay). All RNA samples had RNA integrity numbers (RIN) of at least 9.9 (Supplementary Figure 1).

Samples were then sent to Microsynth (Balgach, Switzerland) for ribosomal RNA depletion using the Ribo-Zero rRNA Removal Kit (Gram-Positive Bacteria, Illumina) and stranded TruSeq library preparation. Triplicate samples were sequenced for all experimental conditions, resulting in a total of 18 samples. Single-read, 75 bp reads were generated by Microsynth using Illumina NextSeq, and read-trimming and demultiplexing were also performed by Microsynth.

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Sequence Analysis

Reads were mapped to either the L. monocytogenes 6179 or R479a chromosomes (HG813249.1 and HG813247.1, respectively) and plasmids

(HG813250.1 and HG813248.1, respectively) using the Burrows-Wheeler aligner mem algorithm (Li and Durbin, 2010). The resulting BAM files were then imported into

ReadXplorer (Hilker et al., 2014), and statistical analysis was performed using the

DeSeq2 package included in ReadXplorer. DeSeq2 utilizes the Benjamini and

Hochberg method for correction for multiple-testing to generate adjusted p-values, also called Q-values (Benjamini and Hochberg, 1995); Q-values lower than 0.05 were considered significant. Principal component analysis (PCA) was performed using R statistical software v3.5.1 (R Development Core Team, 2018) and the packages

DeSeq2 v1.22.2 (Love et al., 2014) and ggplot2 v3.1.0 (Hadley, 2016). Nucleotide and amino acid alignments were done with MAFFT (Katoh et al., 2019), shading of conserved amino acid residues was performed with Boxshade available at: https://embnet.vital-it.ch/software/BOX_form.html. RNA secondary structure predictions were generated using the RNAfold WebServer (Hofacker, 2003).

BLAST Methods

NCBI BLAST (Wheeler et al., 2008) was used to determine homologs between the locus_tags of the 6179 and R479a chromosomes (HG813249.1 and HG813247.1, respectively) as well as homologs between the chromosomal locus_tags of the aforementioned strains and EGD-e locus_tags (NC_003210.1). Protein BLAST searches

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were carried out using the module ncbi-blast (2.4.0). Only those matches with 90% amino acid identity or greater were considered homologs between strains.

Results and Discussion

Chromosomal Gene Expression

Average chromosome coverage for each condition ranged from 165x to 232x with an average of 192x coverage for all 18 samples. On average, 97.3% and 96.4% of the total reads per sample mapped to the 6179 and R479a chromosomes, respectively

(Table 1).

PCA conducted between the replicates demonstrated that for a given strain the replicates of each condition clustered closely together, indicating that there were no major disparities between replicates (Figure 1). In general, changes in the chromosomal transcription patterns of 6179 and R479a were less pronounced in response to 30 minute exposure to 0.01% hydrogen peroxide than in response to 1% lactic acid. The replicates for both lactic acid exposure experiments clustered far from the control, whereas the replicates for the hydrogen peroxide experiments fell closer to the controls

(Figure 1). Based on a Q-value of < 0.05, 102 DE genes were identified in 6179 in the hydrogen peroxide treatment, and 9 DE genes were identified in R479a (Table 2). In contrast, lactic acid exposure resulted in differential expression of 2280 genes in 6179 and 2151 genes in R479a (Table 2).

Table 1. Average transcriptome read statistics for all conditions*

L. Average Chromosome Number of Number of reads Plasmid Number of Number of monocytogenes number of coverage reads mapped mapped to coverage reads mapped reads mapped strain, reads to annotated regions to plasmid to annotated experimental chromosome chromosome regions condition plasmid 6179 control 8,099,939 202x 7,938,845 8,061,656 39x 33,743 32,317 6179 – 0.01% hydrogen 6,636,305 165x 6,494,283 6,545,882 45x 38,436 37,188 peroxide 6179 – 1% lactic 9,442,662 232x 9,089,166 9,073,091 93x 78,113 77,022 acid

R479a control 8,794,964 223x 8,384,647 8,390,921 60x 71,036 69,264 119 R479a – 0.01% hydrogen 6,543,746 167x 6,382,773 6,495,746 52x 61,540 60,538 peroxide R479a – 1% 7,989,763 202x 7,703,457 7,677,857 41x 48,147 47,701 lactic acid

*averages are based on triplicate samples for each condition

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Figure 1. Differences between transcriptome replicates of L. monocytogenes strains 6179 and R479a under oxidative and acid stress conditions based on principal component analysis. For each condition, the three control replicates for each strain were compared to the three treatment replicates. A) 6179 control vs. 6179 exposed to 0.01% H 2O2, B) R479a control vs. R479a exposed to 0.01% H 2O2, C) 6179 control vs. 6179 exposed to 1% lactic acid, D) R479a control vs R479a exposed to 1% lactic acid.

Table 2. Number of differentially expressed (DE) chromosomal and plasmid genes in response to 1% lactic acid and 0.01% hydrogen peroxide

L. monocytogenes strain, Number of Number of Log2fold Number of Number of Log2fold experimental condition chromosomal chromosomal change range plasmid DE plasmid DE change range DE genes DE genes (chromosome) genes genes (plasmid) upregulated downregulated upregulated downregulated 6179 – 0.01% hydrogen 67 35 -1.3 to 1.1 1 0 -0.4 to 1.0 peroxide

6179 – 1% lactic acid 1144 1158 -10.0 to 9.7 16 19 -4.5 to 3.5

R479a – 0.01% hydrogen 9 0 0.8 to 1.6 0 0 -0.5 to 0.4 peroxide

R479a – 1% lactic acid 1042 1096 -9.9 to 10.0 15 13 -4.0 to 3.8

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Further demonstrating the difference in the stress response between the two conditions, the log2fold changes of DE genes ranged from -1.30 to 1.14 in the 6179 hydrogen peroxide treatment and from 0.81 to 1.63 in the R479a hydrogen peroxide treatment. Under lactic acid stress exposure, the log2fold change ranges of 6179 and

R479a were -10.59 to 9.75 and -10.09 to 10.13, respectively (Table 2).

Chromosomal Gene Expression Changes in Response to Hydrogen Peroxide

Although other studies observed pronounced transcriptional shifts (Pleitner et al.,

2014) and increased expression of a functionally characterized oxidative stress survival islet (Harter et al., 2017) following 15 and 10 minute exposures to oxidative stress, respectively, our study observed relatively minor transcriptomic differences even after

30 minutes of oxidative stress exposure. Only 9 DE genes were seen in the R479a hydrogen peroxide treatment (Supplementary Table 5), although 7 of these were also among the 50 DE genes of 6179 with the highest log2fold changes (Supplementary

Table 6). Two of these shared DE genes are annotated as ohrA and ohrR, encoding a peroxiredoxin and its transcription factor which are involved in peroxide resistance in

Bacillus subtilis (Fuangthong et al., 2001). In 6179, ohrA and ohrR were upregulated by log2fold changes of 1.06 and 1.04, respectively, and in R479a by respective log2fold changes of 1.02 and 0.99. In L. monocytogenes , transposon mutagenesis of ohrA resulted in decreased tolerance to hydrogen peroxide, diamide, and cumene hydroperoxide (Reniere et al., 2016). Additionally, exposure to chlorine dioxide resulted in increased levels of ohrA expression (Pleitner et al., 2014). genes annotated as enzymes involved in certain steps of the citric acid cycle (c itZ – citrate synthase, c itB –

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aconitase, and citC – isocitrate dehydrogenase) were also upregulated in both 6179 and

R479a following exposure to hydrogen peroxide. citC and citZ are part of an operon

(Mittal et al., 2009) along with the upstream gene ( lmo1568 homolog), which was upregulated as well. The relevance of these three citric acid cycle genes to stress response is currently unknown.

In 6179, the DE gene with the highest log2fold change (1.14) was bilEA , a gene involved in bile resistance (Sleator et al., 2005). However, bilEA was not differentially expressed in R479a. Another DE gene unique to the 6179 transcriptome in response to hydrogen peroxide exposure was LM6179_3010 , a gene encoding a 141 amino acid protein upregulated by a log2fold change of 1.11. LM6179_3010 shares 100% nucleotide identity with the L. monocytogenes EGD-e gene lmo2230 , a known member of the σ B regulon, the primary stress response regulon in L. monocytogenes

(Raengpradub et al., 2008). LM6179_3010 and lmo2230 show 28% amino acid identity to ArsC, an arsenate reductase from Bacillus (B. ) subtilis (Sato and Kobayashi, 1998).

However, essential cysteine amino acid residues required for function as an arsenate reductase are not conserved (Supplementary Figure S1, (Bennett et al., 2001)), suggesting that Lmo2230 and homologs do not function as arsenate reductases.

Lmo2230 and its homologs are discussed in more detail in the section covering the lactic acid stress response below.

Also unique to the 6719 hydrogen peroxide response was the upregulation of two oxidative stress protection genes: sodA (LM6179_2184 ), a superoxide dismutase upregulated by a log2fold change of 0.79, and LM6179_1300 , annotated as a glutathione peroxidase and upregulated by a log2fold change of 0.56 (Chatterjee et al.,

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2006; Hingston et al., 2015). The importance of the superoxide dismutase in L. monocytogenes stress response has been documented before (Soni et al., 2011; Bucur et al., 2018), and upregulation of the EGD-e homolog of LM6179_1300 , lmo0983 , was observed following exposure to chlorine dioxide (Pleitner et al., 2014). However, stress survival islet 2 (SSI-2), an insert of two genes homologous to the Listeria innocua genes lin0464 and lin0465 (LM6179_0748 and LM6179_0749 ), was not differentially expressed in 6179 despite previous implication in oxidative stress (Harter et al., 2017).

The lack of significant upregulation of the SSI-2 genes under the conditions applied in our study may be explained by the differences in oxidative stress induction between the two studies. Here, 0.01% hydrogen peroxide was used to induce oxidative stress, whereas Harter et al. (2017) used 10 mM cumene hydroperoxide. The lower number of

DE genes in response to hydrogen peroxide treatment in R479a compared to 6179 suggests that R479a, which belongs to a different ST than 6179, may be inherently more tolerant to hydrogen peroxide-induced oxidative stress than 6179. However, it is also possible that the 30 minute oxidative stress exposure period or the concentration of hydrogen peroxide applied in this study may have been too short to induce broader changes in gene expression.

Chromosomal Gene Expression Changes in Response to Lactic Acid Treatment

As a whole, the 6179 and R479a responses to lactic acid stress were quite similar, as was expected based on their chromosomal similarity (Naditz et al., 2019). Of the genes with homologs in both strains, 1742 were differentially expressed in the transcriptomes of both 6179 and R479a (Figure 2). Only 24 of these 1742 shared DE

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genes had inverse expressions (upregulated in 6179 and downregulated in R479a or vice versa), thus demonstrating that the overall lactic acid stress response is highly similar between both strains. The remaining DE genes, 538 in 6179 and 409 in R479a, were unique to the response of each strain. Due to the number of DE genes, we focused further analysis on those DE genes previously implicated in acid stress response and/or with log2fold changes > 2.0.

Figure 2. Number of unique and shared DE genes in L. monocytogenes strains 6179 and R479a exposed to 1% lactic acid.

A few notable patterns of downregulated DE genes were observed in both 6179 and R479a following lactic acid stress. Of these, the most apparent was that a large proportion of the 50 DE genes with the most negative log2fold changes encoded ribosomal proteins (23/50 in 6179 and 24/50 in R479a, Supplementary Table 7, 8).

Additional common patterns included the downregulation of genes encoding tRNAs, flagellar and motility components, chemotaxis proteins, and genes involved in replication and fatty acid biosynthesis. The downregulation of these genes suggests a decrease in general metabolic activity and motility in response to the stress conditions.

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In terms of general stress response, lmo2748 , encoding the general stress protein 26

(GSP26), was upregulated by a log2fold change of 7.96 in 6179 and 7.59 in R479a.

Although a role for GSP26 in osmotic stress response has been determined in L. monocytogenes , the same study demonstrated that a deletion of lmo2748 does not result in deficiencies in survival under acid stress (Abram et al., 2008). Indeed, in B. subtilis , GSP26 is induced in response to glucose and oxygen limitation as well as to heat, osmotic, and, to a lesser degree, oxidative stress (Volker et al., 1994).

The general stress response of L. monocytogenes is connected to the organism’s pathogenic lifestyle by direct interactions with virulence factors. Notably, σ B directly regulates the transcription of two internalins involved in initial host cell invasion, inlA and inlB (Chaturongakul et al., 2008), and a complex regulatory interaction between the σ B and PrfA regulons is necessary for virulence (Chaturongakul et al., 2008; Gaballa et al., 2019). In this study, inlA (which possesses a premature stop codon in 6179 and is therefore annotated as two separate genes) was upregulated by log2fold changes of

7.07 and 6.95 in 6179 and 8.43 in R479a. To a lesser degree, inlB was also upregulated in both 6179 and R479a with log2fold changes of 4.16 and 4.18, respectively. Other notable upregulated virulence genes include prfA (log2fold changes of 3.70 in 6179 and

3.72 in R479a) and hly (log2fold changes of 1.92 in 6179 and 4.58 in R479a). These results demonstrate that even brief exposure to lactic acid at low pH is capable of inducing the expression of virulence genes in L. monocytogenes .

Previous studies have demonstrated that the presence of lysogenic prophages in a genome can confer beneficial effects (Edlin et al., 1977; Burns et al., 2015) including tolerance to acid stress (Wang et al., 2010). In L. monocytogenes , an induction of

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prophage gene expression has recently been reported during intracellular replication

(Hain et al., 2012; Schultze et al., 2015) and for the L. monocytogenes 10403S A118 prophage after acid stress exposure (Ivy et al., 2012). The tRNA-Arg-TCT prophage is found in both 6179 and R479a. This prophage contained 20 DE genes in 6179 (19 upregulated with log2fold changes ranging from 0.79 to 2.98) and 11 DE genes in

R479a (all upregulated ranging in log2fold change from 0.62 to 4.39). 6179 harbors two additional prophages integrated into the tRNA-Arg-CCG and tRNA-Thr-GGT loci. These had 47 DE genes (45 upregulated with log2fold changes ranging from 0.73 to 2.37) and

31 DE genes (29 upregulated with log2fold changes ranging from 1.45 to 3.29), respectively. Notably, a role in biofilm formation for the comK prophage found in certain

L. monocytogenes strains including R479a has previously been supported (Verghese et al., 2011), and in this study, 29 of the 68 comK prophage genes in R479a were differentially expressed (28 upregulated with log2fold changes ranging from 1.10 to

4.12). However, it remains unknown whether the expression of prophage genes under stress conditions merely results in the formation of lytic phage particles or increases stress tolerance. Although not conclusive, the results of this study raise the possibility that the induction of these prophage genes under lactic acid stress confers beneficial effects on 6179 or R479a. However, verifying this hypothesis would require further experimentation.

