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Effects of the Mobile Genetic Element Icebs1 on Bacterial Host Fitness Joshua M. Jones

Effects of the Mobile Genetic Element Icebs1 on Bacterial Host Fitness Joshua M. Jones

Effects of the Mobile Genetic Element ICEBs1 on Bacterial Host Fitness

Joshua M. Jones

B.S. Biochemistry University of Maine, 2014

Submitted to the Microbiology Graduate Program in partial fulfillment of the requirements for the degree of Doctor of Philosophy

at the

Massachusetts Institute of Technology

June 2020

Ⓒ 2020 Massachusetts Institute of Technology. All rights reserved.

Signature of Author:……………………………………………………………………………… Joshua M. Jones Microbiology Graduate Program May 6, 2020

Certified by:……………………………………………………………………………………… Alan D. Grossman Professor of Thesis Supervisor

Accepted by:……………………………………………………………………………………… Jacquin Niles Associate Professor of Biological Engineering Chair of Microbiology Program

1 Effects of the Mobile Genetic Element ICEBs1 on Bacterial Host Fitness

Joshua M. Jones

Submitted to the Microbiology Graduate Program in partial fulfillment of the requirements for the degree of Doctor of Philosophy

ABSTRACT

Mobile genetic elements drive bacterial by mediating horizontal transfer and by carrying cargo that confer important traits to host cells. Traits provided by include resistance, novel metabolic capabilities, factors, and the ability to form symbioses. Mobile genetic elements, especially Integrative Conjugative Elements (ICEs), are abundant in . Many do not contain cargo genes with known functions, but some likely carry novel types of cargo genes that provide traits beyond the scope of those currently attributed to mobile elements. In this thesis I describe the characterization of a fitness benefit provided by the mobile genetic element ICEBs1 to its bacterial host, subtilis. Activation of ICEBs1 conferred a frequency-dependent selective advantage to host cells during formation and sporulation. The advantage was due to inhibition of biofilm- associated and delayed sporulation, which enabled ICEBs1 host cells to exploit their neighbors and grow more prior to sporulation. I identified a single gene within ICEBs1, ydcO, as both necessary and sufficient for the repression of development. Manipulation of host development programs allows ICEBs1 to increase host fitness. These findings highlight that cargo genes can alter existing aspects of rather than providing entirely new traits, broadening our understanding of how mobile genetic elements influence their hosts.

Thesis Supervisor: Alan D. Grossman Title: Praecis Professor of Biology; Department Head

2 Acknowledgements

I am extremely grateful to Alan for his scientific and personal mentorship during the past five years. Alan helped me learn not only how to do good science and communicate it effectively, but also taught by example how to balance calmness with intensity.

Thanks also to my thesis advisory committee members Mike Laub and Jeff Gore for feedback on my projects and encouragement over the years.

Thank you to the members of the Grossman lab, past and present. I’m grateful to have worked alongside close friends who are also excellent scientists. I’ll especially miss our brunches and afternoon Muddy meetings.

Thank you to all of my friends, both within the MIT and beyond. I’m extremely fortunate to have a group of friends that are as ridiculously fun as they are caring and supportive.

I’m particularly grateful for my family, especially my parents, who have always supported me in my interests and encouraged me to pursue excellence.

Finally, a huge thank you to Mónica for being a loving and supportive partner. All the ups and downs of grad school were much better shared with you.

3 Table of Contents

Abstract 2

Acknowledgements 3

List of Figures 5

List of Tables 6

Chapter 1 Introduction 7

Chapter 2 A mobile genetic element increases bacterial host fitness by 45 manipulating development

Appendix A Genetic screen to isolate ydcO suppressor mutants 88

Appendix B yddI is also important for ICEBs1 host fitness 92

Appendix C Prolonged ICEBs1 induction is detrimental to host cells 96

Chapter 3 Conclusions and Perspectives 105

4 List of Figures

Chapter 1 Fig. 1. The three primary forms of HGT in bacteria 10

Fig. 2. The ICE conjugative cycle 14

Fig. 3. Genetic map of ICEBs1 28

Fig. 4. Regulation of ICEBs1 29

Chapter 2 Fig. 1. The fitness of ICEBs1-containing cells during development 81 depends on their initial frequency in the population

Fig. 2. ICEBs1-containing cells delay sporulation in a frequency- 82 dependent manner

Fig. 3. The ICEBs1 - genes, rapI-phrI, are necessary but 83 not sufficient to confer a selective advantage

Fig. 4. Expression from Pxis and ydcO are required for the fitness benefit 84 of ICEBs1

Fig. 5. ydcO alone is sufficient to inhibit sporulation and provide a 85 selective advantage

Fig. 6. ydcO inhibits expression of genes associated with sporulation 86 initiation and biofilm formation

Appendix C Fig. 1. ICEBs1 induction incurs a frequency-dependent fitness cost to host 101 cells

Fig. 2. ICEBs1 induction is detrimental during stationary phase 102

Fig. 3. ICEBs1 replication and conjugation gene expression contribute to 103 growth and stationary phase defects

5 List of Tables

Chapter 2 Table 1. Frequency of transconjugants generated in biofilm matings 80

Table 2. B. subtilis strains used 87

Appendix A Table 1. ydcO suppressor mutants 91

Appendix B Table 1. B. subtilis strains used 94

Appendix C Table 1. B. subtilis strains used 104

6 Chapter 1

Introduction

7 Overview

Bacteria are able to evolve rapidly in part due to their ability to acquire new genetic material through . Mobile genetic elements are important drivers of horizontal gene transfer, as they encode genes to transfer themselves between cells. Mobile genetic elements often encode “cargo genes” that provide novel traits to the host, notably antibiotic resistance genes. In this thesis, I describe the characterization of a fitness benefit provided by a mobile genetic element, ICEBs1, to its bacterial host . We found that a single

ICEBs1 gene, ydcO, provided a fitness benefit by interfering with the host’s developmental pathway that controls biofilm formation and sporulation. When ICEBs1 gene expression is induced in the context of a growing biofilm, ydcO enables cells with ICEBs1 to express costly biofilm-associated genes at lower levels and delay sporulation, both of which contribute to a growth advantage.

Introduction to Horizontal Gene Transfer in Bacteria

Horizontal gene transfer (HGT) is the acquisition of DNA from non-parental origin.

Horizontal gene transfer has been documented in all kingdoms of life, but is by far the most frequent and best characterized in bacteria, where it is a major driving force of evolution (de la

Cruz and Davies, 2000; Soucy et al., 2015). Bacteria can acquire foreign DNA directly from their surrounding environment and from other cells that are not part of a parent-offspring relationship (Thomas and Nielsen, 2005). Horizontal gene transfer exposes bacteria to diverse genetic material, promoting evolution on a rapid timescale. In many cases, horizontally acquired segments of DNA make up a substantial fraction of the in bacteria, and they are often

8 responsible for important differences among otherwise closely related (de la Cruz and

Davies, 2000; Gogarten and Townsend, 2005; Koonin and Wolf, 2008; Ochman et al., 2000).

There are three major, widely recognized mechanisms of horizontal gene transfer in bacteria: conjugation, , and transformation (Fig. 1) (Soucy et al., 2015; Thomas and Nielsen,

2005). Conjugation and transduction are DNA transfer processes mediated by self-transmissible mobile genetic elements (MGEs), which are segments of DNA with the ability to move between cells (Frost et al., 2005). MGEs encode genes that, when expressed inside a bacterial host cell, provide the means of transfer. The biology of mobile genetic elements and their functions will be discussed in detail below. The third main mechanism of HGT is transformation, which is the acquisition of DNA present in the environment (Johnston et al., 2014). The ability to take in

DNA for transformation is called genetic competence. Not all bacteria are known to possess this ability, and among most of those that do, it is positively regulated by conditions such as starvation and stress (Claverys and Martin, 2003; Johnston et al., 2014).

Beyond these canonical forms of horizontal gene transfer, there are less well-characterized mechanisms whose contributions to microbial evolution are not well understood. Gene transfer agents (GTAs) are phage-like particles that transfer random fragments of chromosomal DNA

(Lang et al., 2012). DNA and other can be transferred by nano-tube bridges between adjacent cells (Dubey and Ben-Yehuda, 2011) and by membrane vesicles released from cells

(Domingues and Nielsen, 2017). In , transient cell fusions can lead to exchange of and recombination between (Naor and Gophna, 2012).

9 Conjugation Transduction Transformation

Direct cell-to-cell DNA transfer DNA transfer through Uptake of DNA in the through secretion system viral particles environment

Figure 1. The three primary forms of HGT in bacteria. Conjugation is mediated by integrative conjugative elements (ICEs) and conjugative plasmids. Transduction is mediated by . Organisms that can become genetically competent can undergo transformation with DNA present outside of the cell.

Mobile genetic elements

Mobile genetic elements play an active role in bacterial evolution by transferring DNA between cells. As mentioned above, two major mechanisms of horizontal gene transfer are mediated by mobile genetic elements: transduction and conjugation. Transduction is mediated by bacteriophages (bacterial ), which are MGEs that encode genes to produce viral particles used for cell-to-cell spread by infection (Frost et al., 2005). Conjugation is thought to be the most prevalent form of horizontal gene transfer in bacteria (Guglielmini et al., 2011; von

Wintersdorff et al., 2016). Conjugation is mediated by two classes of MGEs: conjugative plasmids and integrative conjugative elements (ICEs). Both encode DNA secretion systems to

10 transfer directly from one cell to another (Frost et al., 2005). In addition to providing efficient mechanisms of horizontal gene transfer, all classes of MGEs frequently carry “cargo genes,” which provide important traits to the host cell (Frost et al., 2005).