Additionally, both strains analyzed in this study possess different stress survival islets (SSIs) inserted between the homologs of lmo0443 and lmo0449 . As mentioned above, 6179 possesses SSI-2, a two-gene insert with homology to the L. innocua genes lin0464 and lin0465 , a transcriptional regulator and protease involved in oxidative and

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alkaline stress (Harter et al., 2017). Corroborating the conclusion of Harter et al. that

SSI-2 is not involved in acid stress, the transcriptional response of 6179 to lactic acid stress showed a significant decrease in the transcription of the lin0464 homolog (-1.66 log2fold change) and no differential expression of the lin0465 homolog. R479a possesses SSI-1, a five-gene insert homologous to lmo0444 -lmo0448 . Previous results have shown that when all five genes of SSI-1 were deleted, L. monocytogenes growth in pH 4.8 media was significantly decreased (Ryan et al., 2010b). However, only the homologs of lmo0444 and lmo0445 were differentially expressed in the R479a transcriptome following lactic acid stress with log2fold changes of 0.76 and 3.76, respectively.

Two other important acid stress response systems in L. monocytogenes are the glutamate decarboxylase (GAD) and arginine deiminase systems (Soni et al., 2011;

Bucur et al., 2018). Three genes encoding glutamate decarboxylases ( lmo0447 , lmo2363 , and lmo2434 ) and two genes encoding antiporters ( lmo0448 and lmo2362 ), are involved in the glutamate decarboxylase pathway of EGD-e (Cotter et al., 2005).

Notably, because 6179 lacks SSI-1, it lacks the homologs to lmo0447 and lmo0448 found in R479a. However, the homologs of lmo2363 , lmo2434 , and lmo2362 present in both 6179 and R479a were upregulated in both strains, with lmo2434 being upregulated by a log2fold change of 7.28 in 6179 and 8.08 in R479a. The results from this study are in line with the observation that lmo2363 and lmo2362 play a greater role than lmo0447 in acid-stress survival (Cotter et al., 2001; Ryan et al., 2009). The genes encoding the arginine deiminase operon ArcABC (lmo0036, lmo0037, lmo0039, and lmo0043 ), as well as the transcriptional regulator ( lmo1367 ) (Ryan et al., 2009), were all significantly

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upregulated in both 6179 and in R479a. Thus, as expected, two of the well-defined acid stress response mechanisms were upregulated in response to 1% lactic acid exposure.

In both 6179 and R479a, high upregulation of mntABC (lmo1847, lmo1849, lmo1848 ), a set of genes encoding a putative manganese transporter, was observed; the log2fold expression change ranged from 7.93 to 8.32 in 6179 and from 6.65 to 9.89 in R479a (Supplementary Tables S7 and S8). Functional characterization in EGD-e has demonstrated that mntA is involved in host-cell penetration and macrophage survival

(Reglier-Poupet et al., 2003; Bierne and Cossart, 2007). Furthermore, another gene encoding an additional putative manganese transporter, mntH (lmo1424 ), was highly upregulated in both strains (log2fold change of 5.65 in 6179 and 4.32 in R479a). The high upregulation of the manganese transporters mntABC and mntH suggest that manganese transporters may also be important for L. monocytogenes acid stress response in addition to their described contribution in oxidative stress response and virulence.

σB Genes

The sigma factor σ B is an important regulator of stress and virulence genes in L. monocytogenes . Using data from three studies (Raengpradub et al., 2008; Oliver et al.,

2009; Liu et al., 2019), we created a panel of 387 genes representing the σ B regulon

(Supplementary Table 11); of these genes, 6179 and R479a possessed homologs to

380 and 369, respectively (Figure 3). Following the exposure to lactic acid, each strain exhibited differential expression of 311 of the σ B genes found in that strain.

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Figure 3. Mean of normalized read counts of all σB-regulated genes plotted against log2 fold expression value changes for each gene. DE genes are colored red. (A) L. monocytogenes 6179 during H 2O2 stress, (B) L. monocytogenes 6179 during lactic acid stress, (C) L. monocytogenes R479a during H2O2 stress, (D) L. monocytogenes R479a during lactic acid stress.

In contrast, far fewer σ B genes were differentially expressed upon exposure of either strain to hydrogen peroxide. In the case of 6179, only 29 σ B genes were differentially expressed, and no DE genes in the R479a oxidative stress condition were members of the σ B regulon (Figure 3). It should be noted that induction of a select number of σ B-dependent genes was observed in L. monocytogenes following centrifugation before cold shock stress exposure (Chan et al., 2007). Although it is currently unknown how such an induction might affect the L. monocytogenes σB- dependent stress response as a whole and if this induction would also occur during acid or oxidative stress, in the case of this study, a pre-induction of σ B and genes in the σ B

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regulon during centrifugation could have potentially “primed” L. monocytogenes for stress exposure. While the possibility of a pre-induction of σ B following the initial centrifugation step cannot be ruled out, both the control and experimental treatment samples underwent this same centrifugation step, and therefore the differences between the control and treatment transcriptomes should largely be due to stress exposure. Thus, the overall pattern of σ B regulon induction suggests that the immediate response of L. monocytogenes strains 6179 and R479a to lactic acid exposure is at least in part mediated by σ B. In line with this, an upregulation of 45% of the genes from the σ B regulon was recently described in a proteome study analyzing acid stress response in L. monocytogenes Scott A (Bowman et al., 2012).

Members of the σ B regulon also include a number of DE conserved hypothetical proteins which were differentially expressed in the experimental conditions. Additionally, many of these genes were also heavily upregulated. Following lactic acid stress, members of the σ B regulon accounted for 108 and 122 DE genes encoding conserved hypothetical proteins in 6179 and R479a, respectively (the 50 most upregulated are shown in Supplementary Tables S9 and S10). One of these genes, lmo2269

(LM6179_3049 and LMR479a_2383 in 6179 and R479a, respectively), was the DE gene with the third highest log2fold change in 6179 (9.60) and the sixth highest log2fold change in R479a (9.89). lmo2269 has been shown to be upregulated under heat stress in EGD-e (van der Veen et al., 2007). The numerous σ B genes encoding conserved hypothetical proteins, their upregulation, and their high expression levels suggest that additional, uncharacterized σ B-controlled genes may be important in the L. monocytogenes stress response. This is in line with the observation by Oliver et al.

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(2010) that over 30% of the σ B-dependent genes shared by all strains in their study were uncharacterized, and many of them were upregulated in response to oxidative and acid stress.

Notably, the putative arsenate reductase lmo2230 (LM6179_3010 and

LMR479a_2344 in 6179 and R479a, respectively) was highly upregulated in both strains during lactic acid stress. This gene was the fourth highest expressed gene in

6179 and the seventh highest expressed in R479a with log2fold increases of 9.450 in

6179 and 9.85 in R479a. Multiple studies have demonstrated that lmo2230 is a member of the σ B regulon (Raengpradub et al., 2008; Oliver et al., 2009; Toledo-Arana et al.,

2009; Utratna et al., 2011). These studies also showed that lmo2230 is upregulated during early stationary phase (Raengpradub et al., 2008; Oliver et al., 2009), within the murine intestinal lumen (Toledo-Arana et al., 2009) and in response to osmotic and acid stress (Raengpradub et al., 2008; Utratna et al., 2011; Horlbog et al., 2019). Heat and cold stress have also been shown to induce lmo2230 expression (van der Veen et al.,

2007; Hingston et al., 2017a), and lmo2230 is also upregulated following exposure to chlorine dioxide (Pleitner et al., 2014).

However, in contrast to the results from the lactic acid experiments in this study, lmo2230 expression was downregulated or only very weakly upregulated after exposure to the organic acid salts sodium diacetate, potassium lactate, or a combination of the two (Stasiewicz et al., 2011). lmo2230 was also differentially expressed in response to acid stress in brain-heart infusion broth adjusted to pH 5, but the log2fold changes were much lower (1.3 to 2.5) than seen in this study (Tessema et al., 2012). In addition to differences in experimental conditions, the differences in upregulation of lmo2230 could

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be explained by different timepoints, different L. monocytogenes strains, and/or the usage of a microarray compared to RNA sequencing. Although upregulation of lmo2230 in response to stress has been observed in numerous studies (van der Veen et al.,

2007; Raengpradub et al., 2008; Oliver et al., 2009; Toledo-Arana et al., 2009; Utratna et al., 2011; Pleitner et al., 2014; Hingston et al., 2017a; Horlbog et al., 2019), its function in L. monocytogenes remains uncharacterized. The weak amino acid identity of

Lmo2230 to the arsenate reductase ArsC from B. subtilis might suggest a possible role in heavy metal detoxification. However, it remains true that Lmo2230 lacks conserved amino acid residues essential for function as an arsenate reductase (Supplementary

Figure S1), and it is upregulated under the aforementioned stress conditions which do not involve heavy metal (arsenate or arsenic) toxicity. Taken together, these factors indicate that Lmo2230 may have a broader, yet unknown, function in L. monocytogenes stress response.

Interestingly, an extremely high percentage of all transcriptome reads from the lactic acid replicates (averaging 28% and 38% for 6179 and R479a, respectively) mapped to the non-coding RNA rli47 (sbrE ), a member of the σ B regulon (Figure 4)

(Oliver et al., 2009; Toledo-Arana et al., 2009; Mujahid et al., 2012; Liu et al., 2017).

Expression of rli47 significantly increased during exposure to lactic acid, with a log2fold change of 8.47 in 6179 and 9.97 in R479a. Recently, Marinho et al. (2019) determined that rli47 transcripts hinder cell growth under stress conditions by suppressing translation of ilvA , an enzyme involved in isoleucine biosynthesis. Other studies also support the notion that rli47 is important for response to various stressors including stationary phase (Oliver et al., 2009; Toledo-Arana et al., 2009; Mujahid et al., 2012),

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intracellar survival in macrophages (Mraheil et al., 2011), and oxidative stress (Mujahid et al., 2012). Taken together, our data and published gene expression and functional characterization data indicate that rli47 likely plays a vital role in global stress response.

Figure 4. Average mean of normalized transcriptome sequencing reads mapped to the non-coding RNA Rli47 in L. monocytogenes strains 6179 and R479a exposed to control conditions, acid (1% lactic acid, pH 3.4), and oxidative stress (0.01% H2O2). Q-values (<0.001) are indicated by asterisks.

Here, we also observe a potential link between Rli47 and the Listeria adhesion protein (LAP, lmo1634). LAP is a bifunctional, highly conserved alcohol-acetaldehyde dehydrogenase which is essential in promoting the systemic spread of L. monocytogenes during infection (Pandiripally et al., 1999; Drolia et al., 2018). Previous

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research has demonstrated lap upregulation during nutrient starvation and low glucose

(Jaradat and Bhunia, 2002), heme stress (dos Santos et al., 2018), and oxygen limited environments (Burkholder et al., 2009). In our data, LAP was significantly downregulated in both 6179 and R479a with log2fold changes of -5.10 and -2.99, respectively. Interestingly, differential expression analyses, including the one conducted in this study, consistently show a pattern of rli47 upregulation coupled with the concurrent downregulation of lap . This pattern is present during stationary phase (Oliver et al., 2009; Toledo-Arana et al., 2009; Mujahid et al., 2012), the overexpression of σ B

(Liu et al., 2017), multiplication in the gut (Toledo-Arana et al., 2009), and in macrophages (Chatterjee et al., 2006; Mraheil et al., 2011). Furthermore, lap was the highest upregulated gene (29.9 fold) in an rli47 deletion mutant (Marinho et al., 2019), suggesting that Rli47 is somehow involved in lap expression. The aforementioned evidence seems to implicate a relationship between LAP and Rli47 in L. monocytogenes , specifically that Rli47 is involved in the regulation of lap expression during stress conditions.

Plasmid Gene Expression

In addition to analyzing chromosomal gene expression patterns, we sought to provide the first global insights into the differential expression of plasmid genes of L. monocytogenes under stress conditions. Although the plasmids of 6179 and R479a share 46 genes, pLM6179 and pLMR479a harbor a number of strain-specific plasmid genes, some of which are predicted to be involved in stress response (Naditz et al.,

2019). Total plasmid coverage for all 18 samples ranged from 41x to 170x with an

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average of 71x coverage. The average of total reads per sample mapped to pLM6179 and pLMR479a were 74,509 and 60,499, respectively (Table 1).

For both strains, there were far fewer transcriptional changes following the 30 minute exposure to 0.01% hydrogen peroxide than to 1% lactic acid. After hydrogen peroxide treatment, analysis identified a single DE gene in the pLM6179 plasmid transcriptome, whereas no differential expression of any pLMR479a gene was found. In contrast, the 1% lactic acid treatment resulted in 42 DE genes for pLM6179 and 27 DE genes for pLMR479a. Of these, 18 and 13 were upregulated in the 6179 and R479a plasmid transcriptomes, respectively (Table 2, Supplementary Tables 1-4, 13, 14). For the lactic acid treatment, log2fold changes ranged from -4.70 to 3.15 for pLM6179 and from -6.14 to 3.84 for pLMR479a (Table 2, Supplementary Tables 1-4, 13, 14).

Plasmid Gene Expression Changes in Response to Hydrogen Peroxide Treatment

Interestingly, 0.01% hydrogen peroxide exposure resulted in no significant differences in plasmid gene expression between treatment and the control for R479a. In

6179, only one DE gene following 30 minute exposure to 0.01% hydrogen peroxide was observed. This gene, LM6179_RS15385 , was upregulated by a log2fold change of 1.11 and is annotated as encoding a conserved protein of unknown function. The minimal differences between the plasmid transcriptomes of the control and hydrogen peroxide treatments is in line with our chromosomal data and indicates either that 0.01% hydrogen peroxide exposure is not extremely stressful to 6179 or R479a or that the time of exposure to the stressor was insufficient to elicit major differential gene expression. It is interesting to note that our previous study showed a significant contribution of

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pLM6179 and pLMR479a to survival under 0.01% hydrogen peroxide, but the exposure to the stress was longer (2 hours) in our previous study (Naditz et al., 2019). While not significant, the heat shock protein-encoding clpL gene of pLM6179 ( LM6179_RS15400 ) was highly expressed in terms of read counts. An average of 47% of all reads mapped to pLM6179 mapped to clpL under hydrogen peroxide exposure.

Plasmid Gene Expression Changes in Response to Lactic Acid Treatment

Although pLM6179 and pLMR479a are largely distinct, they nevertheless share

46 genes, and we observed some shared expression patterns between these two plasmids in response to lactic acid exposure. First, a membrane protein of unknown function (locus_tags: LM6179_RS15475, LMR479A_RS14845) was upregulated on both plasmids. Next, two genes found on the transposase Tn5422 , cadA and cadC , were downregulated in 6179 and R479a. These two genes encode a cadmium- transporting protein and accessory protein, respectively (Supplementary Table 14, 15)

(Lebrun et al., 1994b). The gene encoding the putative plasmid replication protein repA was also downregulated, indicating a decrease in plasmid replication in response to the stress conditions.