Bacteriophages

Bacteriophages are thought to be the most abundant biological entities on the planet (Clokie et al., 2011; Hatfull, 2015). The lifecycle of a consists of infecting a host cell, hijacking the host’s cellular machinery to replicate its genome and produce its , assembling infectious viruses packed with the bacteriophage genome, and release of viruses

(typically through killing the host cell). Binding of the to specific receptor sites on another suitable host cell initiates the infectious cycle again beginning with transfer of the bacteriophage genome into the cell (Clokie et al., 2011; Olszak et al., 2017). Transduction occurs when segments of the host’s are incorporated into the viral particle and transmitted to another cell upon infection. The host DNA transferred in this manner can be random (generalized transduction) or specific (specialized transduction) (Chiang et al., 2019; Frost et al., 2005).

Specialized transduction is mediated by a class of bacteriophages, lysogenic phages, that can integrate into the host’s chromosome as part of their lifecycle. In this state, virulence genes are repressed, and the phage genome is passively replicated along with the bacterial chromosome

(Howard-Varona et al., 2017; Touchon et al., 2017). Activation of the infectious cycle leads to excision of the phage genome from the chromosome. Imprecise excision beyond the borders of the phage genome leads to of nearby segments of the host chromosome within the viral particle (Howard-Varona et al., 2017; Touchon et al., 2017). Lysogenic phages often include

11 cargo genes and may have profound effects on the host cell while in the integrated state (Argov et al., 2017; Hargreaves et al., 2014).

Conjugative plasmids

Conjugative plasmids are pieces of DNA (usually circular) that are replicated independently from the bacterial chromosome within a host cell and are transferred between cells by the action of a specialized conjugation machinery (Norman et al., 2009; Smillie et al., 2010). Conjugative plasmids contain genes needed for their replication within the host cell (to be inherited during ) and genes for the conjugation machinery. The conjugation machinery is usually of the Type-IV secretion system family, which is assembled on the surface of a donor cell and contains a pore through which the DNA can transfer (Bhatty et al., 2013; Smillie et al.,

2010). Multiple aspects of the conjugation process are performed by a plasmid-encoded called a “.” The relaxase initiates transfer by recognizing a specific sequence in the plasmid, the origin of transfer “oriT”, and cutting one strand of the DNA duplex (de la Cruz et al., 2010; Wong et al., 2012). The relaxase remains bound to the cut end of the now single- stranded DNA, and this relaxase-DNA complex is the substrate recognized by the conjugation machinery (Zechner et al., 2012). The relaxase-DNA complex is transferred through the conjugation machinery into a recipient cell, where the relaxase re-circularizes the plasmid (de la

Cruz et al., 2010).

Conjugation is a powerful method of horizontal gene transfer, in part because non-plasmid

DNA can also transfer through the conjugation machinery during mating. If a stable mating pair is formed between a donor and recipient, very long segments of DNA can be transferred, including an entire bacterial chromosome (Hayes, 1957). Other types of plasmids that lack

12 conjugation functions can also be mobilized into a recipient cell using the conjugation machinery of a conjugative plasmid residing in the same cell (Smillie et al., 2010). All types of plasmids can carry cargo genes, which can be disseminated broadly by conjugation. The most infamous example of this is the spread of plasmid-encoded antibiotic resistance genes (Bennett, 2009).

Integrative conjugative elements

Integrative conjugative elements (ICEs) are thought to be the most abundant type of conjugative element in bacteria (Guglielmini et al., 2011). ICEs normally reside integrated in the chromosome of their host cell, where they are replicated and inherited along with the chromosome during cell division (Johnson and Grossman, 2015). ICEs range in size from ~12 -

500 kb and contain the genes needed to excise from the chromosome, replicate like a plasmid, and transfer by conjugation. (Cury et al., 2017; Johnson and Grossman, 2015; Wozniak and

Waldor, 2010). ICEs are evolutionarily related to both conjugative plasmids and lysogenic phages (Burrus and Waldor, 2004; Carraro and Burrus, 2015; Wozniak and Waldor, 2010). Like conjugative plasmids, ICEs typically transfer through a type-IV secretion system and use a relaxase for DNA processing. ICEs often use similar mechanisms of regulation, integration, and excision as lysogenic phages. ICE conjugation machinery can also mobilize other MGEs that lack dedicated transfer functions (Johnson and Grossman, 2015).

Upon entering a host cell, some ICEs integrate preferentially into specific, often highly conserved sites, while other ICEs integrate with little specificity (Cury et al., 2017; Johnson and

Grossman, 2015; Wozniak and Waldor, 2010). In the integrated state, genes for excision and transfer are usually repressed. ICEs encode regulatory genes that sense various signals to coordinate their activation for conjugative transfer. Conditions that stimulate ICE activation

13 include DNA damage or stress in the host cell, cell-cell communication to sense possible mating partners, and the presence of specific compounds in the environment (Johnson and Grossman,

2015; Wozniak and Waldor, 2010). These signals activate the conjugative lifecycle of the ICE

(Fig. 2), beginning with excision of the linear ICE DNA from the chromosome to form a circular plasmid-like intermediate. ICEs were once thought to be incapable of replication, but autonomous plasmid-like replication has since been characterized in multiple systems and is likely a general feature of the ICE lifecycle (Carraro et al., 2015; Carraro et al., 2016; Lee et al.,

2010; Wright and Grossman, 2016). ICE activation also leads to expression of genes for the conjugation machinery, and the process of ICE DNA transfer is similar to that described above for conjugative plasmids.

Figure 2. The ICE conjugative life cycle. Induction leads to expression of ICE genes and excision from the chromosome. ICE DNA is processed by a relaxase, generating single- stranded ICE DNA bound to the relaxase, which is transferred to a recipient through a type-4 secretion system (T4SS). The ICE integrates into the chromosome of the recipient (now a transconjugant) and reintegrates in the donor.

14 Benefits of mobile genetic elements

In addition to driving evolution of bacteria by providing efficient means of DNA transfer between cells, mobile genetic elements can have profound consequences for the fitness of their host cells. Many MGEs carry cargo genes that are not needed for their lifecycle but provide benefits to the host cell. In many cases, the exact suite of MGEs (and associated cargo genes) in a host cell can determine critical functions and behaviors of the cell (Frost et al., 2005; Ochman et al., 2000). MGEs can be thought of as vehicles that spread these cargo genes and their associated phenotypes throughout bacteria. From the perspective of MGEs, which selfishly use host resources for their maintenance and transfer between cells, cargo genes likely help offset their cost to the host (Baltrus, 2013; Rankin et al., 2011)

Historically, many MGEs in bacteria were discovered because of cargo genes that conferred obvious phenotypes, notably antibiotic resistance (Johnson and Grossman, 2015; Shapiro, 1995).

Many putative MGEs, especially ICEs, have now been identified in genome sequences or metagenomic samples, and the presence of cargo genes can sometimes be inferred based on similarity to known genes (Bi et al., 2012; Cury et al., 2017; Guglielmini et al., 2011; Jørgensen et al., 2014; Leplae et al., 2004). However, in many cases no cargo genes with known functions are apparent. Many of these MGEs likely carry novel cargo genes that perform functions beyond the scope of those currently attributed to cargo genes. The following sections review the main types of benefits currently known to be provided by mobile genetic elements to their hosts, emphasizing less well-characterized benefits.

15 Resistance to antimicrobials

Mobile genetic elements carrying determinants of continue to be identified in virtually any environment where bacteria are exposed to such compounds. Genes conferring resistance to normally lethal concentrations of and toxic heavy metals are found in all types of MGEs. In some cases, a single element carries multiple cargo genes providing multiple resistances. For example, members of the broad host range IncP-1 plasmid family, which can reside in virtually all gram-negative bacteria, frequently provide resistance to multiple antibiotics, heavy metals, and disinfectant ammonium compounds (Popowska and

Krawczyk-Balska, 2013). The great diversity of cargo genes carried by IncP-1 plasmids is in part driven by insertion of other MGEs within the plasmids (Popowska and Krawczyk-Balska, 2013).

Bacteriophages are also implicated in the dissemination of antibiotic resistance genes, not only by encoding resistance genes but also by spreading them through transduction (Brown-Jaque et al., 2015).

In some cases, MGEs are induced to transfer in the presence of the antibiotic to which they provide resistance. The broad host-range ICE Tn916 and other related elements carrying resistance genes are induced to transfer in the presence of tetracycline (Doucet-

Populaire et al., 1991; Showsh and Andrews, 1992). Linking regulation of transfer to cargo gene function increases the likelihood that the new hosts receiving the element will benefit from the cargo genes, despite costs incurred by activation and transfer.

Novel metabolic functions

Cargo genes can also provide novel metabolic functions, enabling the host cell to utilize new nutrient and energy sources. The metabolic substrates can be naturally occurring or man-made

16 compounds foreign or rare in (xenobiotic compounds). An ICE found in ,

CTnscr94, confers its hosts with the ability to ferment sucrose and can be transferred to other

Enterobacteria (Hochhut et al., 1997). Among hundreds of clinical isolates of catheter-associated

Providencia stuartii, certain isolates contained conjugative plasmids that conferred the ability to utilize urea, sucrose, and lactose (Mobley et al., 1985). During experimental evolution of E. coli introduced to the mouse gut, acquisition of two lysogenic phages from the resident flora provided a growth advantage, though it is unclear if this was directly due to cargo genes in the phages (Frazão et al., 2019).

Numerous MGEs enabling the breakdown and utilization of toxic xenobiotic compounds have been identified, largely due to studies of polluted environments and interest in harnessing novel forms of bacterial for bioremediation. In many cases, metabolic pathways are constructed by composites of multiple MGEs, each providing a subset of the needed

(Top and Springael, 2003). For example, the enzymes for different steps of chlorobenzene degradation by sp. P51 are encoded in a plasmid (pP51) and in a (Tn5280) located in pP51 (van der Meer et al., 1991). As described for some MGEs that provide antibiotic resistance, transfer of MGEs carrying metabolic cargo genes may be induced by the substrate of the cargo genes. ICEclc confers Pseudomonas with the ability to degrade chlorocatechols and is stimulated to transfer in their presence (Sentichilo et al., 2003).