In terms of DE genes unique to each strain, in pLM6179, the most highly upregulated and expressed DE gene was clpL , a gene found on pLM6179 but absent from pLMR479a. c lpL was upregulated by a log2fold change of 3.15 (Supplementary

Table 14). The ClpL protein is a member of the HSP100 subgroup of heat shock proteins, and the pLM6179 ClpL protein is identical to a functionally characterized ClpL protein harbored by the L. monocytogenes plasmid pLM58 that functions in heat stress

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response (Pöntinen et al., 2017). In other studies, ClpL proteins which share more than

67% amino acid identity to the protein encoded by the pLM6179 clpL gene have been shown to be involved in acid stress response, tolerance to detergents and penicillin, and long-term survival in different Streptococcus species (Kajfasz et al., 2009; Tran et al.,

2011a). A ClpL homolog in Lactobacillus reuteri sharing 67% amino acid identity to the pLM6179 ClpL was shown to be upregulated in response to acid stress (pH 2.7) (Wall et al., 2007). This, combined with the high expression level and high upregulation of clpL under lactic acid stress seen in this study, further supports the hypothesis that plasmid- borne clpL genes in L. monocytogenes play a role in acid stress response. Furthermore, if clpL is involved in environmental stress response, then this would at least partially explain the observed trend that plasmid-cured L. monocytogenes 6179 is less tolerant towards lactic acid, salt, and oxidative stress (Naditz et al., 2019). Further supporting this putative role for clpL in L. monocytogenes stress response, the upregulation of plasmid-encoded clpL during salt and acid stress was reported recently for two L. monocytogenes plasmids, pLMG1-12 and pLMG1-9 (Hingston et al., 2019a). Other upregulated pLM6179 DE genes include mainly uncharacterized genes, a pair of putative toxin-antitoxin genes (LM6179_RS15455 and LM6179_RS15450), and a gene

(LM6179_RS15380 ) found on many L. monocytogenes plasmids that is putatively involved in UV protection or DNA repair (Kuenne et al., 2010).

In response to 1% lactic acid treatment, the upregulated pLMR479a DE genes primarily consisted of genes encoding uncharacterized proteins for which no potential function can be inferred based on sequence similarity. However, a putative multicopper oxidase gene ( LMR479a_RS15145 ) was also upregulated. The protein encoded by

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LMR479a_RS15145 shows more than 95% amino acid identity to a homolog in

Staphylococcus aureus ATCC12600 which has been demonstrated to be involved in copper resistance and oxidative stress response (Sitthisak et al., 2005). However, the putative multicopper oxidases on pLMR479a and on other Listeria plasmids encode 386 amino acid proteins, which are shorter than the Staphylococcus aureus homologs (462 amino acids). Recently, an identical homolog of the putative multicopper oxidase was also found to be upregulated under mild acid stress (pH 5) in the L. monocytogenes plasmid pLMG1-12 (Hingston et al., 2019a).

Of significant interest was that the majority (65% on average) of the pLMR479a reads during lactic acid stress were mapped to an intergenic region (position 81657 to

81765) between locus_tags LMR479a_RS15240 and LMR479a_RS15245

(Supplementary Figure S3). Additionally, expression of this intergenic region was significantly upregulated by a log2fold change of 3.27. This region shows 56% nucleotide identity to the RFAM family RF02683, a recently described family of nickel- cobalt riboswitches known to be involved in transition metal tolerance (Furukawa et al.,

2015). In Firmicutes , such as Clostridiales and Erysipelotrichaceae , these riboswitches bind to ions such as zinc, copper, nickel, and cobalt, and this binding activity is utilized in the detection of and response to toxic concentrations of transition metals (Furukawa et al., 2015). The putative riboswitch identified on pLMR479a shares 70% nucleotide identity and 90% percent coverage with both the functionally characterized

Erysipelotrichaceae bacterium 3_1_53 NiCo riboswitch and the Clostridium scindens

ATCC 35704 NiCo riboswitch (Furukawa et al., 2015) (Supplementary Figure S4 and

S5). RNA secondary structural predictions revealed that the putative riboswitch from

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pLMR479a possessed a similar secondary structure to those of the functionally characterized NiCo riboswitches showing the four base-paired regions described by

Furukawa et al. (2015) (Supplementary Figure S6).

Furukawa et al. (2015) also identified homologs of these NiCo riboswitches in

Listeria . Interestingly, all homologs in Listeria identified by Furukawa et al. (2015) were found on plasmids, such as the homolog harbored on L. monocytogenes strain 08-5578 plasmid pLM5578. In contrast, the riboswitch homologs in Erysipelotrichaceae bacterium 3_1_53 and C. scindens are located on the chromosome. Notably, while the putative pLMR479a riboswitch shows high similarity to the characterized riboswitch homologs in Erysipelotrichaceae bacterium 3_1_53 and C. scindens , the transporter genes located upstream of these riboswitches show no similarity. All of the characterized NiCo riboswitches identified by Furukawa et al. (2015), including the putative Listeria riboswitches, are located upstream of putative heavy or transition metal transporters that correspond with the transition metal ions bound by the associated riboswitch. Similarly, the characterized riboswitches in Erysipelotrichaceae bacterium

3_1_53 and C. scindens are associated with a NiCo transporter. In pLMR479a, the putative riboswitch is located upstream of a putative zinc-transporter

(LMR479a_RS15240 ), showing 41% amino acid identity to ZosA from B. subtilis , a zinc transporter important in oxidative stress response (Gaballa and Helmann, 2002).

However, it should be noted that the zosA -like gene was not a DE gene under lactic acid stress applied here. The pLMR479a ZosA-like transporter is a 627 amino acid protein and belongs to the P-type ATPase superfamily 3.A.3 of the transporter classification database (Saier et al., 2016), whereas the transporters upstream of

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Erysipelotrichaceae bacterium 3_1_53 and C. scindens riboswitches are 308 amino acid residues in length and belong to the cation diffusion facilitator (CDF) family 2.A.4.

This suggests that although transition metal-binding riboswitches may be similar, their associated transition metal transporters can be highly variable. BlastN searches against

NCBI Genbank using the putative pLMR479a riboswitch as query revealed 44 identical homologs in different L. monocytogenes plasmids (Supplementary Table 14, suggesting that homologs of these riboswitches are found on a number of L. monocytogenes plasmids. Here, we provide the first gene expression data for this putative, plasmid- borne riboswitch in L. monocytogenes. This data shows that transcription of this riboswitch accounts for up to 73% of the transcripts of pLMR479a during 1% lactic acid stress. In the future, the potential function of this putative riboswitch in acid stress response and its regulation of the upstream ZosA-like transporter will need to be verified.

Conclusion

Here, we provide detailed insights into the short-term stress response of two L. monocytogenes strains, 6179 and R479a, to oxidative and acid stress. In particular, the lactic acid exposure resulted in striking changes in chromosomal gene expression. Our results provide more evidence for the importance of σ B-regulated genes in stress response, including lmo2230 and its homologs, and show massive transcription of the noncoding RNA Rli47. Additionally, our results demonstrate that the overall chromosomal responses to the aforementioned stress conditions are highly similar between 6179 and R479a. This study also provides the first in-depth analyses of

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plasmid gene expression in L. monocytogenes. Our results from two L. monocytogenes strains carrying distinct plasmids show that known and putative plasmid-encoded stress response genes such as clpL and the multi-copper oxidase are highly expressed and upregulated in response to stress conditions. We also reveal evidence in support of an important role of a putative, uncharacterized riboswitch in lactic acid stress response in pLMR479a based on its high expression level and significant upregulation during acid stress.

Availability of Sequencing Data

The raw sequencing data were deposited at the NCBI Sequence Read Archive under bioproject no. PRJNA563702.

Author Contributions

SSE, BWC, and ALN were involved in study design and conceived the experiments. BWC and ALN performed experiments, BWC, ALN, JMA, and SSE analyzed the data. BWC, ALN, JMA and SSE wrote the manuscript. All authors approved the final manuscript.

Acknowledgements

SSE, JMA, and ALN are supported by the USDA National Institute of Food and

Agriculture Hatch projects no. 1011114 and 1018898 and by the USDA National

Institute of Food and Agriculture, Agricultural and Food Research Initiative Competitive

Program, grant number: 2019-67017-29687. BWC is supported by the National Science

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Foundation Graduate Research Fellowship Program. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. 1744592. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

Conflicts of Interests

The authors declare that they have no competing interests.

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CHAPTER 5. GENERAL CONCLUSION

Conclusion

Here, we present a comprehensive examination of L. monocytogenes plasmids, their distribution in different STs, their contribution to survival in food and FPEs, and candidate genes that may aid in this contribution. To date, this is by far the largest survey of plasmids in L. monocytogenes , with more than 100 times more genomes than

Kuenne et al. (2010), and 10 times more strains than Hingston et al. (2017b). Our study provides an in-depth comparative analysis on plasmid presence on a broad and representative data set that includes genomes from 14 different STs, 32 countries, 7 decades and 3 isolation sources, utilizing 1924 L. monocytogenes sequences from published, peer-reviewed articles, and including the most commonly found L. monocytogenes STs. One requirement of the dataset was the restriction to assembled genomes from published articles. However, we are confident that the assembled genome set is comprehensive. Analyzing the metadata of the collected genomes indicated a large dataset for ST5, ST9 and ST121, which is in line with previously published epidemiological studies (Maury et al., 2016; Moura et al., 2017). Metadata analyses also indicated an increase in number of isolates as time increases. This is most probably due to the increase in reporting as well as the 2006 advent of next generation sequencing technology and the decreasing costs associated with whole- genome sequencing.

The distribution of plasmids among strains tested is variable dependent upon the

ST of the L. monocytogenes strain, with some STs having a low abundance of plasmids, while other STs have a very high percentage of plasmids. Here we show

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evidence that plasmid presence is more ubiquitous than previously described, with 53% of all isolates containing a plasmid strongly suggesting that plasmids are commonly found in L. monocytogenes . For example, ST121 has the highest percentage of plasmid-carrying isolates, with 92% of all strains harboring plasmids, followed by ST5 with 81% of the dataset including plasmids. Interestingly, there were no plasmids identified within ST4 strains, which historically, are predominantly clinical isolates

(Moura et al., 2017). This variability is possibly due to the different niche environments of particular isolates, as well as the environment stresses L. monocytogenes strains are exposed to.

In line with previous studies , L. monocytogenes plasmids have a modular structure and these modules or even entire plasmids can be highly conserved. The plasmids range from 3 kb to 176 kb in size. Within most STs, L. monocytogenes plasmids have a minimum of two subtypes based upon plasmid sizes, and the plasmids were placed in at least two groupings according to their size and two groupings based on their RepA variant. Additionally, we found that the genetic content across L. monocytogenes ST plasmids was very diverse but between STs, plasmid genes were highly similar. For example, examination of L. monocytogenes ST5 plasmids showed clear divergence between two subtypes, but a high similarity between plasmids within the same subtype. In three representative ST5 plasmids examined, all appear to be of the same subgroup with nearly identical sequences with a high percentage of similarity, despite the differences in plasmid sizes. Moreover, for both ST5 and ST9, the related plasmids within the same subgroups show a high degree of conservation of genetic content, indicating these plasmids are highly similar, if not identical. That the Mauve

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alignments indicate the relatedness of plasmids within the groups but show the distinct variability across the groupings is concurrent with other studies (Kuenne et al., 2010).

Just as with the presence of plasmids, the genetic content of the plasmids is most likely dependent upon the selective environments and subsequent stressors present. Stress response genes analyzed here were highly conserved but also show different abundance patterns in different STs. For example, evaluation of the plasmid dataset for the presence of stress-related genes in L monocytogenes ST155 plasmids found nearly or all ST155 plasmids carried the stress-related genes for NADH peroxidase npx , zinc riboswitch or the QAC operon bcrABC . However, none of the

ST155 plasmids had genes for the multicopper oxidase mco or the cadmium efflux genes cadABC . Conversely, at least 23% of ST7 plasmids carried every examined stress-related gene, but no more than 57% of the ST7 strains carried these genes, indicating for ST7 plasmids, gene content was highly variable. These data therefore indicates that many unknown plasmid-encoded genes may be beneficial to L. monocytogenes strains and their functions may be highly dependent upon on the niche where these strains are found.

The dataset not only allows searching for genes of interest, but also the identification of novel features, for example, a putative mercury resistance locus.

Review of the Patric annotation and Uniprot protein analysis indicates a novel mercury operon containing seven genes possibly involved in increasing the tolerance to mercury, a heavy metal often associated with detergents and other cleaning products.

Identification of this novel operon shows that this comprehensive dataset can be utilized for the identification of novel stress response genes.

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Within our dataset, each source had an equitable distribution of overall isolates, but the distribution of plasmids was highest among environmental sources, followed by food isolates, which is in line with previous reports (Hingston et al., 2019, Moura et al.,

2016). Future studies could examine this data set to analyze the potential plasmid presence in clinical vs. food vs. environmental strains and examine the plasmid and genetic conservation based upon variable sources.

Similar functional studies on stress-responses attributed to plasmid-encoded genes have been conducted by previous studies, although for some studies without the use of plasmid-cured or complement strains (Hingston et al., 2017b). This thesis significantly advances our knowledge about the contribution of L. monocytogenes plasmids to stress response and survival. For the first time, revealed that specific L. monocytogenes plasmids confer increased tolerance to different stress conditions that could be present within different food and FPEs. This includes stress induced by a BC disinfectant, heat, acidic stress, and high salt concentrations combined with mild acidic stress. Furthermore, the increased tolerance was examined for the first time using three different plasmids from three different sequence types, using isogenic pairs of plasmid- containing and plasmid-cured strains. Due to the exhibited conservation of genetic content among the three different L. monocytogenes ST plasmids, these results may indicate the stress response encoded by these plasmids may also apply to other L. monocytogenes plasmids as well.

Food and FPE environments that are susceptible to L. monocytogenes contamination have a variety of stress factors that could play a role in L. monocytogenes persistence. Consequently, future studies should examine these

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isogenic sets under additional conditions for the contribution of plasmids to survival, such as minimal media/low nutrient concentrations, logarithmic vs stationary growth phase, low temperatures and others.

Finally, we provide the first detailed analyses of gene expression patterns in L monocytogenes plasmids as well as insights into the specific short-term stress response of two L. monocytogenes strains, 6179 and R479a, when exposed to oxidative and acidic stress, and the differential expression of plasmid genes of L. monocytogenes under those stress conditions. This is vital for research as most L. monocytogenes plasmid genes are still uncharacterized. Our results reveal candidate genes possibly involved in stress response, such as genes with predicted functions in stress survival such as clpL, mco, as well as a putative zinc riboswitch and a number of conserved hypothetical proteins. An involvement of the clpL and mco genes in L. monocytogenes stress response is consistent with the literature.

Even as pLM6179 and pLMR479a share a subset of genes, each plasmid harbors several strain-specific genes, some of which are predicted to be involved in stress response (Naditz et al., 2019). Our results indicate an overall chromosomal response to the aforementioned stress conditions that is highly similar between the two strains. Surprisingly, when exposed to oxidative stress, there was no differential expression in the pLMR479a transcriptome and only a single DE gene in the pLM6179 transcriptome. The minimal changes in expression between the control transcriptomes and the oxidative treatment transcriptome indicates the 0.01% hydrogen peroxide treatment may not have caused stress to the two strains, either because of low hydrogen peroxide concentration, high cell density reducing the effective hydrogen

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peroxide concentration, or insufficient stress exposure time. It should be noted that results from Chapter 3 showed a significant contribution of the plasmids pLM6179 and pLMR479a to survival under 0.01% hydrogen peroxide. However, the period of stress exposure conducted in that study was longer and at lower cell densities (Naditz et al.,

2019). Although there was no significant change in expression, the gene encoding the

HSP ClpL in pLM6179 was both upregulated and highly expressed in terms of read counts. Alteration of the concentration of hydrogen peroxide or extension of stress exposure may result in a significant change in differential expression for this and other plasmid-encoded genes.

In contrast, the lactic acid stress response was striking in terms of changes in chromosomal but also for plasmid gene expression. The stress exposure data indicate a number of shared plasmid gene expression patterns, including upregulation and high expression of the transposase for Tn 5422 and a membrane protein of unknown function. Conversely, the genes encoding a cadmium-transporting protein ( cadA ) and accessory protein (cadC ) were downregulated, as was the plasmid replication protein encoding gene repA . This indicates a decrease in the production of plasmid proteins that may be unnecessary for survival in the stress environment, as well as a decrease in overall plasmid replication. The two L. monocytogenes strains utilized in this study carry plasmids that have contain both known and putative plasmid-encoded stress response genes, such as clpL and multicopper oxidase (Sitthisak et al., 2005; Wall et al., 2007;

Hingston et al., 2019a). Our results show that these stress-response genes are highly expressed and upregulated in response to lactic acid exposure.

We also provide data in support of a novel, uncharacterized riboswitch in

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pLMR479a and its role in lactic acid stress response, due to its high expression level and significant upregulation while under acidic stress. After lactic acid exposure the majority (up to 70%) of the pLMR479a reads mapped to this intergenic region.

Furthermore, the intergenic region was also significantly upregulated. This indicates both the high volume and increased expression of this region in comparison to control conditions. Examination of this region indicated a 56% nucleotide identity to a family of nickel-cobalt riboswitches that are involved in heavy metal resistance (Furukawa et al.,

2015). These riboswitches are typically associated with a transporter of the same heavy metal, but the function of the riboswitch itself is unclear.

RNA secondary structure and examination of the riboswitch family indicate a similar structure to functionally characterized riboswitches identified in other Firmicutes .

The differences in the potentially related transporter between the L. monocytogenes riboswitch and the described ones, as well as BLASTn searches for related homologs indicate this is to be a zinc transporter, and not a nickel-cobalt transporter. The putative zinc transporter was not a DE gene during lactic acid stress exposure. The identification of the putative zinc riboswitch in L. monocytogenes plasmids reveals the usefulness of an approach like transcriptome sequencing for identifying novel candidate genes involved in a certain phenotype. The potential for this riboswitch and others will need to be examined further, perhaps by testing additional potential plasmid genes during lactic acid stress for similar riboswitch expression or utilizing cloning and expression in plasmid-free L. monocytogenes .

Overall, this thesis presents a large addition to the knowledge on L. monocytogenes plasmids. We provide a comprehensive compilation of L.

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monocytogenes sequences that is useful for examination of a variety of interests, including the largest dataset of L. monocytogenes plasmids with a great availability of metadata. Such a dataset would be very useful in examining the epidemiology of L. monocytogenes plasmids based on a source location, type, and collection date.

Furthermore, as all genomes presented in the dataset are assembled with accession numbers provided, deeper studies on gene content could be readily examined. We provide the first examination of stress-response in L. monocytogenes plasmids, showing the contribution of these mobile genetic elements in increased tolerance to environmental stressors. The protocols developed under these studies could be utilized to examine the stress response of other stress-related genes, using these same cured strains. Finally, by providing the first transcriptomic data for L. monocytogenes plasmids, we both confirm the activation of stress-related genes in response to analyzed stress conditions, but also identified novel genes that could be utilized for examination of stress response, either with plasmid-containing and plasmid-cured strains or by cloning and expressing these genes in plasmid-free strains of L. monocytogenes, or other Firmicutes, or E. coli. This thesis provides more avenues for examination of the role of plasmids in L. monocytogenes survival.

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APPENDIX. SUPPLEMENTARY FIGURES

6179 Control - 1 6179 Lactic Acid - 1 6179 H O - 1 2 2

R479a Control - 1 R479a Lactic Acid - 1 R479a H 2O2 - 1

6179 Control - 2 6179 Lactic Acid - 2 6179 H 2O2 - 2

R479a Control - 2 R479a Lactic Acid - 2 R479a H O - 2 2 2

6179 Control - 3 6179 Lactic Acid - 3 6179 H O - 3 2 2

R479a Control - 3 R479a Lactic Acid - 3 R479a H 2O2 - 3

Supplementary Figure 1. Electropherogram results of extracted RNA samples. The RNA integrity of the samples was measured using an RNA 6000 Nano chip via an Agilent 2100 Bioanalyzer (Prokaryote Total RNA Nano assay).

184

lmo2230_EGDe 1 MTQKLIYFL -SQTHIRSAIAEAWAKRLSLSNVKFISGSWHKSKSTPFIAEALNEFAIEPP LM6179_3010_6179 1 MTQKLIYFL -SQTHIRSAIAEAWAKRLSLSNVKFISGSWHKSKSTPFIAEALNEFAIEPP LMR479A_2344_R479 1 MTQKLIYFL -SQTHIRSAIAEAWAKRLSLSNVKFISGSWHKSKSTPFIAEALNEFAIEPP P45947_ARSC 1 MENKIIYFL CTGNSCRSQMAEGWAK QYLGDEW KVY SAGIEAHGLN PNAVK AMKEVGIDIS consensus 1 *..*.**** .....**..**.***...... *..*...... *....*..*..*...

lmo2230_ EGDe 60 ESLSYSPSSELLADADLIVTIYDSAHETAPKFPANIQEKIIYWDIDDPEQEIAL -PQKWA LM6179_3010_6179 60 ESLSYSPSSELLADADLIVTIYDSAHETAPKFPANIQEKIIYWDIDDPEQEIAL -PQKWA LMR479A_2344_R479 60 ESLSYSPSSELLADADLIVTIYDSAHETAPKFPANIQEKIIYWDIDDPEQEIAL -PQKWA P45947_ARSC 61 NQT SDIID SDILNN ADLVVTLCGD AADKCPMT PPH VKRE--HWGF DDP ARAQ GTEEE KWA consensus 61 ...*....*..*..***.**....*....*..*...... *..***...... ***

lmo2230_ EGDe 119 SYQEVCDNIALSVKNLEHVLIEA LM6179_3010_6179 119 SYQEVCDNIALSVKNLEHVLIEA LMR479A_2344_R479 119 SYQEVCDNIALSVKNLEHVLIEA P45947_ARSC 119 F FQRVRDEIGNR LKEFAET--GK consensus 121 ..*.*.*.*....*......

Supplementary Figure 2. L. monocytogenes with the arsenate reductase ArsC from Bacillus subtilis . The alignment was done with MAFFT available at: https://mafft.cbrc.jp/alignment/server/ , shading of conserved amino acid residues was performed with Boxshade available at: https://embnet.vital-it.ch/software/BOX_form.html . Asterisks indicate identical positions, dots indicate similar positions. Amino acid residues essential for function od ArsC from Bacillus subtilis based on (Bennett et al., Proc Natl Acad Sci U S A. 2001;98(24):13577-82) are highlighted in red. Abbreviations : lmo2230_ EGDe: L. monocytogenes EGDe, LM6179_3010_6179: L. monocytogenes 6179, LMR479A_2344_R479: L. monocytogenes R479a, and P45947_ARSC: Bacillus subtilis ArsC

185

Supplementary Figure 3. Mean of normalized transcriptome sequencing reads mapped to the noncoding RNA in L. monocytogenes strain R479a plasmid exposed to control conditions, oxidative stress (0.01% H2O2), and acidic stress (1% lactic acid). Q-values (<0.001) are indicated by asterisks.

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Erysipelotrichaceae bacterium 3_1_53

Cation diffusion transporter Locus_tag:

NiCo riboswitch

Clostridium scindens ATCC 35704

Cation diffusion transporter

Locus_tag: NiCo riboswitch

L. monocytogenes 08-5578 pLM5578

+ zosA – putative Zn transporter Locus_tag: LM5578_RS15705

Putative riboswitch

L. monocytogenes R479a pLMR479a

+ zosA – putative Zn transporter Locus_tag: LMR479a_RS15240

Putative riboswitch

Supplementary Figure 4. Visual representation of the genomic organization of functionally characterized NiCo riboswitches from Erysipelotrichaceae bacterium 3_1_53 and Clostridium scindens ATCC 35704 and putative riboswitches found on Listeria plasmids.

C_scindens 1 A CA GUACAAACUGAGCAGGCGAUGAAC CUUUA----- GGUCAUG GGGC CGGGCCGC UU-- Eba 1 A CA GUACAAACUGAGCAGGCAAUGA CCAGAGC----- GGUCAUGCAGC CGGGCUGCGA-- pLM5578 1 U CA UUGUGAUCUGAACAGGCGGUGAACGUAACACGAGG UUCAUGCAGC UGGGCUGCAAUU pLMR479a 1 U CA UUGUGAUCUGAACAGGCGGUGAACGUAACACGAGG UUCAUGCAGC UGGGCUGCAAUU consensus 1 ** *. .* ****.*****..***.*...... * *****..** ****.**....

C_scindens 54 -- UUG UGGCAGCAGA UUGCAAU UUC ---- AGCA CAUCUGUGGGACAGUU Eba 54 --AA GCGGCAACAGA UG AU UACAC------GCA CAUCUGUGGGACAGUU pLM5578 61 AUUUGCGGCAGCAGA CUAUG UAUUCUAAGGGCA UAUCUGUGGGACAGUU pLMR479a 61 AUUUGCGGCAGCAGA CUAUG UAUUCUAAGGGCA UAUCUGUGGGACAGUU consensus 61 ....*.****.**** ...... *** ***************

Supplementary Figure 5. Nucleotide alignment of functionally characterized NiCo riboswitches and putative riboswitch from Listeria monocytogenes plasmid pLMR479a. The alignment was done with MAFFT available at: https://mafft.cbrc.jp/alignment/server/ , shading of conserved nucleotide residues was performed with Boxshade available at: https://embnet.vital-it.ch/software/BOX_form.html .

Asterisks indicate identical positions, dots indicate similar positions. Nucleotide positions conserved in more than 97% of the 187 sequences are highlighted in blue, positions conserved in more than 90% of the sequences are highlighted in teal. Conservation of nucleotide positions is based on (Furukawa et al., Mol Cell 2015; 57:1088-1098). Abbreviations : C_scindens: C. scindens (Accession number: NZ_ACTJ00000000.1) , Eba: Erysipelotrichaceae bacterium 3_1_53 (Accession number: NZ_ABFY00000000.2), pLM5578: L. monocytogenes 08-5578 (Accession number: GCA_ 000093125.2), and pLMR479a: L. monocytogenes R479a.

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A B

C D

Supplementary Figure 6. Predicted secondary structures of the putative riboswitches from A) L. monocytogenes pLMR479a, B) L. monocytogenes pLM5578, and the functionally characterized NiCo riboswitches from C) C. scindens ATCC35704, and D) Erysipelotrichaceae bacterium 3_1_53. Secondary structure predictions were generated using the RNAfold WebServer (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi ) (Hofacker, Nucleic Acids Res 2003; 31(13): 3429-3431).

Supplementary Table 1. DeSeq2 results from L. monocytogenes 6179 plasmid genes in response to 30 minutes exposure to hydrogen peroxide stress.

Feature Gene Product log2Fold Change Q-Value LM6179_RS15185 conserved protein of unknown function -0.13 0.900597676 LM6179_RS15190 conserved protein of unknown function -0.06 0.900597676 LM6179_RS15195 conserved protein of unknown function 0.01 0.989455652 LM6179_RS15200 conserved protein of unknown function 0.21 0.900597676 LM6179_RS15205 conserved protein of unknown function 0.58 0.673179351 LM6179_RS15210 conserved protein of unknown function 0.26 0.900597676 LM6179_RS15215 conserved protein of unknown function 0.26 0.900597676 LM6179_RS15220 conserved exported protein of unknown function 0.70 0.55418383 LM6179_RS15225 yddH putative cell wall hydrolase -0.15 0.900597676

LM6179_RS15230 conserved protein of unknown function 0.06 0.919222401 189 LM6179_RS15235 conserved protein of unknown function -0.18 0.900597676 LM6179_RS15240 conserved protein of unknown function -0.05 0.927516023 LM6179_RS15245 conserved membrane protein of unknown function -0.12 0.900597676 LM6179_RS15255 conserved protein of unknown function 0.40 0.900597676 LM6179_RS15260 traI DNA topoisomerase 0.68 0.55418383 LM6179_RS15270 conserved protein of unknown function -0.13 0.900597676 LM6179_RS15275 0.48 0.55418383 LM6179_RS15280 conserved protein of unknown function -0.09 0.900597676 LM6179_RS15290 conserved protein of unknown function -0.36 0.871935685 LM6179_RS15295 conserved protein of unknown function -0.13 0.900597676 LM6179_RS15300 conserved protein of unknown function 0.07 0.919222401 LM6179_RS15305 conserved protein of unknown function 0.13 0.900597676 LM6179_RS15310 conserved protein of unknown function -0.07 0.90500286 LM6179_RS15315 conserved protein of unknown function -0.06 0.90500286 LM6179_RS15320 conserved membrane protein of unknown function -0.15 0.900597676

LM6179_RS15325 Putative typeII/typeIV secretion system protein -0.13 0.900597676 LM6179_RS15330 conserved protein of unknown function -0.22 0.900597676 Supplementary Table 1 (cont.) Feature Gene Product log2Fold Change Q-Value LM6179_RS15335 conserved protein of unknown function -0.36 0.900420654 LM6179_RS15340 conserved protein of unknown function -0.13 0.900597676 LM6179_RS15345 cadA cadmium-transporting ATPase Tn5422 -0.12 0.900597676 LM6179_RS15350 cadC Cadmium efflux system accessory protein Tn5422 -0.18 0.900597676 LM6179_RS15355 tnpR transposase Tn5422 0.10 0.900597676 LM6179_RS15360 tnpA transposase Tn5422 0.04 0.90500286 LM6179_RS15365 repA Plasmid replication protein -0.42 0.55418383 LM6179_RS15370 repB parA family protein 0.40 0.55418383 LM6179_RS15375 conserved protein of unknown function 0.36 0.55418383 LM6179_RS15380 uvrX putative DNA repair protein 0.67 0.55418383