Altered host lifestyle: and pathogenesis

Among the most dramatic phenotypes MGEs can provide through their cargo genes is the enabling of symbioses and pathogenesis. In these cases, a resident MGE influences how its bacterial host interacts with a or host. The presence or absence of specific MGEs

17 can be the main difference between a and an otherwise harmless . This is in contrast to MGEs like those described above that can enhance pathogenesis by providing antibiotic resistance or metabolic benefits.

In , two plasmids each contribute critical virulence factors; pXO1 encodes the genes for anthrax toxin (Mikesell et al., 1983), and pXO2 carries a group of genes needed to produce a poly-D-glutamic acid capsule around the cell (Green et al., 1985). In a mouse infection model, the capsule genes were required in order to establish an infection and kill the host

(Drysdale et al., 2005). Several important depend largely on toxin genes encoded by lysogenic bacteriophages for their virulence (Brüssow et al., 2004; Penadés et al.,

2015). For example, is an aquatic organism that when infected with the toxin- encoding phage CTXΦ is capable of a pathogenic lifestyle in human hosts (Waldor and

Mekalanos, 1996).

Mobile genetic elements can also enable their bacterial hosts to form symbioses. The presence of a large ICE (~500 kb) identified in Mesorhizobium loti enables otherwise non- symbiotic hosts to form nitrogen-fixing root nodules on Lotus plant (Sullivan and

Ronson, 1998; Sullivan et al., 2002). A bacterial of , , defends the host from parasitic wasps that lay inside the aphid. This protection is the consequence of a toxin encoded by a lysogenic phage present in the Hamiltonella defensa genome (Oliver et al., 2009). Symbiosis-promoting elements benefit the immediate host (the bacteria), which in turn provide a benefit to a plant or animal host.

18 Interactions between mobile genetic elements

Mobile genetic elements can function as barriers to the acquisition of other MGEs (Thomas and Nielsen, 2005). In some cases, blocking other MGEs might provide a benefit to the host if the incoming element is costly or lethal like a lytic bacteriophage. Alternatively, a MGE could prevent acquisition of a potentially beneficial element. Generally speaking, interactions between

MGEs likely plays an important role in shaping the exact suite of MGEs (and cargo genes) present in cell, thereby affecting host fitness. Some MGEs intrinsically block other elements without the use of cargo genes. For example, plasmid incompatibility and bacteriophage immunity are intrinsic effects of genes directly involved in the lifecycle of the element (Novick,

1987; Ptashne, 1967). Cargo genes can also provide barriers to the acquisition of other MGEs.

Most conjugative plasmids and some ICEs encode entry exclusion genes that block incoming transfer of themselves and other elements (Garcillán-Barcia and de la Cruz, 2008). Exclusion can protect cells from lethality caused by excessive transfer. Death of cells due to high levels of conjugation was first described in matings of the F-plasmid (Alfoldi et al., 1957). Exclusion genes carried by the F-plasmid conferred protection, preventing damage to the by conjugation (Ou, 1980). An exclusion gene encoded by ICEBs1 from Bacillus subtilis also provides a selective advantage to its host cells under conditions where donors are attempting to mate into each other (Avello et al., 2019).

Abortive infection systems protect populations from bacteriophage by sacrificing infected cells to prevent phage spread (Dy et al., 2014). Abortive infection systems use diverse mechanisms, but in all cases they trigger the altruistic “suicide” of infected cells, leading to the release of few, if any, virus particles (Dy et al., 2014). These systems are often found encoded by

19 mobile elements. Numerous abortive infection systems have been discovered in Lactococcus lactis due to its importance in cheesemaking and the economic impact of phage infection in this industry; most of these are encoded by plasmids (Chopin et al., 2005). ICEBs1 from Bacillus subtilis encodes an abortive infection gene that targets a lysogenic phage SPβ (Johnson et al.,

2020, manuscript in preparation). Bacteriophages also carry abortive infection systems; the lysogenic phage λ in E. coli provides abortive infection against lytic phages (Parma et al., 1992).

A recently discovered type of mobile element in Vibrio cholerae called PLE (phage-inducible chromosomal island-like elements) function as abortive infection systems against the phage ICP1

(O’Hara et al., 2017).

Though CRISPR-Cas systems are typically regarded as host-encoded defenses against bacteriophage and other MGEs, they have also been identified within MGEs (Dy et al., 2014). A conjugative plasmid in Lactococcus lactis carries a CRISPR-Cas system that provides the host cell with resistance against some phages (Millen et al., 2012). In the Vibrio cholerae phage-PLE interaction described above, the phage ICP1 counteracts the PLE defense system by carrying a

CRISPR-Cas system that specifically targets the PLE (McKitterick et al., 2019; O’Hara et al.,

2017; Seed et al., 2013).

Manipulation of existing host functions

Mobile genetic elements can also encode genes that manipulate existing aspects of host physiology rather than providing entirely new phenotypes. Relatively few cargo genes like this have been characterized, in part because they may not be easily selectable under known conditions. MGEs frequently encode genes that more closely resemble typical bacterial genes than MGE genes. In some cases, these can regulate host functions and provide benefits. In B.

20 anthracis, multiple MGEs contribute to the regulation of host development. -encoded sigma-factors regulate sporulation and promote biofilm formation, and the presence of certain enhanced long-term colonization of soil and the earthworm gut (Schuch and Fischetti,

2009). The two virulence plasmids described previously, pXO1 and pXO2, play dual roles in virulence and regulation of host development. The pXO2 plasmid encodes a gene with similarity to the sensor of a chromosome-encoded kinase that promotes sporulation. The pXO2- encoded sensor domain converts the host kinase from an activator of sporulation to an inhibitor of sporulation (White et al., 2006). Expression of the pXO2-encoded sensor domain, and subsequent inhibition of sporulation, is dependent on a pXO1 gene, atxA, which encodes the master regulator of B. anthracis virulence (Dale et al., 2018). Co-regulation of production and sporulation is likely important during pathogenesis, as anthrax are thought to be more susceptible to the than vegetative cells (Mock and Fouet, 2001).

Studies of the effects of resident lysogenic phages on host cells have demonstrated that many host genes are differentially regulated in phage-containing cells, however, it is not always clear if any of the changes are mediated by cargo genes or if they confer a benefit. The presence of a

Stx-2 family lysogenic phage in E. coli conferred increased acid tolerance and altered ; cells were enhanced in swimming motility but not swarming motility (Su et al., 2010). Another study also reported that a phage altered host motility, in this case repressing swarming motility

(Jian et al., 2013). Defective, or “cryptic”, phages are common in bacterial . These are phages that remain stuck in the chromosome due to inactivating . In E. coli, removal of

9 cryptic phages (3.6% of the genome) had many physiological effects, including reduced biofilm formation and reduced resistance to stress and antibiotics (Wang et al., 2010).

21 Chromosomal regions derived from defective mobile elements may be reservoirs of potentially beneficial genes.

We suspect that many MGEs without obvious cargo genes may have novel cargo genes that modulate aspects of host physiology to provide a benefit. Such cargo genes may be difficult to identify because they may not be widely conserved or easily selectable under known conditions.

Additionally, the organism in which a MGE is identified may not be the “original” host. MGEs undergo extensive co-evolution with their hosts, therefore cargo gene function may be host dependent.

Biofilms and Horizontal Gene Transfer

Biofilms are thought to be the predominant form of microbial growth in natural environments

(Flemming and Wuertz, 2019; Hall-Stoodley et al., 2004). The characteristics of biofilms formed by different organisms vary, but generally speaking, biofilms are surface-associated groups of cells encased in a protective polymeric matrix produced by the cells within (Flemming and

Wingender, 2010; Flemming et al., 2016). Biofilms are considered hotspots of horizontal gene transfer by transformation and the spread of mobile genetic elements (Aminov, 2011; Madsen et al., 2012; Molin and Tolker-Nielsen, 2003; Elsas and Bailey, 2002). Additionally, biofilms in many natural settings are polymicrobial, increasing the possibility of exposure to foreign DNA.

A critical feature of biofilms with respect to HGT is high cell density. For conjugative elements that rely on cell-cell contact for transfer, growth in a biofilm provides the necessary spatial proximity required for stable cell-cell interactions. Some conjugative elements promote biofilm formation, likely as a secondary effect of aggregation strategies to stabilize mating pairs

22 of cells (Madsen et al., 2012). On the other hand, the heterogeneity inherent to biofilms can also be a limitation to transfer of conjugative elements (Flemming et al., 2016; Stalder and Top,

2016). The spatial organization and lack of mixing in biofilms limits the number of unique cell- cell interactions and opportunities for mating. Some cells in biofilms may also be relatively inactive metabolically (stationary phase and semi-dormant persisters) and may not be suitable donors or recipients for conjugation.

Organisms with the ability to become genetically competent can be transformed with extracellular DNA present in biofilms. Extracellular DNA is generally abundant in biofilms, and some organisms actively secrete DNA as a critical component of the biofilm matrix (Ibáñez de

Aldecoa et al., 2017; Jakubovics et al., 2013; Okshevsky and Meyer, 2015). Regulated release of

DNA (as opposed to DNA released by cell death) coincides with the development of competence in some cases, suggesting that another role of DNA release is to promote transformation. In B. subtilis, competence development is linked to DNA release (Zafra et al., 2012). Natural competence of Vibrio cholerae is stimulated during growth on chitin, on which it grows as biofilms in nature (Meibom et al., 2005). Under the same conditions, competent V. cholerae cells use a toxin secretion system to kill neighboring cells, making DNA available for uptake

(Borgeaud et al., 2015).

A variety of interactions between bacteriophage and cells growing as biofilms are possible. In some cases, biofilm growth can be protective against phage predation, possibly due to reduced mixing and diffusivity of the phage particles (Simmons et al., 2017; Vidakovic et al., 2018).

Interestingly, bacteriophages are critical for biofilm formation by ; phage-mediated killing releases DNA into the biofilm matrix, and phage particles themselves

23 play a structural role in the matrix (Rice et al., 2009; Secor et al., 2015). The outcomes of phage- biofilm interactions are likely highly dependent on the stage of biofilm growth as well as the bacterial and phage composition of the community (Hansen et al., 2019).

Development in Bacillus subtilis

Bacillus subtilis performs dramatic developmental transitions in response to environmental and social cues (Aguilar et al., 2007; Lopez et al., 2009; Shank and Kolter, 2011). As cells in a growing population sense starvation as well as crowding, they differentiate heterogeneously into three main developmental states: competence, biofilms, and dormant spores. Entry into these states is regulated in part by a common factor, Spo0A, which directly and indirectly controls the expression of hundreds of genes (Fujita et al., 2005; Molle et al., 2003). Regulation of development has important consequences for the population as well as individual fitness, as cells in different states can have advantages or disadvantages relative to their peers.

Competence

Cells in the competent state can take in DNA present in their environment and incorporate it into their genome by recombination (Dubnau and Blokesch, 2019). Under conditions optimized to induce B. subtilis competence in the laboratory, only about 10% of cells in the population become competent. Cells in this state produce DNA uptake machinery as well as DNA repair and recombination proteins needed to incorporate the acquired DNA into the chromosome (Berka et al., 2002). Many physiological changes are co-regulated with the competent state in addition to transformability (Berka et al., 2002; Ogura et al., 2002). Notably, competent cells are in a growth arrested state; DNA replication slows prior to transformability, and the cells experience a

24 prolonged lag before resuming growth after transformation (Nester and Stocker, 1963; Hahn et al., 2015; Mirouze et al., 2015). Competent cells temporarily sacrifice growth but may ultimately benefit from DNA uptake and transformation as well as transient antibiotic resistance in the growth arrested state (Johnsen et al., 2009).

Biofilms

B. subtilis forms architecturally complex biofilms on the surface of solid growth media and at the liquid-air interface in unmixed liquid media (Branda et al., 2001; Vlamakis et al., 2013). This ability has largely been lost in laboratory domesticated strains, but is retained by “wild” strains.

Formation of the biofilm is driven by production of the extracellular matrix, which provides structural integrity, protection, and plays roles in signaling and migration (Aguilar et al., 2010;

Branda et al., 2006; Vlamakis et al., 2013. Notably, not all of the cells growing in the biofilm contribute to production of matrix components (Chai et al., 2008; Vlamakis et al., 2008). Some cells benefit from growth in the biofilm without incurring the metabolic cost of matrix production, even in an isogenic population (Dragoš et al., 2018).

Sporulation

In response to starvation and high cell density, B. subtilis forms metabolically dormant and highly stress-resistant spores to preserve the genome in a protected state until nutrients are available again (Grossman, 1995; Grossman and Losick, 1988; Piggot and Hilbert, 2004; Tan and Ramamurthi, 2014). Sporulation is the final outcome of full activation of the Spo0A pathway

(Chung et al., 1994; Fujita et al., 2005; Fujita and Losick, 2005). Later stages of growth in biofilms involves high levels of sporulation, but the timing of sporulation of individual cells in a population is highly heterogenous (Branda et al., 2001; Chastanet et al., 2010; Vlamakis et al.,

25 2008). It is advantageous for cells to delay sporulation and continue growing as long as nutrients are still available, but cells that do not sporulate eventually will die. In early studies of sporulation during continuous culture, spontaneous sporulation-defective mutants routinely arose and were highly selected for (Dawes and Mandelstam, 1970). B. subtilis also releases toxins to kill neighboring cells, a behavior called “cannibalism,” which provides nutrients to delay the initiation of sporulation and allow more growth (González-Pastor, 2011; López et al., 2009).

Regulation of development

The central regulator of development is the Spo0A. Spo0A binds to DNA and either represses or stimulates expression of its target genes depending on the location of the

Spo0A binding site relative to the target gene promoter (Strauch et al., 1990; Trach et al., 1991).

Activity of Spo0A requires its phosphorylation, which is regulated by a pathway known as the phosphorelay (Trach et al., 1991). The phosphorelay integrates information about environmental conditions and cell density to control Spo0A activity (Bischofs et al., 2009; Grossman, 1995).

The target genes of Spo0A vary in their sensitivity to Spo0A, therefore different groups of genes are differentially expressed depending on the amount of phosphorylated Spo0A (Spo0A~P) in the cell (Fujita et al., 2005). Moderate levels of Spo0A~P are sufficient for competence and biofilm formation, but higher levels of Spo0A~P are needed to initiate sporulation (Chung et al.,

1994; Fujita et al., 2005; Hamon and Lazazzera, 2002). The primary role of Spo0A~P in competence and biofilm formation is repression of other developmental regulators, abrB and sinR (Bai et al., 1993; Hahn et al., 1995; Hamon and Lazazzera, 2002; Hamon et al., 2004).

These are also transcription factors, and they repress genes needed for competence and biofilm formation as well as sporulation. Shared regulation by Spo0A~P creates a general progression of

26 development, reserving sporulation as the final transition. In the context of a biofilm, this progression appears important. Biofilm architecture likely plays a role in dispersal through formation of elevated structures which are preferential sites for sporulation (Branda et al., 2001).

Mutants that are defective in aspects of biofilm architecture also produce defective spores

(Veening et al., 2006).

Introduction to ICEBs1

ICEBs1 is an integrative and conjugative element found in most strains of B. subtilis (Fig. 3).

It was discovered both bioinformatically by homology to another ICE (Burrus et al., 2002) and by its mode of regulation by cell-cell signaling (Auchtung et al., 2005). ICEBs1 transfers efficiently to numerous other Bacillus species (Auchtung et al., 2005; Brophy et al., 2018). The full possible host range of ICEBs1 is not known, but transfer at low efficiencies is possible into other gram-positive bacteria including Enterococcus, , and species

(Brophy et al., 2018).

ICEBs1 typically resides integrated into a leucine-tRNA gene (trnS-leu2) in the B. subtilis chromosome (Auchtung et al., 2005). Expression of conjugation genes is controlled by the Pxis promoter, which is kept off in the integrated state by the ICEBs1 repressor ImmR (Auchtung et al., 2005; Auchtung et al., 2007). Two known conditions activate the conjugative lifecycle of

ICEBs1 (Fig. 4). 1) ICEBs1 uses cell-cell signaling to sense the availability of potential mating partners that lack ICEBs1. 2) ICEBs1 is induced by RecA during the host DNA damage response

(Auchtung et al., 2005). In both cases, activation of conjugation gene expression is stimulated by degrading the repressor ImmR (Bose et al., 2008; Bose and Grossman, 2011).

27 Figure 3. Genetic map of ICEBs1. Genes are represented by horizontal block arrows indicating the direction of transcription. Vertical right-angle arrows mark the positions of promoters, and the arrowhead indicates the direction of transcription. The 60 bp attachment (att) sites marking the ends of ICEBs1 are shown as black rectangles.

ICEBs1 encodes cell-cell signaling genes, rapI and phrI, which provide two levels of control over activation of conjugation. 1) RapI stimulates conjugation at high cell density, when the likelihood of contacting a potential mating partner is high. 2) PhrI inhibits activation if those potential mating partners already contain ICEBs1 (Auchtung et al., 2005). Expression of rapI is repressed by AbrB, which is in turn repressed at high cell density, allowing production of RapI

(Phillips and Strauch, 2002). RapI induces conjugation by stimulating the activity of ImmA, a protease that degrades the repressor ImmR (Bose et al., 2008; Bose and Grossman, 2011). RapI is inhibited by PhrI, a secreted peptide produced only by cells that contain ICEBs1. PhrI serves as an indicator of the abundance of other ICEBs1-containing cells and prevents induction of

ICEBs1 if a large fraction of the available potential mating partners already contain ICEBs1

(Auchtung et al., 2005). The combined effect of rapI and phrI is that ICEBs1 conjugation is induced preferentially when the total density of cells is high but most cells do not already have

ICEBs1, increasing the likelihood that induction will lead to successful to transfer to new hosts.

28 Figure 4. Regulation of ICEBs1. ICEBs1 is regulated by two paths that lead to de-repression of Pxis and expression of genes needed for transfer. See text for details. An abbreviated genetic map of ICEBs1 is shown to highlight factors involved in regulation. Block arrows represent genes, with black indicating DNA processing genes, dark grey indicating regulatory genes, and white indicating unknown function. Vertical right-angle arrows indicate promoters. In the genetic pathways, black arrows indicate activation and red bars indicate inhibition.

Induction of ICEBs1 conjugation gene expression results in excision from the chromosome by site-specific recombination between identical sequences flanking the integrated element (Lee et al., 2007). The circular form of ICEBs1 produced by excision is capable of plasmid-like replication using proteins encoded by ICEBs1 and host proteins (Lee and Grossman, 2007; Lee et al., 2010). Induction also leads to expression of conjugation genes and assembly of the conjugation machinery, a type IV secretion system. When a donor cell contacts a potential

29 recipient, ICEBs1 DNA (and certain plasmids if present in the cell) can pass through the conjugation machinery into the recipient. Recipients that stably acquire ICEBs1 (by integration into the chromosome) are called transconjugants. Transconjugants can function as donors shortly after receiving ICEBs1, since the repressor ImmR is not present yet (Babic et al., 2011). Donors can mate multiple times into different cells, and when cells are growing as chains (a hallmark feature of biofilm growth), ICEBs1 can spread rapidly through the connected cells in the chain

(Babic et al., 2011).