LM6179_RS15385 conserved protein of unknown function 1.11 0.000114303 190 LM6179_RS15390 conserved protein of unknown function 0.20 0.900597676 LM6179_RS15400 clpL Heat shock protein clpL – stress response 0.44 0.505469968 LM6179_RS15405 Predicted protein 0.20 0.900597676 LM6179_RS15410 -0.23 0.900597676 LM6179_RS15415 putative resolvase 0.04 0.919222401 LM6179_RS15420 conserved protein of unknown function -0.12 0.900597676 LM6179_RS15425 conserved protein of unknown function -0.15 0.900597676 LM6179_RS15430 conserved protein of unknown function -0.50 0.59961754 LM6179_RS15435 Predicted protein -0.13 0.900597676 LM6179_RS15440 Predicted protein -0.25 0.900597676 LM6179_RS15445 conserved protein of unknown function -0.21 0.900597676 LM6179_RS15450 Putative toxin-antitoxin system protein -0.05 0.90500286 LM6179_RS15455 Putative toxin-antitoxin system protein -0.09 0.900597676 LM6179_RS15460 conserved protein of unknown function -0.24 0.750191409 LM6179_RS15465 Putative HTH transcriptional regulator -0.26 0.900420654

Helicase conserved C-terminal domain protein LM6179_RS15470 (fragment) -0.12 0.900597676 LM6179_RS15475 conserved membrane protein of unknown function -0.16 0.900597676 Supplementary Table 1 (cont.) Feature Gene Product log2Fold Change Q-Value LM6179_RS15480 conserved protein of unknown function -0.01 0.982377743 LM6179_RS15485 conserved protein of unknown function -0.03 0.919222401 LM6179_RS15515 conserved protein of unknown function -0.11 0.900597676 LM6179_RS15565 transposase 0.29 0.673179351 LM6179_RS15905 0.20 0.900597676 LM6179_RS15910 conserved protein of unknown function -0.08 0.900597676

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Supplementary Table 2. DeSeq2 results from L. monocytogenes 6179 plasmid genes in response to 30 minutes exposure to lactic acid stress. Feature Gene Product log2Fold Change Q-Value LM6179_RS15185 conserved protein of unknown function -0.62 0.607223075 LM6179_RS15190 conserved protein of unknown function 0.30 0.128373392 LM6179_RS15195 conserved protein of unknown function 1.60 0.001826538 LM6179_RS15200 conserved protein of unknown function 1.40 0.008991885 LM6179_RS15205 conserved protein of unknown function 1.76 0.005359391 LM6179_RS15210 conserved protein of unknown function 0.97 0.252360022 LM6179_RS15215 conserved protein of unknown function 1.38 0.00315053 LM6179_RS15220 conserved exported protein of unknown function 2.73 0.001826538 LM6179_RS15225 yddH putative cell wall hydrolase 0.98 0.250316872 LM6179_RS15230 conserved protein of unknown function 0.42 0.236869841 LM6179_RS15235 conserved protein of unknown function -0.82 0.399261654 LM6179_RS15240 conserved protein of unknown function 0.98 0.195460564 LM6179_RS15245 conserved membrane protein of unknown function 2.05 1.48344E-05 LM6179_RS15255 conserved protein of unknown function 1.51 0.08888664 192 LM6179_RS15260 traI DNA topoisomerase 0.60 0.512261308 LM6179_RS15265 conserved protein of unknown function 1.98 0.114936162 LM6179_RS15270 conserved protein of unknown function -0.24 0.787444058 LM6179_RS15280 conserved protein of unknown function 1.79 0.134601263 LM6179_RS15290 conserved protein of unknown function -2.22 3.06296E-07 LM6179_RS15295 conserved protein of unknown function -2.87 3.06296E-07 LM6179_RS15300 conserved protein of unknown function -3.61 0.000291078 LM6179_RS15305 conserved protein of unknown function -3.85 0.000107493 LM6179_RS15310 conserved protein of unknown function -3.09 4.0868E-14 LM6179_RS15315 conserved protein of unknown function -2.75 9.8398E-18 LM6179_RS15320 conserved membrane protein of unknown function -2.58 6.55475E-12 LM6179_RS15325 Putative typeII/typeIV secretion system protein -2.68 6.20004E-20 LM6179_RS15330 conserved protein of unknown function -2.13 3.62982E-08 LM6179_RS15335 conserved protein of unknown function -2.71 8.03668E-07 LM6179_RS15340 conserved protein of unknown function -1.47 1.77078E-05 LM6179_RS15345 cadA cadmium-transporting ATPase Tn5422 -1.37 4.09011E-07 LM6179_RS15350 cadC Cadmium efflux system accessory protein Tn5422 -1.10 6.49795E-05

Supplementary Table 2 (Cont.) Feature Gene Product log2Fold Change Q-Value LM6179_RS15355 tnpR transposase Tn5422 1.41 3.00211E-11 LM6179_RS15360 tnpA transposase Tn5422 1.54 3.01776E-16 LM6179_RS15365 repA Plasmid replication protein -2.72 3.0628E-33 LM6179_RS15370 repB parA family protein 2.10 6.34862E-15 LM6179_RS15375 conserved protein of unknown function 2.20 7.67871E-23 LM6179_RS15380 uvrX putative DNA repair protein 0.72 0.029142952 LM6179_RS15385 conserved protein of unknown function 0.74 0.004179584 LM6179_RS15390 conserved protein of unknown function -0.69 0.058769569 LM6179_RS15400 clpL Heat shock protein clpL – stress response 3.15 2.50254E-29 LM6179_RS15405 Predicted protein 1.49 0.164160899 LM6179_RS15410 -0.15 0.564622469 LM6179_RS15415 putative resolvase 0.79 0.000648951 LM6179_RS15420 conserved protein of unknown function -0.27 0.308543607 LM6179_RS15425 conserved protein of unknown function -1.27 0.017555867 193 LM6179_RS15430 conserved protein of unknown function -2.18 2.20455E-05 LM6179_RS15435 Predicted protein -4.56 2.94187E-10 LM6179_RS15440 Predicted protein -4.70 2.63117E-07 LM6179_RS15445 conserved protein of unknown function -0.96 1.6214E-05 LM6179_RS15450 Putative toxin-antitoxin system protein 1.04 1.83879E-05 LM6179_RS15455 Putative toxin-antitoxin system protein 1.24 2.20455E-05 LM6179_RS15460 conserved protein of unknown function -1.93 3.01776E-16 LM6179_RS15465 Putative HTH transcriptional regulator -2.02 1.49857E-10 Helicase conserved C-terminal domain protein LM6179_RS15470 (fragment) 0.25 0.316346613 LM6179_RS15475 conserved membrane protein of unknown function 0.80 0.00069187 LM6179_RS15480 conserved protein of unknown function -0.62 0.000574688 LM6179_RS15485 conserved protein of unknown function -1.09 1.21404E-06 LM6179_RS15515 conserved protein of unknown function 0.19 0.650196684 LM6179_RS15565 transposase -0.76 0.002757249 LM6179_RS15905 -1.65 0.182533794 LM6179_RS15910 conserved protein of unknown function 1.29 2.11142E-09

Supplementary Table 3. DeSeq2 results from L. monocytogenes R479a plasmid genes in response to 30 minutes exposure to hydrogen peroxide stress. Feature Gene Product log2FoldChange Q-Value LMR479a_misc_RNA_rli Putative riboswitch 0.08 0.976 LMR479A_RS14825 n/a -0.41 0.976 LMR479A_RS14830 Transposase, IS6 family 0.20 0.976 LMR479A_RS14835 Integrase, catalytic region -0.17 0.976 LMR479A_RS14840 conserved protein of unknown function 0.09 0.976 LMR479A_RS14845 conserved membrane protein of unknown function 0.22 0.976 LMR479A_RS14850 conserved protein of unknown function 0.07 0.976 LMR479A_RS14855 conserved protein of unknown function -0.17 0.976 LMR479A_RS14860 conserved protein of unknown function 0.16 0.976 LMR479A_RS14865 conserved protein of unknown function 0.08 0.976 LMR479A_RS14870 conserved protein of unknown function 0.26 0.976 LMR479A_RS14875 conserved protein of unknown function 0.02 0.986 LMR479A_RS14880 conserved protein of unknown function 0.24 0.976

LMR479A_RS14885 conserved protein of unknown function 0.06 0.976 194 LMR479A_RS14890 conserved exported protein of unknown function 0.30 0.976 LMR479A_RS14900 yddH putative cell wall hydrolase -0.11 0.976 LMR479A_RS14905 conserved protein of unknown function 0.05 0.986 LMR479A_RS14910 conserved protein of unknown function 0.16 0.976 LMR479A_RS14915 conserved protein of unknown function 0.03 0.986 LMR479A_RS14920 conserved membrane protein of unknown function 0.02 0.986 LMR479A_RS14930 conserved protein of unknown function -0.01 0.986 LMR479A_RS14935 traI DNA topoisomerase -0.24 0.976 LMR479A_RS14940 conserved protein of unknown function -0.14 0.976 LMR479A_RS14945 conserved protein of unknown function 0.12 0.976 LMR479A_RS14950 conserved membrane protein of unknown function -0.06 0.976 LMR479A_RS14955 conserved protein of unknown function -0.06 0.976 LMR479A_RS14960 conserved protein of unknown function -0.51 0.976 LMR479A_RS14965 conserved protein of unknown function -0.09 0.976 LMR479A_RS14970 conserved protein of unknown function -0.24 0.976 LMR479A_RS14975 conserved protein of unknown function -0.07 0.976

Supplementary Table 3 (Cont.) Feature Gene Product log2FoldChange Q-Value LMR479A_RS14980 conserved protein of unknown function 0.05 0.976 LMR479A_RS14985 conserved protein of unknown function -0.09 0.976 LMR479A_RS14990 conserved protein of unknown function -0.15 0.976 LMR479A_RS14995 conserved membrane protein of unknown function -0.30 0.976 LMR479A_RS15000 conserved protein of unknown function -0.23 0.976 LMR479A_RS15005 conserved protein of unknown function 0.18 0.976 LMR479A_RS15015 conserved exported protein of unknown function 0.02 0.986 LMR479A_RS15020 conserved exported protein of unknown function 0.21 0.976 LMR479A_RS15025 conserved exported protein of unknown function 0.30 0.976 LMR479A_RS15030 conserved exported protein of unknown function 0.06 0.976 LMR479A_RS15035 conserved protein of unknown function -0.12 0.976 LMR479A_RS15040 conserved protein of unknown function -0.03 0.986 LMR479A_RS15045 conserved exported protein of unknown function -0.25 0.976 LMR479A_RS15050 conserved protein of unknown function -0.24 0.976 LMR479A_RS15055 protein of unknown function -0.08 0.976 195 LMR479A_RS15060 conserved protein of unknown function -0.19 0.976 LMR479A_RS15065 repA Plasmid replication protein repA -0.03 0.986 LMR479A_RS15070 conserved protein of unknown function -0.16 0.976 LMR479A_RS15075 Cobyrinic acid a,c-diamide synthase -0.08 0.976 LMR479A_RS15080 uvrX putative DNA repair protein 0.22 0.976 LMR479A_RS15085 conserved protein of unknown function 0.31 0.976 LMR479A_RS15090 exported protein of unknown function -0.30 0.976 LMR479A_RS15095 PcfY -0.16 0.976 LMR479A_RS15100 protein of unknown function -0.20 0.976 LMR479A_RS15105 protein of unknown function 0.07 0.976 LMR479A_RS15110 conserved protein of unknown function -0.12 0.976 LMR479A_RS15115 conserved protein of unknown function -0.11 0.976 LMR479A_RS15120 SNF2 family protein (fragment) 0.04 0.986 LMR479A_RS15125 cadA cadmium-transporting ATPase Tn5422 0.15 0.976 LMR479A_RS15130 cadC Cadmium efflux system accessory protein Tn5422 -0.33 0.976 LMR479A_RS15135 tnpR transposase 0.24 0.976

Supplementary Table 3 (Cont.) Feature Gene Product log2FoldChange Q-Value LMR479A_RS15140 tnpA transposase -0.10 0.976 LMR479A_RS15145 Putative multicopper oxidase 0.14 0.976 LMR479A_RS15150 copA copper-transporting P-type ATPase B 0.20 0.976 LMR479A_RS15155 conserved protein of unknown function 0.02 0.986 LMR479A_RS15160 conserved protein of unknown function -0.08 0.976 LMR479A_RS15165 conserved protein of unknown function -0.01 0.986 LMR479A_RS15170 Lead, cadmium, zinc and mercury transporting ATPase 0.38 0.976 LMR479A_RS15175 MerTP family mercury (Hg2+) permease, binding protein MerP 0.00 0.986 LMR479A_RS15180 n/a 0.18 0.976 LMR479A_RS15185 Transcriptional regulator 0.25 0.976 LMR479A_RS15190 conserved protein of unknown function -0.09 0.976 LMR479A_RS15195 transposase 0.09 0.976 LMR479A_RS15200 ybbM conserved membrane protein of unknown function 0.08 0.976 LMR479A_RS15205 ybbL Uncharacterized ABC transporter ATP-binding protein ybbL 0.12 0.976 LMR479A_RS15210 conserved protein of unknown function 0.36 0.976 196 LMR479A_RS15215 n/a -0.16 0.976 LMR479A_RS15220 DNA topoisomerase (fragment) -0.02 0.986 LMR479A_RS15225 conserved protein of unknown function 0.01 0.986 LMR479A_RS15230 conserved protein of unknown function -0.24 0.976 LMR479A_RS15235 tnp transposase 0.10 0.976 LMR479A_RS15240 zosA Zinc-transporting ATPase -0.15 0.976 LMR479A_RS15245 transposase -0.12 0.976 LMR479A_RS15250 conserved protein of unknown function -0.02 0.986 LMR479A_RS15255 Glycine betaine ABC transporter glycine betaine-binding protein 0.06 0.976 LMR479A_RS15260 npr NADH peroxidase 0.16 0.976 LMR479A_RS15265 conserved protein of unknown function -0.11 0.976 LMR479A_RS15615 n/a -0.08 0.976 LMR479A_RS15620 n/a 0.04 0.976 LMR479A_RS15625 Predicted protein 0.13 0.976

Supplementary Table 4. DeSeq2 results from L. monocytogenes R479a plasmid genes in response to 30 minutes exposure to lactic acid stress. Feature Gene Product log2Fold Change Q-Value LMR479a_misc_RNA_rli Putative riboswitch 3.27 5.16133E-19 LMR479A_RS14825 n/a -0.18 0.718178503 LMR479A_RS14830 Transposase, IS6 family 0.37 0.404368625 LMR479A_RS14835 Integrase, catalytic region -0.76 0.542647658 LMR479A_RS14840 conserved protein of unknown function 1.91 3.08834E-07 LMR479A_RS14845 conserved membrane protein of unknown function 1.31 0.001173151 LMR479A_RS14850 conserved protein of unknown function -1.66 0.000214392 LMR479A_RS14855 conserved protein of unknown function 2.33 0.003581443 LMR479A_RS14860 conserved protein of unknown function 0.29 0.524710549 LMR479A_RS14865 conserved protein of unknown function 1.44 0.055123835 LMR479A_RS14870 conserved protein of unknown function 0.06 0.954003817 LMR479A_RS14875 conserved protein of unknown function -1.00 0.537460433 LMR479A_RS14880 conserved protein of unknown function -0.19 #NUM!