ICEBs1 carries cargo genes that encode functions not directly related to its horizontal transfer by conjugation. These benefit or protect the host cell under certain conditions. yddJ is always expressed and encodes an exclusion protein. Exclusion proteins prevent acquisition of a targeted element, often the element encoding the protein. YddJ not only prevents redundant acquisition of another copy of ICEBs1 (which could lead to recombination and instability of the element), but also protects donors from lethal excessive transfer (Avello et al., 2019). spbK (yddK) encodes an abortive infection gene that protects populations of ICEBs1-containing cells from a lysogenic phage SPβ. Cells containing ICEBs1 express spbK constantly, and if they become infected SpbK interacts with a phage protein and triggers cell death to prevent production and spread of the phage (Johnson et al., 2020, manuscript in preparation).

In Ch. 2 of this thesis, I describe the identification of a third cargo gene, ydcO, which provides a growth advantage to cells containing ICEBs1 during biofilm growth and sporulation.

The benefit was due to inhibition of development, enabling more growth compared to cells lacking ICEBs1. Appendix A explains a genetic screen performed to identify mutants of host genes that reduce ydcO-mediated developmental inhibition. In Appendix B, I describe an

30 additional ICEBs1 gene that impacts the fitness of cells. Appendix C describes experiments demonstrating that prolonged induction of ICEBs1 gene expression is highly detrimental to host cells under certain conditions.

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44 Chapter 2

A mobile genetic element increases bacterial host fitness

by manipulating development

Joshua M. Jones1, Ilana Grinberg2, Avigdor Eldar2, and Alan D. Grossman1,*

1Department of Biology

Massachusetts Institute of Technology

Cambridge, MA 02139 USA

2Department of Molecular Microbiology and Biotechnology

Faculty of Life Sciences

Tel Aviv University

Tel Aviv, Israel

This chapter is being prepared for publication.

45 Abstract

Horizontal gene transfer is a major force in bacterial evolution. Mobile genetic elements are responsible for much of horizontal gene transfer and also carry beneficial cargo genes.

Uncovering strategies used by mobile genetic elements to benefit the host is crucial for understanding their stability and spread in populations. We describe a benefit that ICEBs1, an integrative and conjugative element of Bacillus subtilis, provides to its host cells. We found that activation of ICEBs1 conferred a frequency-dependent selective advantage to its host cells during two different developmental processes: biofilm formation and sporulation. These benefits were due to a delay in biofilm-associated gene expression and sporulation by ICEBs1-containing cells, enabling ICEBs1 host cells to exploit their neighbors and grow more prior to development. A single ICEBs1 gene, ydcO (re-named here to devI for development inhibitor), was both necessary and sufficient for the delay in biofilm gene expression and sporulation. Manipulation of host developmental programs allows ICEBs1 to increase host fitness, thereby increasing propagation of the element.

46 Introduction

Conjugative elements and phages are abundant mobile genetic elements in bacteria, capable of transferring DNA between cells (Frost et al., 2005). Integrative and conjugative elements

(ICEs) appear to be the most widespread type of conjugative element (Guglielmini et al., 2011).

They are found integrated in a host genome. When activated, they excise and produce conjugation machinery that transfers the element DNA from the host cell to recipients (Carraro and Burrus, 2015; Johnson and Grossman, 2015; Wozniak and Waldor, 2010).

ICEs often carry “cargo” genes that are not necessary for transfer, but confer a phenotype to host cells. In fact, ICEs (conjugative transposons) were first identified because of the phenotypes conferred by cargo genes (Franke and Clewell, 1981). Cargo genes include those encoding antibiotic resistances, metabolic pathways, and determinants of pathogenesis and symbiosis

(Johnson and Grossman, 2015). Transfer of mobile elements between cells contributes to rapid evolution and spread of associated genes and phenotypes (Frost et al., 2005; Treangen and

Rocha, 2011).

Despite the benefits cargo genes can provide, the maintenance and transfer of mobile genetic elements requires host cellular resources and in some cases is lethal (Baltrus, 2013). Persistence of a mobile genetic element in natural populations requires balancing the cost to the host with benefits and/or a sufficiently high rate of transfer. Many mobile elements, especially ICEs, have now been identified bioinformatically (Bi et al., 2012; Guglielmini et al., 2011) or by their mechanisms of regulation (Auchtung et al., 2005). Many of these ICEs do not contain obvious cargo genes that can be inferred from sequence or easily detected experimentally (Cury et al.,

2017).

47 ICEBs1, a relatively small (~20 kb) ICE found in most strains of Bacillus subtilis, was identified bioinformatically (Burrus et al., 2002) and experimentally based on its regulation by cell-cell signaling (Auchtung et al., 2005). Most of the ICEBs1 genes needed for conjugation are grouped together in an operon that is repressed until activating signals are sensed. Two pathways activate ICEBs1, both of which lead to of the repressor ImmR by the protease and anti- repressor ImmA (Auchtung et al., 2007; Bose et al., 2008; Bose and Grossman, 2011). ICEBs1 contains the cell-cell signaling genes, rapI and phrI, which regulate ICEBs1 activation by sensing population density and the relative abundance of ICEBs1-containing host cells

(Auchtung et al., 2005). RapI is produced at high cell density and during the transition to stationary phase and stimulates the proteolytic cleavage of the repressor ImmR by the protease

ImmA (Bose and Grossman, 2011). Overproduction of RapI stimulates activation of ICEBs1 in

>90% of cells (Auchtung et al., 2005). RapI activity (and therefore ICEBs1 activation) is inhibited by PhrI, a peptide that is secreted by cells that contain ICEBs1. PhrI levels indicate the relative abundance of ICEBs1-containing cells in the population, preventing the activation and possible redundant transfer of ICEBs1 if most neighboring cells already contain the element.

ICEBs1 is also activated during the RecA-dependent DNA damage response (Auchtung et al.,

2005).

In addition to conjugation genes, ICEBs1 contains two recently characterized cargo genes that can benefit host cells. 1) yddJ is expressed even when the ICE conjugation genes are not. It encodes an exclusion protein that enables ICEBs1-containing cells to block incoming transfer of additional copies of ICEBs1 from other would-be donor cells (Avello et al., 2019). Without

YddJ, excessive attempted conjugation from surrounding cells can be lethal (Avello et al., 2019).

48 2) spbK (yddK) is also expressed when the conjugation genes are repressed. It protects ICEBs1- containing cells from predation by the lysogenic phage SPß via an abortive infection mechanism

(Johnson et al., 2020, manuscript in preparation).

Biofilms appear to be hotspots of horizontal gene transfer for bacteria growing in natural settings (Madsen et al., 2012; Molin and Tolker-Nielsen, 2003). Undomesticated strains of B. subtilis form complex biofilms on agar plates and at the air-liquid interface in standing cultures

(Vlamakis et al., 2013). There is also extensive spore formation in B. subtilis biofilms (Branda et al., 2001; Vlamakis et al., 2008). In addition, during growth in a biofilm, ICEBs1 is naturally activated and transfers efficiently, generating on the order of 10 new ICEBs1-containing host cells (transconjugants) per donor cell (Lécuyer et al. 2018). B. subtilis biofilms are held together by a matrix composed of secreted exopolysaccharides, protein fibers, and DNA (Vlamakis et al.,

2013). This matrix reinforces cell-cell contacts, likely promoting rapid spread of ICEBs1 by conjugation. Additionally, the conditions that promote biofilm-related gene expression (high cell density) also promote activation and transfer of ICEBs1 and sporulation (Auchtung et al., 2005;

Grossman and Losick, 1988). Though biofilm growth is clearly beneficial to conjugation, it is unknown how ICEBs1 impacts its host cells under these conditions.

In this study, we describe a selective advantage provided by ICEBs1 to its host cells during growth in biofilms. This fitness benefit was due to inhibition of host biofilm and spore development. We identified the ICEBs1 gene ydcO as necessary and sufficient to inhibit host development and provide a selective advantage to ICEBs1-containing cells. We also provide evidence that ydcO inhibits the key developmental transcription factor Spo0A, reducing its ability to stimulate biofilm and sporulation gene expression. ydcO is conserved in other ICEBs1-

49 like elements, indicating that manipulation of host development may be a conserved strategy among this family of mobile genetic elements. We postulate that manipulation of host pathways may be a common function of many of the as yet uncharacterized cargo genes in ICEs.

Results

ICEBs1 spreads efficiently in biofilms by conjugation

Biofilm formation is characteristic of many bacteria growing in natural settings, including B. subtilis. We used biofilm growth to ask whether ICEBs1 affected the fitness of its host cells under conditions that naturally promote its spread. We performed competition experiments in biofilms using strains of undomesticated B. subtilis (NCIB3610 plasmid-free) with or without

ICEBs1.

We observed highly efficient spread of ICEBs1 at low donor to recipient ratios (Table 1) during growth in biofilms, similar to results reported previously (Lécuyer et al. 2018). To measure mating we mixed ICEBs1-containing cells (potential donors) with cells that did not contain ICEBs1 (ICEBs10, potential recipients) and co-cultured the mix on standard biofilm- stimulating (MSgg agar) (Branda et al., 2001). Since ICEBs1 induction is regulated by cell-cell signaling, we varied the initial frequency of ICEBs1+ cells between approximately 0.01 and 0.9. We inserted unique selectable markers (antibiotic resistances) in the chromosomes of the donors and recipients as well as within ICEBs1. After four days of growth at

30ºC, biofilms were disrupted and the number of transconjugants was determined by selective plating.