LMR479A_RS14885 conserved protein of unknown function 0.40 0.718178503 197 LMR479A_RS14890 conserved exported protein of unknown function -1.25 #NUM! LMR479A_RS14900 yddH putative cell wall hydrolase -1.14 0.524710549 LMR479A_RS14905 conserved protein of unknown function 0.35 0.580035619 LMR479A_RS14910 conserved protein of unknown function -0.57 0.718178503 LMR479A_RS14915 conserved protein of unknown function -0.19 0.903994662 LMR479A_RS14920 conserved membrane protein of unknown function 0.94 0.18379552 LMR479A_RS14930 conserved protein of unknown function 1.85 0.061297211 LMR479A_RS14935 traI DNA topoisomerase -0.44 0.657677882 LMR479A_RS14940 conserved protein of unknown function -1.25 #NUM! LMR479A_RS14945 conserved protein of unknown function 2.13 0.157192912 LMR479A_RS14950 conserved membrane protein of unknown function -0.68 #NUM! LMR479A_RS14955 conserved protein of unknown function 3.60 3.69713E-15 LMR479A_RS14960 conserved protein of unknown function 3.33 7.14107E-13 LMR479A_RS14965 conserved protein of unknown function -0.53 0.537460433 LMR479A_RS14970 conserved protein of unknown function -1.57 0.18379552 LMR479A_RS14975 conserved protein of unknown function -2.19 0.201786707

Supplementary Table 4 (Cont.) Feature Gene Product log2Fold Change Q-Value LMR479A_RS14980 conserved protein of unknown function -1.93 #NUM! LMR479A_RS14985 conserved protein of unknown function -2.52 0.062592086 LMR479A_RS14990 conserved protein of unknown function -1.75 0.127700231 LMR479A_RS14995 conserved membrane protein of unknown function 0.54 0.586331398 LMR479A_RS15000 conserved protein of unknown function 0.66 0.548423529 LMR479A_RS15005 conserved protein of unknown function 0.80 #NUM! LMR479A_RS15015 conserved exported protein of unknown function 3.84 5.16133E-19 LMR479A_RS15020 conserved exported protein of unknown function 1.17 0.258456181 LMR479A_RS15025 conserved exported protein of unknown function 3.15 0.000241623 LMR479A_RS15030 conserved exported protein of unknown function 3.55 2.29757E-10 LMR479A_RS15035 conserved protein of unknown function -0.83 #NUM! LMR479A_RS15040 conserved protein of unknown function 0.41 0.744703554 LMR479A_RS15045 conserved exported protein of unknown function 2.06 4.24943E-06 LMR479A_RS15050 conserved protein of unknown function -3.69 4.52829E-06 LMR479A_RS15055 protein of unknown function -3.34 0.000101922 198 LMR479A_RS15060 conserved protein of unknown function -2.91 1.35472E-05 LMR479A_RS15065 repA Plasmid replication protein repA -4.01 6.56711E-28 LMR479A_RS15070 conserved protein of unknown function 0.00 0.997366926 LMR479A_RS15075 Cobyrinic acid a,c-diamide synthase -0.27 0.592690848 LMR479A_RS15080 uvrX putative DNA repair protein -1.35 0.006764221 LMR479A_RS15085 conserved protein of unknown function -0.84 0.081721647 LMR479A_RS15090 exported protein of unknown function 1.42 0.000398147 LMR479A_RS15095 PcfY 0.83 0.065700492 LMR479A_RS15100 protein of unknown function -3.81 3.67088E-11 LMR479A_RS15105 protein of unknown function -0.25 0.657677882 LMR479A_RS15110 conserved protein of unknown function -0.87 0.067481223 LMR479A_RS15115 conserved protein of unknown function -1.10 0.053156282 LMR479A_RS15120 SNF2 family protein (fragment) -1.71 4.0399E-07 LMR479A_RS15125 cadA cadmium-transporting ATPase Tn5422 -2.07 5.44758E-10 LMR479A_RS15130 cadC Cadmium efflux system accessory protein Tn5422 -2.16 2.04942E-08 LMR479A_RS15135 tnpR transposase 0.38 0.358544922

Supplementary Table 4 (Cont.) Feature Gene Product log2Fold Change Q-Value LMR479A_RS15140 tnpA transposase 0.38 0.319194603 LMR479A_RS15145 Putative multicopper oxidase 1.68 0.000238571 LMR479A_RS15150 copA copper-transporting P-type ATPase B 0.56 0.126442661 LMR479A_RS15155 conserved protein of unknown function -1.48 0.000298656 LMR479A_RS15160 conserved protein of unknown function -0.33 0.625165163 LMR479A_RS15165 conserved protein of unknown function 0.36 0.542647658 LMR479A_RS15170 Lead, cadmium, zinc and mercury transporting ATPase -0.64 0.404368625 LMR479A_RS15175 MerTP family mercury (Hg2+) permease, binding protein MerP -0.62 0.319194603 LMR479A_RS15180 n/a -0.91 0.085773176 LMR479A_RS15185 Transcriptional regulator -1.19 0.053156282 LMR479A_RS15190 conserved protein of unknown function -0.63 0.121601939 LMR479A_RS15195 transposase -0.97 0.065700492 LMR479A_RS15200 ybbM conserved membrane protein of unknown function -0.84 0.053156282 LMR479A_RS15205 ybbL Uncharacterized ABC transporter ATP-binding protein ybbL -0.79 0.053156282 LMR479A_RS15210 conserved protein of unknown function 0.78 0.087153084 199 LMR479A_RS15215 n/a -2.33 0.087153084 LMR479A_RS15220 DNA topoisomerase (fragment) -3.23 0.000374214 LMR479A_RS15225 conserved protein of unknown function 2.70 3.42848E-07 LMR479A_RS15230 conserved protein of unknown function -0.67 0.198245236 LMR479A_RS15235 tnp transposase -0.05 0.91460489 LMR479A_RS15240 zosA Zinc-transporting ATPase 0.22 0.649769299 LMR479A_RS15245 transposase -1.04 0.43944026 LMR479A_RS15250 conserved protein of unknown function -1.67 0.081721647 Glycine betaine ABC transporter glycine betaine-binding LMR479A_RS15255 protein -6.14 9.55792E-13 LMR479A_RS15260 npr NADH peroxidase -0.93 0.047025679 LMR479A_RS15265 conserved protein of unknown function -0.87 0.286500035 LMR479A_RS15615 n/a -1.17 0.095577002 LMR479A_RS15620 n/a 1.86 #NUM! LMR479A_RS15625 Predicted protein -1.27 0.247554758

Supplementary Table 5. Chromosomal DE genes in the L. monocytogenes R479a transcriptome after 30 min exposure to 0.01% hydrogen peroxide. DE genes which are also upregulated DE genes in 6179 under hydrogen peroxide treatment are highlighted in bold.

L. L. L. monocytogenes monocytogenes monocytogenes log2fold R479a locus_tag Gene Product EGDe locus_tag 6179 locus_tag change Q value LMR479A_1738 citB aconitate hydratase (aconitase) lmo1641 LM6179_2393 1.63 2.38E-09 LMR479A_1661 citZ citrate synthase II lmo1567 LM6179_2318 1.21 1.69E-07 LMR479A_1662 ytwI putative integral membrane protein lmo1568 LM6179_2319 1.30 2.65E-07 LMR479A_1660 citC isocitrate dehydrogenase lmo1566 LM6179_2317 1.33 4.94E-06 LMR479A_1994 xpt xanthine phosphoribosyltransferase lmo1885 LM6179_2654 1.07 7.17E-05 High affinity arginine ABC LMR479A_0866 artR transporter (ATP-binding protein) lmo0848 LM6179_1160 0.81 4.55E-03 LMR479A_1993 pbuX xanthine permease lmo1884 LM6179_2653 0.88 8.25E-03 LMR479A_2312 ohrA peroxiredoxin lmo2199 LM6179_2978 1.02 1.38E-02 transcriptional regulator sensing LMR479A_2313 organic peroxides lmo2200 LM6179_2979 0.99 1.55E-02

ohrR 200

Supplementary Table 6. The 50 most upregulated chromosomal DE genes in the L. monocytogenes 6179 transcriptome after 30 min exposure to 0.01% hydrogen peroxide. Genes that are part of the Sigma B regulon are highlighted in blue. DE genes which are also upregulated DE genes in R479a under hydrogen peroxide treatment are highlighted in bold.

L. monocytogenes L. monocytogenes L. monocytogenes log2fold 6179 locus_tag Gene Product EGDe locus_tag R479a locus_tag change Q value LM6179_2165 bilEA BilEA lmo1421 LMR479a_1510 1.143 1.90E-06 LM6179_3010 putative Arsenate reductase lmo2230 LMR479a_2344 1.113 5.71E-05 LM6179_2317 citC isocitrate dehydrogenase lmo1566 LMR479a_1660 1.074 1.32E-05 LM6179_2978 ohrA peroxiredoxin lmo2199 LMR479a_2312 1.056 1.82E-04 transcriptional regulator sensing LM6179_2979 ohrR organic peroxides lmo2200 LMR479a_2313 1.041 5.31E-05 LM6179_2390 yocD putative carboxypeptidase lmo1638 LMR479a_1735 1.018 9.08E-06 conserved membrane protein of LM6179_2166 unknown function lmo1422 LMR479a_1511 1.014 3.99E-05 conserved protein of unknown LM6179_1818 function lmo2454 LMR479a_2579 1.012 1.45E-07 LM6179_2319 ytwI putative integral membrane protein lmo1568 LMR479a_1662 1.005 4.57E-08 201 LM6179_2318 citZ citrate synthase II lmo1567 LMR479a_1661 0.980 7.80E-05 LM6179_2393 citB aconitate hydratase (aconitase) lmo1641 LMR479a_1738 0.968 1.63E-03 conserved membrane protein of LM6179_2392 unknown function lmo1640 LMR479a_1737 0.914 2.43E-03 conserved protein of unknown LM6179_0248 function lmo2828 LMR479a_2966 0.903 1.32E-03 conserved protein of unknown LM6179_3001 function lmo2221 LMR479a_2335 0.861 2.81E-06 LM6179_2391 DNA-3-methyladenine glycosylase I lmo1639 LMR479a_1736 0.849 8.53E-03 LM6179_misc_RNA_43 LhrC 0.833 3.29E-03 LM6179_2637 ppdK Pyruvate, phosphate dikinase lmo1867 LMR479a_1977 0.830 7.16E-04 LM6179_2745 dinB DNA polymerase IV lmo1975 LMR479a_2085 0.829 9.17E-04 LM6179_1716 uvrA excinuclease ABC (subunit A) lmo2488 LMR479a_2613 0.824 1.66E-05 LM6179_2184 sodA superoxide dismutase lmo1439 LMR479a_1528 0.793 8.02E-03 LM6179_2635 ccpN negative regulator of gluconeogenesis lmo1865 LMR479a_1975 0.783 2.29E-04 conserved protein of unknown LM6179_3002 function lmo2222 LMR479a_2336 0.781 8.99E-04

Supplementary Table 6 (Cont.) L. monocytogenes L. monocytogenes L. monocytogenes log2fold 6179 locus_tag Gene Product EGDe locus_tag R479a locus_tag change Q value PTS-dependent dihydroxyacetone kinase, phosphotransferase subunit LM6179_0112 dhaM dhaM lmo2697 LMR479a_2835 0.771 3.61E-03 LM6179_1715 uvrB excinuclease ABC (subunit B) lmo2489 LMR479a_2614 0.771 3.88E-04 LM6179_2044 yneA Cell division suppressor protein YneA lmo1303 LMR479a_1387 0.757 4.01E-02 PTS-dependent dihydroxyacetone LM6179_0111 dhaL kinase, ADP-binding subunit dhaL lmo2696 LMR479a_2834 0.733 1.55E-02 conserved protein of unknown LM6179_1447 function lmo1140 LMR479a_1161 0.710 6.45E-04 dihydroxyacetone kinase, N-terminal LM6179_0110 dhaK domain lmo2695 LMR479a_2833 0.709 1.59E-02 conserved protein of unknown LM6179_0907 function lmo0602 LMR479a_0614 0.684 1.19E-02 transcriptional repressor of the SOS

LM6179_2042 lexA regulon lmo1302 LMR479a_1386 0.678 3.21E-03 202 LM6179_2142 recA multifunctional SOS repair factor lmo1398 LMR479a_1486 0.677 1.27E-02 conserved protein of unknown LM6179_1912 function 0.664 3.30E-02 conserved protein of unknown LM6179_1628 function lmo2571 LMR479a_2699 0.661 3.82E-02 LM6179_2934 BDS Alkyl/aryl-sulfatase BDS1 lmo2157 LMR479a_2269 0.639 4.56E-02 conserved protein of unknown LM6179_2990 function lmo2210 LMR479a_2324 0.624 1.19E-02 LM6179_2636 yqfL positive regulator of gluconeogenesis lmo1866 LMR479a_1976 0.622 3.53E-02 LM6179_2871 Transcriptional regulator lmo2100 LMR479a_2211 0.621 9.81E-03 conserved protein of unknown LM6179_2821 function lmo2050 LMR479a_2161 0.612 3.46E-02 LM6179_2550 exoA apurinic/apyrimidinic endonuclease lmo1782 LMR479a_1890 0.601 2.89E-02 conserved protein of unknown LM6179_0156 function lmo2742 LMR479a_2879 0.600 3.85E-02 LM6179_1107 ywnB putative oxidoreductase lmo0794 LMR479a_0813 0.585 1.48E-02 Fumarylacetoacetate hydrolase family LM6179_3046 protein lmo2266 LMR479a_2380 0.577 1.53E-02

Supplementary Table 6 (Cont.) L. monocytogenes L. monocytogenes L. monocytogenes log2fold 6179 locus_tag Gene Product EGDe locus_tag R479a locus_tag change Q value LM6179_0514 cysK cysteine synthase lmo0223 LMR479a_0232 0.576 1.48E-02 fragment of phosphoribosyl-AMP cyclohydrolase; phosphoribosyl-ATP LM6179_0867 hisI pyrophosphohydrolase (part 1) lmo0562 LMR479a_0574 0.573 3.13E-02 LM6179_0962 Acetyltransferase lmo0652 LMR479a_0669 0.572 1.47E-04 conserved protein of unknown LM6179_0447 function lmo0157 LMR479a_0166 0.567 6.45E-04 LM6179_1300 gpo Glutathione peroxidase lmo0983 LMR479a_1009 0.562 4.31E-02 conserved protein of unknown LM6179_2264 function lmo1518 LMR479a_1611 0.558 6.95E-04 conserved protein of unknown LM6179_1625 function lmo2574 LMR479a_2702 0.545 2.43E-03 LM6179_2122 lisK LisK lmo1378 LMR479a_1466 0.545 1.24E-02

203

Supplementary Table 7. The 50 most downregulated chromosomal DE genes in the L. monocytogenes 6179 transcriptome after 30 min exposure to 1% lactic acid. Genes that are part of the Sigma B regulon are highlighted in blue. DE genes which are also downregulated DE genes in R479a under lactic acid treatment are highlighted in bold.