50 We found that after four days of biofilm growth, the frequency of transconjugants ranged from ~40 – 65% of total cells in the biofilm for starting donor frequencies of ~1 – 50% (Table 1).

The highest frequency of transconjugants was observed when the starting frequency of ICE+ cells was ~10%. Enhanced conjugation at low donor to recipient ratios is likely due to regulation of ICEBs1 by cell-cell signaling (induction is inhibited by the presence of other potential donors) and the higher likelihood of contacting potential recipients at low frequencies of donors.

The high frequency of ICEBs1 conjugation during growth in biofilms presented a challenge for quantifying the fitness of ICEBs1-containing host cells relative to ICEBs10 cells. Mating converts a large fraction of ICEBs10 cells to transconjugants (ICEBs1-containing), reducing the

ICEBs10 proportion of the population in a manner unrelated to fitness. To measure the effect of

ICEBs1 on host fitness, we blocked conjugation using the conEK476E mutation (Berkmen et al.,

2010). We then compared the proportion of ICEBs1-containing hosts to ICEBs10 cells without the confounding influence of conjugation.

ICEBs1 provides a frequency-dependent selective advantage in biofilms

We found that cells containing ICEBs1 that is incapable of conjugation

{ICEBs1(conEK476E)} had a strong fitness benefit in biofilms when they were initially present as a minority of the population. As before, we varied the initial frequency of ICEBs1-containing host cells in the inoculum between approximately 0.01 and 0.9. To measure fitness we determined the frequency of ICEBs1-containing cells (fICE) and ICEBs10 (fNULL) cells in the initial inoculum and in mature biofilms by selective plating. The relative fitness of the ICEBs- containing cells was calculated as the fold change in the ratio of fICE / fNULL over the course of the competition.

51 We found that the fitness of ICEBs1-containing host cells was dependent on their initial frequency in the population (Fig. 1A). Cells containing ICEBs1 had a selective advantage at low frequencies (0.01 or 0.1) when the element is most strongly activated. At high frequencies in the population (0.5 or 0.9), when there is little or no activation, fitness of ICEBs1-containing cells was approximately neutral (Fig. 1A). The total growth yields of the populations were equal regardless of the frequency of ICEBs1-containing cells (Fig. 1C).

The frequency-dependence was most likely due to regulation of ICEBs1 gene expression by the cell-cell signaling genes rapI-phrI or some other function of rapI. We performed control competitions of two differentially marked ICEBs10 strains to verify that the enhanced fitness we observed was due to the presence of ICEBs1 rather than an inherent fitness difference associated with antibiotic resistance markers (see Methods and Materials). There was a small cost associated with the marker used to select cells containing ICEBs1 (relative fitness 0.7 ± 0.09), leading to a slight underestimate of the selective advantage to these cells.

There is a large amount of sporulation in B. subtilis biofilms (Branda et al., 2001; Vlamakis et al., 2008). Consistent with this, we found that approximately 80% of viable colony-forming units (CFUs) in a mature biofilm after 3 days were from spores. The selective advantage to cells containing ICEBs1 growing in biofilms could be due to sporulation and-or biofilm development.

ICEBs1 confers a selective advantage in biofilms without sporulation

We found that ICEBs1 conferred a selective advantage in biofilms in the absence of any sporulation. We blocked sporulation using a mutation that causes a reduction in the amount of the transcription factor Spo0A that is required for spore formation. The spo0A∆Ps mutation is a deletion of the sigma-H-dependent promoter upstream of spo0A (Siranosian and Grossman,

52 1994). This mutation reduces production of Spo0A, and cells do not achieve the threshold concentration required to initiate sporulation (Chung et al., 1994). spo0A∆Ps mutant cells formed biofilms that were morphologically similar to those formed by wild type cells.

In biofilms without sporulation, spo0A∆Ps mutant cells containing ICEBs1 (JMJ788) had a selective advantage compared to spo0A∆Ps mutant cells without ICEBs1 (JMJ786). Notably, the median fitness for spo0A∆Ps mutant cells containing ICEBs1 at a low frequency in the population was approximately six (5.90 ± 1.00). Thus, sporulation was not required for a fitness benefit to ICEBs1-containing cells in biofilms.

Fitness of cells in biofilms can be affected by production of the biofilm matrix. For example, cells that “cheat” by contributing less to biofilm matrix production reap the benefits of growing with other cells that incur the cost of matrix gene expression (Dragoš et al., 2018). We suspect that cells containing ICEBs1 “cheat" by decreasing or delaying expression of biofilm matrix genes compared to cells without ICEBs1.

ICEBs1 confers a selective advantage during sporulation, in the absence of biofilms

We found that cells containing ICEBs1 also had a frequency-dependent selective advantage during sporulation, in the absence of biofilms. We prepared mixtures of cells with and without

ICEBs1 as described above. These mixtures were spotted onto medium (DSM agar) that promotes high levels of sporulation. During growth on this medium, there are no complex colony features found in biofilms. As in the biofilm competitions, cells containing ICEBs1 had a frequency-dependent selective advantage during sporulation (Fig. 1B). At an initial frequency of approximately 0.01 the median relative fitness of the ICEBs1-containing cells was approximately

14 (14.48 ± 4.27). As before, the total growth yields of the populations were equal (Fig. 1D).

53 These results demonstrate that ICEBs1 confers a selective advantage to cells growing on DSM agar, outside the context of biofilms. This could be due to sporulation or growth under these specific conditions.

We found that sporulation was required for the strong selective advantage during growth on sporulation medium (DSM agar). The fitness benefit associated with the ICEBs1-containing cells at a low frequency in the population was greatly reduced in the spo0A∆Ps mutant (no sporulation). The sporulation mutant with ICEBs1 had a median fitness of approximately 1.5 compared to approximately 9 for wild-type. Based on these results, we conclude that the presence of ICEBs1 confers a frequency-dependent selective advantage during sporulation.

Together, our results demonstrate that cells containing ICEBs1 have a frequency-dependent selective advantage during sporulation and in biofilms. This selective advantage is independent of the ability of ICEBs1 to actually transfer from one cell to another. Biofilm formation and sporulation are both regulated by the transcription factor Spo0A (Hamon and Lazazzera, 2001).

Our results indicate that the presence of ICEBs1 could somehow be inhibiting the activity or activation of Spo0A.

ICEBs1-containing cells have a frequency-dependent delay in sporulation

We hypothesized that some ICEBs1-encoded gene(s) inhibited host cell development. This inhibition could delay development and enable cells to continue growth for a small number of generations. This model derives from analogous phenotypes of mutants that do not enter the sporulation pathway (Dawes and Mandelstam, 1970). Mutants that delay the start of sporulation have a growth advantage as they are able to divide one or a few more times while other cells in the population stop growing and start to sporulate. Likewise, cells that have reduced production

54 of biofilm matrix have a selective advantage in biofilms compared to matrix-producing cells

(Dragoš et al., 2018).

We found that in mixed populations sporulation was delayed in cells containing ICEBs1 compared to cells without ICEBs1. As above, we used an ICEBs1 mutant that is incapable of conjugation {ICEBs1(conEK476E)}, and the phenotype was dependent on the initial frequency of the ICEBs1-containing cells in the population. Sampling to quantify CFUs (spores and cells) over time disrupts and prevents monitoring a single population over time. We started several replicate populations, each of which we sampled once at different times to create a time-course.

Sporulation frequency was determined by measuring heat-resistant CFUs as a fraction of total

CFUs for ICEBs1-containing and ICEBs1-cured strains that contained different antibiotic resistance markers to distinguish the strains.

Sporulation is delayed in ICEBs1 host cells during biofilm formation. We found that sporulation of ICEBs1-containing cells was delayed in a frequency-dependent manner during growth in biofilms (Fig. 2A, 2B). When ICEBs1-containing cells were started at approximately

1% of the total population, they reached their maximum sporulation frequency (>80% spores) roughly 17 hours later than the cells without ICEBs1 (Fig. 2A). After three days of biofilm growth, the sporulation frequency of ICEBs1-containing and ICEBs1-cured cells was indistinguishable. Over this period of time the total frequency of ICEBs1-containing cells in the population typically rose from ~1% to ~3%, giving a relative fitness (~3) consistent with results above (Fig. 1A). When the ICEBs1-containing cells were the majority in the population

(approximately 90%) the timing and sporulation frequencies of the ICEBs1-containing and

ICEBs1-cured cells were indistinguishable (Fig. 2B).

55 Sporulation is delayed in ICEBs1 host cells during sporulation in the absence of biofilms.

We also found that sporulation of ICEBs1-containing cells was delayed in a frequency-dependent manner during sporulation in the absence of biofilm formation (Fig. 2C, 2D). When the ICEBs1- containing cells were inoculated at a low frequency (approximately 1%), the delay in sporulation was qualitatively similar to that observed in biofilms (Fig. 2C). However, the increase in the total frequency of ICE-containing cells in the population was approximately 10-fold, giving a relative fitness of approximately 10, consistent with results described above (Fig. 1B). This increase was much greater than the approximately 3-fold increase during biofilm formation. Sporulation under the conditions used here, without biofilms, begins at about 16 hrs into growth, earlier than sporulation in biofilms.