L. monocytogenes L. monocytogenes L. monocytogenes log2fold 6179 locus_tag Gene Product EGDe locus_tag R479a locus_tag change Q value LM6179_1852 Cation efflux family protein lmo2423 LMR479a_2546 -10.59 1.54E-70 LM6179_1681 yocH Cell wall-binding protein yocH lmo2522 LMR479a_2648 -10.12 2.35E-154 LM6179_0443 znuA Zn(II)-binding lipoprotein lmo0153 LMR479a_0162 -9.47 4.57E-09 conserved protein of unknown LM6179_0986 function lmo0675 LMR479a_0693 -8.57 4.91E-21 conserved protein of unknown LM6179_0476 function lmo0186 LMR479a_0195 -8.33 1.09E-240 cell -division ABC transporter LM6179_1697 ftsE (ATP-binding protein) lmo2507 LMR479a_2632 -8.17 7.94E-234 LM6179_1698 ftsX cell-division ABC transporter lmo2506 LMR479a_2631 -8.15 2.28E-270 conserved protein of unknown LM6179_2555 function LMR479a_1895 -7.87 6.44E-06 204 LM6179_1700 Peptidase lmo2504 LMR479a_2629 -7.83 1.33E-190 LM6179_1699 spl P45 lmo2505 LMR479a_2630 -7.75 1.42E-297 LM6179_0697 NLP/P60 family protein lmo0394 LMR479a_0405 -7.71 4.83E-146 LM6179_0987 Flagellar biosynthesis protein FliP lmo0676 LMR479a_0694 -7.38 1.13E-62 LM6179_misc_RNA_26 Purine -7.33 4.70E-05 LM6179_0032 rplX ribosomal protein L24 (BL23) lmo2621 LMR479a_2756 -7.28 3.15E-193 LM6179_0547 rplJ ribosomal protein L10 (BL5) lmo0250 LMR479a_0262 -7.28 1.76E-178 conserved membrane protein of LM6179_1181 unknown function lmo0867 LMR479a_0887 -7.15 2.47E-104 LM6179_0030 rpsNA ribosomal protein S14 lmo2619 LMR479a_2754 -7.08 8.38E-79 N-acetylmuramoyl -L-alanine LM6179_1523 amidase, family 4 lmo1216 LMR479a_1237 -7.03 1.54E-250 putative ABC lipid transporter LM6179_1149 ywjA (ATP-binding protein) lmo0837 LMR479a_0855 -6.91 4.99E-95 LM6179_0698 Acetyltransferase lmo0395 LMR479a_0406 -6.90 4.83E-17 LM6179_0038 rplV ribosomal protein L22 (BL17) lmo2627 LMR479a_2762 -6.89 1.67E-112 LM6179_0548 rplL ribosomal protein L12 (BL9) lmo0251 LMR479a_0263 -6.89 1.85E-123

Supplementary Table 7 (Cont.) L. monocytogenes L. monocytogenes L. monocytogenes log2fold 6179 locus_tag Gene Product EGDe locus_tag R479a locus_tag change Q value Flagellar biosynthesis protein LM6179_0990 FlhB lmo0679 LMR479a_0697 -6.88 1.09E-45 LM6179_0029 rpsH ribosomal protein S8 (BS8) lmo2618 LMR479a_2753 -6.85 5.55E-90 LM6179_0031 rplE ribosomal protein L5 (BL6) lmo2620 LMR479a_2755 -6.84 1.62E-75 LM6179_0044 rpsJ ribosomal protein S10 (BS13) lmo2633 LMR479a_2768 -6.83 3.10E-75 LM6179_0034 rpsQ ribosomal protein S17 (BS16) lmo2623 LMR479a_2758 -6.81 8.08E-92 LM6179_0027 rplR ribosomal protein L18 lmo2616 LMR479a_2751 -6.81 3.80E-94 LM6179_0039 rpsS ribosomal protein S19 (BS19) lmo2628 LMR479a_2763 -6.80 3.88E-143 LM6179_0026 rpsE ribosomal protein S5 lmo2615 LMR479a_2750 -6.77 1.76E-84 LM6179_0028 rplF ribosomal protein L6 (BL8) lmo2617 LMR479a_2752 -6.76 3.65E-89 conserved membrane protein of LM6179_0828 unknown function lmo0523 LMR479a_0535 -6.75 2.29E-12 conserved protein of unknown

LM6179_2821 function lmo2050 LMR479a_2161 -6.75 4.12E-140 205 LM6179_0033 rplNA ribosomal protein L14 lmo2622 LMR479a_2757 -6.73 1.24E-72 LM6179_0043 rplC ribosomal protein L3 (BL3) lmo2632 LMR479a_2767 -6.71 2.90E-76 LM6179_0035 rpmC ribosomal protein L29 lmo2624 LMR479a_2759 -6.69 1.60E-74 Cell wall surface anchor family LM6179_0811 protein lmo0514 LMR479a_0521 -6.69 2.56E-56 transcriptional regulator (DeoR LM6179_3054 fruR family) lmo2337 LMR479a_2456 -6.68 5.78E-53 Zn(II) transporter (ATP -binding LM6179_0444 znuC protein) lmo0154 LMR479a_0163 -6.68 3.39E-08 LM6179_0036 rplP ribosomal protein L16 lmo2625 LMR479a_2760 -6.68 1.55E-58 LM6179_0041 rplW ribosomal protein L23 lmo2630 LMR479a_2765 -6.68 2.78E-97 LM6179_2301 folC folyl-polyglutamate synthase lmo1551 LMR479a_1644 -6.66 2.31E-68 LM6179_0042 rplD ribosomal protein L4 lmo2631 LMR479a_2766 -6.65 6.80E-74 glycine betaine ABC transporter LM6179_1332 opuAB (permease) lmo1015 LMR479a_1041 -6.62 7.13E-127 LM6179_0989 Flagellar biosynthesis protein FliR lmo0678 LMR479a_0696 -6.61 2.21E-39 LM6179_1308 Membrane protein lmo0991 LMR479a_1017 -6.60 4.60E-67

Supplementary Table 7 (Cont.) L. monocytogenes L. monocytogenes L. monocytogenes log2fold 6179 locus_tag Gene Product EGDe locus_tag R479a locus_tag change Q value LM6179_0037 rpsC ribosomal protein S3 (BS3) lmo2626 LMR479a_2761 -6.58 4.69E-86 LM6179_0040 rplB ribosomal protein L2 (BL2) lmo2629 LMR479a_2764 -6.58 5.20E-89 LM6179_0025 rpmD ribosomal protein L30 (BL27) lmo2614 LMR479a_2749 -6.50 2.96E-66 LM6179_2586 rpmB ribosomal protein L28 lmo1816 LMR479a_1926 -6.45 1.05E-60

206

Supplementary Table 8. The 50 most downregulated chromosomal DE genes in the L. monocytogenes R479a transcriptome after 30 min exposure to 1% lactic acid. Genes that are part of the Sigma B regulon are highlighted in blue. DE genes which are also d downregulated DE genes in 6179 under lactic acid treatment are highlighted in bold.

L. monocytogenes L. monocytogenes L. monocytogenes log2fold R479a locus_tag Gene Product EGDe locus_tag 6179 locus_tag change Q value LMR479a_2546 Cation efflux family protein lmo2423 LM6179_1852 -10.09 2.43E-27 LMR479a_2648 yocH Cell wall-binding protein yocH lmo2522 LM6179_1681 -9.97 7.91E-157 LMR479a_2664 Predicted protein (modular protein) -9.08 1.02E-22 conserved protein of unknown LMR479a_0195 function lmo0186 LM6179_0476 -8.79 2.81E-208 cell -division ABC transporter LMR479a_2632 ftsE (ATP-binding protein) lmo2507 LM6179_1697 -8.73 1.10E-94 LMR479a_tRNA4 Glu tRNA -8.50 2.90E-06 glycine betaine ABC transporter LMR479a_1040 opuAA (ATP-binding protein) lmo1014 LM6179_1331 -8.40 5.00E-72 putative metal -dependent

LMR479a_1711 ytnP hydrolase lmo1614 LM6179_2365 -8.23 2.17E-17 207 LMR479a_2631 ftsX cell-division ABC transporter lmo2506 LM6179_1698 -8.09 5.95E-149 LMR479a_0262 rplJ ribosomal protein L10 (BL5) lmo0250 LM6179_0547 -7.93 3.06E-182 conserved membrane protein of LMR479a_1330 unknown function lmo1250 LM6179_1984 -7.89 3.23E-11 LMR479a_1926 rpmB ribosomal protein L28 lmo1816 LM6179_2586 -7.81 9.33E-93 conserved protein of unknown LMR479a_0406 function lmo0395 LM6179_0698 -7.72 3.71E-05 LMR479a_0405 NLP/P60 family protein lmo0394 LM6179_0697 -7.66 9.06E-80 LMR479a_2755 rplE ribosomal protein L5 (BL6) lmo2620 LM6179_0031 -7.64 8.02E-81 LMR479a_0263 rplL ribosomal protein L12 (BL9) lmo0251 LM6179_0548 -7.60 1.16E-146 conserved protein of unknown LMR479a_2863 function lmo2726 LM6179_0140 -7.59 5.49E-05 LMR479a_2765 rplW ribosomal protein L23 lmo2630 LM6179_0041 -7.58 3.68E-168 LMR479a_2768 rpsJ ribosomal protein S10 (BS13) lmo2633 LM6179_0044 -7.56 9.89E-62 LMR479a_2756 rplX ribosomal protein L24 (BL23) lmo2621 LM6179_0032 -7.51 4.22E-100 conserved exported protein of LMR479a_1770 unknown function lmo1671 LM6179_2422 -7.50 1.21E-29

Supplementary Table 8 (Cont.) L. monocytogenes L. monocytogenes L. monocytogenes log2fold R479a locus_tag Gene Product EGDe locus_tag 6179 locus_tag change Q value LMR479a_2766 rplD ribosomal protein L4 lmo2631 LM6179_0042 -7.44 2.29E-122 conserved protein of unknown LMR479a_0851 function lmo0833 LM6179_1145 -7.40 1.79E-09 LMR479a_2763 rpsS ribosomal protein S19 (BS19) lmo2628 LM6179_0039 -7.40 3.52E-104 LMR479a_2762 rplV ribosomal protein L22 (BL17) lmo2627 LM6179_0038 -7.39 9.61E-67 LMR479a_2767 rplC ribosomal protein L3 (BL3) lmo2632 LM6179_0043 -7.35 8.46E-111 LMR479a_2764 rplB ribosomal protein L2 (BL2) lmo2629 LM6179_0040 -7.34 1.83E-70 LMR479a_0694 Flagellar biosynthesis protein FliP lmo0676 LM6179_0987 -7.33 2.60E-14 LMR479a_2759 rpmC ribosomal protein L29 lmo2624 LM6179_0035 -7.32 6.97E-61 LMR479a_2751 rplR ribosomal protein L18 lmo2616 LM6179_0027 -7.30 1.89E-105 conserved membrane protein of LMR479a_0887 unknown function lmo0867 LM6179_1181 -7.26 9.80E-62

LMR479a_1906 ylqC putative RNA binding protein lmo1796 LM6179_2565 -7.25 1.59E-108 208 LMR479a_1644 folC folyl-polyglutamate synthase lmo1551 LM6179_2301 -7.25 4.02E-27 LMR479a_2630 spl P45 lmo2505 LM6179_1699 -7.23 4.24E-109 LMR479a_2752 rplF ribosomal protein L6 (BL8) lmo2617 LM6179_0028 -7.21 3.57E-128 LMR479a_2754 rpsNA ribosomal protein S14 lmo2619 LM6179_0030 -7.21 5.08E-59 conserved protein of unknown LMR479a_2475 function LISIN_2428 LM6179_3073 -7.19 1.87E-04 LMR479a_2761 rpsC ribosomal protein S3 (BS3) lmo2626 LM6179_0037 -7.19 8.92E-57 LMR479a_2753 rpsH ribosomal protein S8 (BS8) lmo2618 -7.13 2.22E-48 LMR479a_2760 rplP ribosomal protein L16 lmo2625 LM6179_0036 -7.12 2.63E-44 LMR479a_2757 rplNA ribosomal protein L14 lmo2622 LM6179_0033 -7.08 2.37E-45 LMR479a_2750 rpsE ribosomal protein S5 lmo2615 LM6179_0026 -7.06 7.13E-69 LMR479a_1907 rpsP ribosomal protein S16 (BS17) lmo1797 LM6179_2566 -7.06 1.48E-98 conserved exported protein of LMR479a_2629 unknown function lmo2504 LM6179_1700 -7.05 1.86E-74 glycine betaine ABC transporter LMR479a_1041 opuAB (permease) lmo1015 LM6179_1332 -6.99 3.40E-104

Supplementary Table 8 (Cont.) L. monocytogenes L. monocytogenes L. monocytogenes log2fold R479a locus_tag Gene Product EGDe locus_tag 6179 locus_tag change Q value LMR479a_2749 rpmD ribosomal protein L30 (BL27) lmo2614 LM6179_0025 -6.97 1.02E-68 LMR479a_1232 putative Membrane protein lmo1211 LM6179_1518 -6.94 2.29E-18 LMR479a_tRNA38 Thr tRNA -6.92 2.82E-08 conserved protein of unknown LMR479a_2989 function lmo2852 LM6179_0272 -6.90 2.03E-12 LMR479a_2758 rpsQ ribosomal protein S17 (BS16) lmo2623 LM6179_0034 -6.85 7.40E-39

209

Supplementary Table 9. The 50 most upregulated chromosomal DE genes in the L. monocytogenes 6179 transcriptome after 30 min exposure to 1% lactic acid. Genes that are part of the Sigma B regulon are highlighted in blue. DE genes which are also upregulated DE genes in R479a under lactic acid treatment are highlighted in bold.