We suspect that the stronger selective advantage of ICEBs1-containing cells in the absence of biofilm formation is due to the earlier onset of sporulation. By 16 hours of growth without biofilms (on DSM), spores made up about 40% of the total CFUs. By the same time in biofilms spores were undetectable (limit of detection ~0.03% spores).

rapI-phrI are necessary but not sufficient for enhanced fitness

The fitness benefits provided by ICEBs1 were dependent on the relative abundance of

ICEBs1-containing cells, indicating that the cell-cell signaling genes rapI-phrI in ICEBs1 were involved, either directly or indirectly. Other Rap proteins in B. subtilis are known to regulate development by inhibiting phosphorylation (activation) of the transcription factor Spo0A. RapI, like other Rap proteins in B. subtilis, can inhibit the pathway needed to phosphorylate (activate) the transcription factor Spo0A, and overexpression of rapI in vivo inhibits sporulation (Parashar et al., 2013, Singh et al., 2013). However, it was unknown whether RapI regulates development

56 in vivo under physiological conditions. Results described below demonstrate that the rapI-phrI system is required for the fitness advantage of ICEBs1-containing cells, but that this requirement is by virtue of causing induction of ICEBs1 gene expression. Another gene in ICEBs1 is both necessary and sufficient for the selective advantage of ICEBs1-containing cells during development.

rapI-phrI are required for fitness benefit of cells containing ICEBs1. We deleted rapI-phrI

(∆rapI-phrI) in ICEBs1 and compared the fitness conferred by this mutant to that conferred by

ICEBs1 with rapI-phrI. Because loss of rapI prevents induction of gene expression, excision, and replication of ICEBs1, we used ICEBs1 mutants (‘locked-in’) that are incapable of excision or replication (see Methods and Materials), regardless of the presence or absence of rapI.

Preventing excision and replication of ICEBs1 allowed us to compare the fitness of wild-type

ICEBs1 to ICEBs1∆rapI-phrI (and other mutants), which would otherwise have a lower gene copy number due to a lower frequency of induction.

We verified that locked-in ICEBs1 still conferred a fitness benefit to host cells. During sporulation in biofilms (MSgg agar), cells containing locked-in ICEBs1 had a relative fitness of approximately 14 when they were started at a low frequency in the population (~0.01) (Fig. 3A).

This benefit was much greater than that conferred by wild-type ICEBs1 that can excise and replicate. We suspect that replication of ICEBs1 incurs a fitness cost to the host cell that reduces the apparent benefit. The sources of this burden could include use of host resources, the host’s response to single-stranded DNA produced by rolling-circle-replication of ICEBs1, and increases in ICEBs1 gene expression due to increased copy number.

57 We found that rapI-phrI were required for the fitness benefit conferred by ICEBs1. During sporulation in biofilms (MSgg agar), the relative fitness of the ∆(rapI-phrI) host strain was approximately neutral (Fig. 3A), in contrast to the high fitness (median ~14) provided by ICEBs1 containing rapI-phrI when the ICEBs1-containing cells were started at a low frequency (~0.01).

The requirement for rapI-phrI could be due to a direct role for one of these, likely RapI, or an indirect role in activating expression of ICE genes.

rapI-phrI are not sufficient in the absence of other ICEBs1 genes to provide a fitness benefit.

We found that rapI-phrI alone were not sufficient to provide a fitness benefit during sporulation or during sporulation in biofilms. We cloned rapI-phrI and their native promoters and inserted them in an ectopic (bcaP) in a strain that did not contain ICEBs1. When this strain was started at a low frequency (~0.01), fitness of this strain was neutral relative to a control strain without rapI-phrI (Fig. 3B). To verify that rapI-phrI were functional, we added back ICEBs1 that was missing rapI-phrI. Adding the rest of ICEBs1 restored the fitness advantage during sporulation and in biofilms, indicating that the ectopic copy of rapI-phrI was functional (Fig.

3B). The requirement for rapI-phrI and some other ICEBs1 gene(s) indicated that the selective advantage was likely dependent on induction of ICEBs1 by RapI.

Activation of ICEBs1 is required for the fitness benefit. We found that expression of one or more ICEBs1 genes controlled by the promoter Pxis was required for the selective advantage in biofilms (with sporulation). Pxis drives most of the gene in ICEBs1 and is indirectly activated by

RapI in a frequency-dependent manner (Auchtung et al., 2005; Bose and Grossman, 2011). We deleted Pxis in a strain in which ICEBs1 was unable to excise or replicate (locked-in-ICEBs1). In this strain, only genes not dependent on Pxis could be expressed, including rapI-phrI. Fitness of

58 this strain was neutral during sporulation in biofilms (Fig. 4). This indicated that expression of one or more genes controlled by Pxis, either alone or in combination with rapI, was required for the fitness benefit conferred by ICEBs1.

Since most of the genes controlled by Pxis have known roles in the conjugative life cycle, we focused our search on genes without a known function. Deletions of genes near Pxis that disrupted ydcO reduced fitness. Results described below demonstrate that a single ICEBs1 gene, ydcO, is necessary and sufficient to inhibit development and provide a selective advantage. The primary role of rapI appears to be in the induction of ydcO expression.

ydcO is necessary for the fitness benefit conferred by ICEBs1

We found that an ICEBs1 gene of unknown function, ydcO, was necessary for the fitness advantage of ICEBs1 host cells. We constructed a deletion of ydcO in the locked-in-ICEBs1 strain. When started at a low frequency in the population (~0.01) the relative fitness of the ydcO mutant was approximately 3.5 (Fig. 4), significantly less that of the isogenic ydcO+ cells

(median fitness ~14) in biofilms with sporulation (Fig. 4). Interestingly, the deletion of ydcO did not reduce fitness fully to neutral, indicating a possible role for other ICEBs1 genes.

ydcO is predicted to encode an 86 amino acid protein. A search for conserved motifs and structural similarity between YdcO and other proteins did not significantly inform our understanding of YdcO function. However, ydcO homologs are found in other Bacillus species

(see below).

ydcO is sufficient to inhibit sporulation and provide a fitness benefit

We found that when expressed constitutively, ydcO alone, in the absence of all other ICEBs1 genes, was sufficient to inhibit sporulation and provide a selective advantage. We cloned ydcO

59 under the control of Pxis at an ectopic locus (lacA) in a strain without ICEBs1. In the absence of

ICEBs1 (and its repressor ImmR), Pxis is constitutively active (Auchtung et al., 2007). Fitness was measured relative to a control strain that had Pxis with no gene downstream.

Sporulation of the Pxis-ydcO-containing strain was strongly inhibited under conditions that normally support robust sporulation, including in biofilms (Fig. 5). During sporulation either with (Fig. 5A) or without biofilm formation (Fig. 5B), the frequency of the Pxis-ydcO strain in the population rose from ~0.01 to ~0.05, giving a relative fitness of ~5. This is greater than the typical fitness conferred by ICEBs1 in biofilms, but less than that observed during sporulation without biofilms. We suspect these differences are due to constitutive expression of ydcO in the absence of ICEBs1's regulatory systems and the earlier onset of starvation on DSM agar compared to MSgg agar; cells that are unable to sporulate eventually die.

YdcO likely targets the developmental transcription factor Spo0A

Results described above demonstrated that ydcO is a robust inhibitor of sporulation.

Sporulation is controlled by the transcription factor Spo0A, which both directly and indirectly regulates the expression of many genes needed for development, including biofilm formation.

The results described below indicate that YdcO most likely targets Spo0A.

ydcO inhibits early sporulation gene expression. We found that ydcO inhibits expression of genes normally activated early during sporulation. Sporulation is initiated when Spo0A~P directly stimulates transcription of several genes, including the three sporulation operons, spoIIA, spoIIE, and spoIIG (Sonenshein, 2000). Using lacZ fusions to the promoters of each of these operons, we found that Pxis-ydcO inhibited activity of each promoter compared to wild-type

60 during sporulation in liquid sporulation medium (Fig. 6A). This indicates that YdcO inhibits the initiation of sporulation.

ydcO inhibits biofilm gene expression. We also found that ydcO inhibits expression of genes needed for extracellular matrix production during biofilm formation. We measured expression of biofilm matrix genes epsB and tasA by RT-qPCR with primers internal to each gene. During late exponential and early stationary phase in liquid sporulation medium, transcript levels of both genes were reduced by about 50% in the Pxis-ydcO strain compared to wild-type (Fig. 6B). We suspect that inhibition of biofilm matrix genes, in addition to delaying sporulation, is an important mechanism of selection for ICEBs1 host cells during growth in a biofilm. This is consistent with the selective advantage of ICEBs1 host cells in biofilms without sporulation described earlier.

Expression of the eps and tasA operons is inhibited directly by AbrB and SinR and indirectly activated by Spo0A~P (via the direct effect of Spo0A~P on abrB and the indirect effect of

Spo0A~P on sinR by stimulating sinI) (Bai et al., 1993; Hahn et al., 1995; Hamon et al., 2004;

Kearns et al., 2005). We also found that ydcO stimulates expression of abrB, which is inhibited by Spo0A~P. Transcript levels of abrB were increased by approximately 6-fold in stationary phase in the Pxis-ydcO strain compared to wild-type (Fig. 6B). Stimulation of abrB and inhibition of biofilm and early sporulation genes is consistent with YdcO functioning as an inhibitor of Spo0A or its activation by phosphorylation. Results described below indicate that

YdcO is not inhibiting phosphorylation of Spo0A.

61 YdcO does not target the phosphorelay

We found that expression of ydcO inhibited sporulation by approximately 100-fold (~106 spores/ml compared to ~108) in otherwise wild type cells in the absence of ICEBs1. Because expression of ydcO inhibited expression of early sporulation genes (above), we postulated that

YdcO was inhibiting the transcription factor Spo0A that is needed for the initiation of sporulation. Activation (phosphorylation) of Spo0A occurs through a pathway known as the phosphorelay (Sonenshein, 2000; Trach et al., 1991). The phosphorelay is composed of several kinases and two phosphotransfer proteins. The phosphorelay and Spo0A~P are inhibited by multiple phosphatases. YdcO could affect any step involved in regulating Spo0A. To narrow down the possible targets of YdcO, we bypassed the requirement of the phosphorelay for sporulation using a constitutively active form of Spo0A called Sad67. Sad67 is constitutively active due to an internal deletion and does not require phosphorylation for its activity (Ireton et al., 1993). We used an allele of sad67 that cannot be phosphorylated (sad67-D56N) and that is expressed from the LacI-repressible-IPTG-inducible promoter Pspac (Ireton et al., 1993).