L. monocytogenes L. monocytogenes L. monocytogenes log2fold 6179 locus_tag Gene Product EGDe locus_tag R479a locus_tag change Q value conserved protein of unknown LM6179_2993 function lmo2213 LMR479a_2327 9.75 4.68E-108 putative succinyl -diaminopimelate LM6179_0569 dapE desuccinylase lmo0265 LMR479a_0284 9.65 1.56E-211 LM6179_3049 YhzC protein (fragment) lmo2269 LMR479a_2383 9.60 7.85E-104 LM6179_3010 putative Arsenate reductase lmo2230 LMR479a_2344 9.50 2.41E-224 conserved protein of unknown LM6179_1049 function lmo0737 LMR479a_0756 9.48 6.57E-211 LM6179_0917 protein of unknown function LISIN_0633 LMR479a_0623 9.26 5.59E-25 ribose 5 -phosphate isomerase LM6179_1048 rpiB B/allose 6-phosphate isomerase lmo0736 LMR479a_0755 9.10 3.53E-118

conserved protein of unknown 210 LM6179_0743 function lmo0439 LMR479a_0448 9.00 3.87E-141 LM6179_1047 rpe Ribulose-phosphate 3-epimerase lmo0735 LMR479a_0754 8.98 1.04E-136 LysM domain protein (LPXTG LM6179_1194 motif) lmo0880 LMR479a_0901 8.97 2.56E-177 LM6179_2600 Short chain dehydrogenase lmo1830 LMR479a_1940 8.73 2.25E-155 conserved protein of unknown LM6179_0086 function lmo2673 LMR479a_2809 8.69 1.85E-123 LM6179_2838 cbh Choloylglycine hydrolase lmo2067 LMR479a_2178 8.52 1.10E-150 LM6179_misc_ R479a_misc_ RNA_rli47 rli47 Rli47 RNA_rli47 8.47 6.53E-18 phosphotransferase system (PTS) beta-glucoside-specific enzyme LM6179_1050 bglP IIBCA component lmo0738 LMR479a_0757 8.44 7.67E-246 LM6179_0427 yjdI conserved hypothetical protein lmo0133 LMR479a_0141 8.37 1.66E-45 Manganese transport system LM6179_2618 mntC membrane protein mntC lmo1848 LMR479a_1958 8.32 5.86E-140 conserved membrane protein of LM6179_1311 unknown function lmo0994 LMR479a_1020 8.26 3.69E-166

Supplementary Table 9 (Cont.) L. monocytogenes L. monocytogenes L. monocytogenes log2fold 6179 locus_tag Gene Product EGDe locus_tag R479a locus_tag change Q value fragment of fused mannose - specific PTS enzymes: IIA LM6179_1096 manX component ; IIB component (part 1) lmo0784 LMR479a_0803 8.22 2.37E-144 fragment of fused mannose - specific PTS enzymes: IIA LM6179_1095 manX component ; IIB component (part 2) lmo0783 LMR479a_0802 8.20 1.58E-112 conserved protein of unknown LM6179_2451 function lmo1694 LMR479a_1797 8.17 1.24E-261 LM6179_0567 Internalin C2 lmo0263 LMR479a_0281 8.16 1.11E-164 conserved protein of unknown LM6179_0935 function lmo0628 LMR479a_0641 8.14 6.76E-54 LM6179_1230 PTS system protein lmo0914 LMR479a_0937 8.11 1.23E-29 Manganese -binding lipoprotein LM6179_2617 mntA mntA lmo1847 LMR479a_1957 8.10 3.69E-254 conserved exported protein of LM6179_0916 unknown function lmo0610 LMR479a_0622 8.08 5.59E-123 211 conserved membrane protein of LM6179_0014 unknown function lmo2602 LMR479a_2738 8.03 4.60E-44 conserved exported protein of LM6179_0298 unknown function lmo0019 LMR479a_0019 7.99 4.75E-88 LM6179_0162 ydaG General stress protein 26 lmo2748 LMR479a_2885 7.96 1.65E-201 manganese ABC transporter (ATP - LM6179_2619 mntB binding protein) lmo1849 LMR479a_1959 7.93 2.99E-222 succinate -semialdehyde LM6179_1229 gabD dehydrogenase lmo0913 LMR479a_0936 7.80 2.27E-112 conserved protein of unknown LM6179_1975 function lmo1241 LMR479a_1321 7.69 0.00E+00 putative acyltransferase with acyl - LM6179_0428 yjdJ CoA N-acyltransferase domain lmo0134 LMR479a_0142 7.57 1.41E-69 LM6179_0980 yhxD putative oxidoreductase lmo0669 LMR479a_0687 7.57 1.30E-108 conserved protein of unknown LM6179_1626 function lmo2573 LMR479a_2701 7.52 3.25E-112 LM6179_1231 IIC component PTS system lmo0915 LMR479a_0938 7.52 1.02E-74 LM6179_1051 bglH aryl-phospho-beta-d-glucosidase lmo0739 LMR479a_0758 7.51 2.37E-294

Supplementary Table 9 (Cont.) L. monocytogenes L. monocytogenes L. monocytogenes log2fold 6179 locus_tag Gene Product EGDe locus_tag R479a locus_tag change Q value Uncharacterized sugar epimerase LM6179_1880 yhfK yhfK lmo2391 LMR479a_2515 7.49 3.29E-102 LM6179_1107 ywnB putative oxidoreductase lmo0794 LMR479a_0813 7.45 1.78E-139 Cell wall surface anchor family LM6179_2857 protein lmo2085 LMR479a_2197 7.43 1.88E-124 putative enzyme with pyruvate as LM6179_1033 ydaP substrate lmo0722 LMR479a_0740 7.36 4.96E-110 putative glutamate decarboxylase LM6179_1841 gamma lmo2434 LMR479a_2558 7.28 5.75E-166 conserved membrane protein of LM6179_1884 unknown function lmo2387 LMR479a_2511 7.26 1.83E-76 putative PTS system, cellobiose - LM6179_0123 specific, IIC component lmo2708 LMR479a_2846 7.26 1.14E-81 conserved protein of unknown LM6179_1627 function lmo2572 LMR479a_2700 7.23 9.26E-78 LM6179_1629 putative Membrane protein lmo2570 LMR479a_2698 7.16 8.83E-105 212 conserved membrane protein of LM6179_0901 unknown function lmo0596 LMR479a_0608 7.12 3.06E-161 conserved protein of unknown LM6179_1252 function lmo0937 LMR479a_0959 7.08 4.50E-43 LM6179_0734 Internalin-A (fragment) lmo0433 LMR479a_0443 7.07 1.31E-92 conserved protein of unknown LM6179_1232 function lmo0916 LMR479a_0939 7.05 3.13E-41

Supplementary Table 10. The 50 most upregulated chromosomal DE genes in the L. monocytogenes R479a transcriptome after 30 min exposure to 1% lactic acid. Genes that are part of the Sigma B regulon are highlighted in blue. DE genes which are also upregulated DE genes in 6179 under hydrogen peroxide treatment are highlighted in bold. L. monocytogenes L. monocytogenes L. monocytogenes log2fold R479a locus_tag Gene Product EGDe locus_tag 6179 locus_tag change Q value LMR479a_0356 rpiB Ribose-5-phosphate isomerase B lmo0345 LM6179_0645 10.13 4.87E-94 putative succinyl -diaminopimelate LMR479a_0284 dapE desuccinylase LM6179_0569 10.00 4.43E-174 R479a_misc_ LM6179_misc_ RNA_rli47 rli47 Rli47 RNA_rli47 9.97 1.88E-86 conserved membrane protein of LMR479a_0360 unknown function lmo0349 LM6179_0649 9.97 2.78E-49 Manganese -binding lipoprotein LMR479a_1957 mntA mntA lmo1847 LM6179_2617 9.89 1.22E-197 LMR479a_2383 YhzC protein (fragment) lmo2269 LM6179_3049 9.89 4.51E-113 conserved protein of unknown LMR479a_2344 function lmo2230 LM6179_3010 9.85 0 conserved protein of unknown 213 LMR479a_0641 function lmo0628 LM6179_0935 9.75 1.23E-40 Manganese transport system LMR479a_1958 mntC membrane protein mntC lmo1848 LM6179_2618 9.70 5.85E-254 conserved membrane protein of LMR479a_0361 unknown function lmo0350 LM6179_0650 9.59 1.35E-46 conserved protein of unknown LMR479a_2327 function lmo2213 LM6179_2993 9.57 1.73E-141 LMR479a_2715 yrhE putative oxido-reductase lmo2586 LM6179_1612 9.54 6.78E-261 conserved protein of unknown LMR479a_2809 function lmo2673 LM6179_0086 9.37 1.52E-234 conserved exported protein of LMR479a_0900 unknown function lmo0880 LM6179_1194 9.26 2.95E-106 conserved protein of unknown LMR479a_0359 function lmo0348 LM6179_0648 9.11 3.80E-25 LMR479a_0281 inlC2 Internalin C2 lmo0263 LM6179_0567 9.04 9.83E-255 LMR479a_0355 Short chain dehydrogenase lmo0344 LM6179_0644 8.90 4.08E-100 LMR479a_2178 cbh Choloylglycine hydrolase lmo2067 LM6179_2838 8.89 1.81E-135 conserved protein of unknown LMR479a_0901 function lmo0880 LM6179_1194 8.73 1.58E-179

Supplementary Table 10 (Cont.) L. monocytogenes L. monocytogenes L. monocytogenes log2fold R479a locus_tag Gene Product EGDe locus_tag 6179 locus_tag change Q value LMR479a_0354 ywjH putative transaldolase lmo0343 LM6179_0643 8.65 1.04E-68 PTS -dependent dihydroxyacetone LMR479a_0358 dhaL kinase, ADP-binding subunit dhaL lmo0347 LM6179_0647 8.60 1.69E-140 conserved exported protein of LMR479a_0622 unknown function lmo0610 LM6179_0916 8.48 7.21E-267 conserved membrane protein of LMR479a_0663 unknown function lmo0647 LM6179_0957 8.44 6.12E-61 oligo -alpha -mannoside phosphotransferase system LMR479a_2917 gmuA enzyme IIA lmo2780 LM6179_0201 8.43 3.75E-116 LMR479a_0443 inlA Internalin-A lmo0433 LM6179_0734 8.43 4.16E-235 conserved exported protein of LMR479a_0937 unknown function lmo0914 LM6179_1230 8.38 6.69E-30 LMR479a_2713 fdhD Protein fdhD homolog lmo2584 LM6179_1614 8.38 3.24E-89

LMR479a_0623 protein of unknown function LISIN_0633 LM6179_0917 8.37 2.99E-29 214 conserved protein of unknown LMR479a_2714 function lmo2585 LM6179_1613 8.25 1.90E-124 conserved exported protein of LMR479a_0336 unknown function lmo0324 LM6179_0623 8.24 1.75E-23 conserved membrane protein of LMR479a_2846 unknown function lmo2708 LM6179_0123 8.23 1.43E-122 phosphotransferase system (PTS) lichenan-specific enzyme IIA LMR479a_2822 licA component lmo2685 LM6179_0099 8.21 1.01E-112 LMR479a_0938 PTS system protein lmo0915 LM6179_1231 8.17 1.38E-84 conserved protein of unknown LMR479a_2579 function lmo2454 LM6179_1818 8.16 5.28E-243 putative glutamate decarboxylase LMR479a_2558 gamma lmo2434 LM6179_1841 8.08 3.84E-154 LMR479a_2197 Peptidoglycan binding protein lmo2085 LM6179_2857 8.00 8.01E-170 conserved protein of unknown LMR479a_2701 function lmo2573 LM6179_1626 8.00 1.67E-108 conserved protein of unknown LMR479a_0939 function lmo0916 LM6179_1232 8.00 5.46E-53

Supplementary Table 10 (Cont.) L. monocytogenes L. monocytogenes L. monocytogenes log2fold R479a locus_tag Gene Product EGDe locus_tag 6179 locus_tag change Q value conserved membrane protein of LMR479a_2738 unknown function lmo2602 LM6179_0014 7.94 1.57E-77 conserved protein of unknown LMR479a_2918 function lmo2781 LM6179_0202 7.93 3.42E-241 Uncharacterized sugar epimerase LMR479a_2515 yhfK yhfK lmo2391 LM6179_1880 7.93 2.46E-130 conserved protein of unknown LMR479a_2700 function lmo2572 LM6179_1627 7.79 2.74E-104 phosphotransferase system (PTS) lichenan-specific enzyme IIC LMR479a_2821 licC component lmo2684 LM6179_0098 7.76 5.07E-176 LMR479a_2919 PTS system protein lmo2782 LM6179_0203 7.75 2.46E-98 conserved membrane protein of LMR479a_1020 unknown function lmo0994 LM6179_1311 7.71 1.69E-87 conserved protein of unknown

LMR479a_0141 function lmo0133 LM6179_0427 7.70 6.33E-36 215 LMR479a_2885 ydaG General stress protein 26 lmo2748 LM6179_0162 7.59 1.32E-106 conserved protein of unknown LMR479a_1321 function lmo1241 LM6179_1975 7.52 8.19E-164 LMR479a_0353 tkt transketolase lmo0342 LM6179_0642 7.48 3.22E-62 LMR479a_0282 inlD Internalin D 7.48 5.50E-191

216

Supplementary Table 11. Genes used to compose the σB panel. Genes in the table are composed of all genes from Raengpradub et al. predicted to have σB promoters based on a Hidden Markov model, the genes upregulated by σB based on RNA-Seq conducted by Oliver et al., and all members of the σ B regulon with EGD-e locus tags found via literature review conducted by Liu et al. An “X” denotes that the gene was found to be σ B-dependent in the given study. Raengpradub et al., 2008 Oliver et al., 2009 ( BMC Liu et al., 2019 (Appl Environ Microbiol ) – Genomics ) – genes (Future Microbiol ) – genes with σ B-dependent upregulated by σ B genes identified in promoters based on based on RNA-Seq of literature review with Hidden Markov model stationary phase cells EGD-e locus tags lmo0007 X

lmo0013 X X

lmo0014 X

lmo0015 X

lmo0016 X

lmo0019 X X

lmo0036 X

lmo0037 X X

lmo0043 X X

lmo0052 X

lmo0060 X

lmo0071 X

lmo0075 X

lmo0076 X

lmo0079 X

lmo0080 X

lmo0091 X

lmo0095 X

lmo0105 X

lmo0122 X lmo0133 X X X

lmo0134 X X

lmo0169 X X

lmo0170 X X

lmo0186 X

lmo0200 X X

lmo0210 X X

lmo0211 X

lmo0220 X

lmo0221 X

lmo0222 X

lmo0228 X

lmo0230 X X

217