We found that ydcO was still capable of inhibiting sporulation when the phosphorelay was bypassed by expression of sad67-D56N. During growth in liquid sporulation medium we triggered sporulation by inducing expression of sad67-D56N near the end of and measured the sporulation frequency 24 hours later. The sad67-D56N strain (JMJ770) produced ~105 spores/ml. In contrast, we were unable to detect spores of the sad67-D56N strain containing Pxis-ydcO (JMJ769) (limit of detection 10 spores/ml). These results demonstrate that the function of ydcO is independent of the phosphorelay that is needed to activate Spo0A. We

62 hypothesize that YdcO targets Spo0A directly, blocking its effects on developmental gene expression.

ydcO is conserved among ICEs homologous to ICEBs1

We found that ydcO is conserved among Bacillus species and in many cases is located within what appear to be ICEs similar to ICEBs1. We used NCBI BLAST to search for homologous protein sequences using both pBLAST (protein database) and tBLASTn (translated database). Homologs with 100% sequence coverage and greater than 70% identity to YdcO from

B. subtilis NCIB3610 were found in dozens of other B. subtilis strains and in closely related species including B. licheniformis, B. atrophaeus, and B. amyloliquefaciens. Excluding Bacillus species from the searches to possibly identify more distantly related proteins with known functions produced no hits.

We analyzed the sequence surrounding the ydcO homologs identified to determine if there is similarity to ICEBs1. All of the ydcO homologs appear to be within mobile element regions similar to ICEBs1, though some are clearly missing genes present in ICEBs1. Although we cannot infer whether any of these regions are functional mobile elements, we suspect that the ability to inhibit host development may be a conserved strategy among ICEBs1-like elements and possible other ICEs with cargo genes of unknown function.

Discussion

Our work demonstrates that ICEBs1 confers a selective advantage on its host cells by delaying biofilm and spore development, enabling the host to grow more than cells without

ICEBs1. When ICEBs1-containing cells are the minority in a mixed population, ICEBs1 genes

63 are induced. One of these genes, ydcO, is necessary and sufficient to inhibit biofilm- and sporulation-associated gene expression, likely by inhibiting the key developmental regulator

Spo0A. Together with previous findings we conclude that ICEBs1 encodes at least three distinct strategies to benefit its host cells. 1) Inhibition of development (described here) provides a growth advantage in biofilms and during sporulation. 2) Exclusion, mediated by yddJ, blocks the conjugation machinery and protects the host cell from lethal excessive transfer (Avello et al.,

2019). 3) An abortive infection mechanism, mediated by spbK (yddK) protects populations of

ICEBs1 host cells from predation by the lysogenic phage SPβ (Johnson et al., 2020, manuscript in preparation). We propose that all three strategies provide a competitive advantage for ICEBs1 and its host cells in different conditions.

ICEBs1 stability during sporulation

We hypothesize that in addition to providing a fitness advantage to its host cell, delaying sporulation may also improve stability of ICEBs1 in the host during development. Sporulation involves the formation of an asymmetric division septum generating the larger mother cell and the smaller forespore (Errington, 2001; Higgins and Dworkin, 2012). Sporulation is induced when cells are at a high population density and running out of nutrients, conditions that also activate ICEBs1 (Auchtung et al., 2005; Grossman and Losick, 1988). The plasmid form of

ICEBs1 that is generated after excision from the chromosome is not known to have a mechanism for active partitioning, and is likely to remain in the larger mother cell if the cells do enter the sporulation pathway and divide asymmetrically. Therefore, the ability of ICEBs1 to delay the initiation of sporulation under conditions when the element is activated could help prevent loss of the element and maintain ICEBs1 in host cells.

64 Mobile genetic elements employ various strategies to promote their maintenance during sporulation. Rates of curing during sporulation for various plasmids in Bacillus species vary widely and do not necessarily correlate with their stability during normal cell division (Tokuda et al., 1993; Turgeon et al., 2008). Mechanisms encoded by plasmids to promote their stability during growth and sporulation include the production of dynamic cytoskeletal filaments (Becker et al., 2006) and post-segregational killing of plasmid-cured pre-spores with toxin-antitoxin systems (Short et al., 2015). Interestingly, even lytic phage genomes can be incorporated into spores (first described in the 1960s) by co-opting the host’s chromosomal partitioning system

(Meijer et al., 2005).

Diversity of cargo genes and associated phenotypes

Mobile genetic elements, especially ICEs, are widespread in bacteria (Frost et al., 2005;

Guglielmini et al., 2011). Many known mobile genetic elements encode cargo genes that confer easily recognizable phenotypes, notably antibiotic resistance. Other cargo genes provide less obvious phenotypes but still fundamentally alter the physiology of the host cell. A large (500-kb)

ICE was discovered in Mesorhizobium loti because its horizontal transfer conferred the ability to form nitrogen-fixing symbiotic rood nodules on Lotus plant species (Sullivan and Ronson, 1988).

In many pathogens, cargo genes in mobile elements are largely responsible for virulence. For example, Vibrio cholerae is capable of a pathogenic lifestyle in human hosts due a to the toxin- encoding phage CTXΦ (Waldor and Mekalanos, 1996). In the sporulating pathogen Bacillus anthracis, mobile genetic elements regulate both virulence and host development. Two plasmids, pXO1 and pXO2, provide the genes for toxin synthesis and production of a protective capsule, respectively (Green et al., 1985; Mikesell et al., 1983). pXO1 also contains a regulatory gene,

65 atxA, that regulates virulence factor production and also inhibits host cell sporulation (Dale et al.,

2018). Co-regulation of virulence factors and sporulation is likely important during infection, as

B. anthracis spores are thought to be more susceptible than vegetative cells to clearance by the immune system (Mock and Fouet, 2001).

Mobile elements are also known to alter the host’s interaction with other horizontally acquired DNA, which has implications for the fitness and evolvability of the host. For example, the plasmid pBS32 in B. subtilis encodes an inhibitor of the host’s DNA uptake machinery, blocking natural transformation (Konkol et al., 2013). Interestingly, genes with roles in defense against foreign DNA, CRISPR-Cas systems, are also identified within mobile elements (Faure et al., 2019; McDonald et al., 2019; Millen et al., 2012). Competition between mobile elements not only shapes the repertoire of cargo genes in a given cell, but it may also protect the host from harmful elements.

Many mobile genetic elements have been identified bioinformatically from genome sequences or discovered by means other than the phenotypes they provide (Bi et al., 2012;

Guglielmini et al., 2011; Johnson and Grossman, 2015). Many elements lack obvious cargo genes, or at least lack cargo genes that have recognizable functions (Cury et al., 2017). We suspect that many elements with uncharacterized cargo genes provide important traits to their hosts beyond the scope of the phenotypes currently attributed to mobile elements. Though mobile genetic elements can have remarkably broad host ranges, such as the Tn916-Tn1545 group of ICEs (Clewell et al., 1995; Roberts and Mullany, 2009) and the IncP-1 group of plasmids (Popowska and Krawczyk-Balska, 2013), cargo genes and their associated functions could be highly specific to certain hosts.

66 Characterization of unknown cargo genes is likely to expand the diversity of traits currently attributed to mobile genetic elements. We speculate that many of these genes modulate normal host functions rather than provide entirely new phenotypes. Understanding cargo gene function is critical for understanding interactions between and co-evolution of mobile elements and their hosts.

Methods and Materials

Media and growth conditions

Prior to competition experiments, cells were grown as light lawns for approximately 20 hours at room temperature on 1.5% agar plates containing 1% w/v , 0.1% w/v monopotassium glutamate, and 1x Spizizen’s salts (2 g/l (NH4)SO4, 14 g/l K2HPO4, 6 g/l KH2PO4, 1 g/l

Na3citrate-2H2O, and 0.2 g/l MgSO4-7H2O) (Harwood and Cutting, 1990). Cells were resuspended from light lawns and grown at 37ºC with shaking in S750 defined minimal medium

(Jaacks et al., 1989) with 1% w/v glucose and 0.1% w/v monopotassium glutamate. Biofilms were grown at 30ºC on MSgg agar plates (as defined in Branda et al., 2001 with the exception of and phenylalanine, which we did not include). The sporulation medium used was

DSM (liquid or plates solidified with 1.5% agar) (Harwood and Cutting, 1990). MSgg agar and

DSM agar plates were dried for 20-24 hours at 37ºC prior to use. Antibiotics were used at the following concentrations for selection on LB agar plates: (5 µg/ml), kanamycin

(5 µg/ml), spectinomycin (100 µg/ml), tetracycline (12.5 µg/ml), and a combination of erythromycin (0.5 µg/ml) and lincomycin (12.5 µg/ml) to select for macrolide-lincosamide-

67 streptogramin (MLS) resistance. Isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma) used at a final concentration of 1 mM to induce expression from the Pspac promoter.

Strains and alleles

The B. subtilis strains used are listed in Table 2. The strain background used in most experiments was a derivative of the undomesticated strain NCIB3610 lacking its endogenous plasmid pBS32 (NCIB3610 plasmid-free). Some experiments were performed in the laboratory- domesticated background JH642 strain background and contain pheA1 and trpC2 mutation

(Perego et al., 1988). ICEBs10 indicates the strain is cured of ICEBs1. Standard techniques were used for cloning and strain construction (Harwood and Cutting 1990). Some alleles were previously described and are summarized below. Variants of ICEBs1 that were blocked for transfer contained the conEK476E mutation derived from MMB1118 (Berkmen et al., 2010). The spo0A∆Ps allele was derived from AG1242 (Siranosian and Grossman, 1994). Strains JMJ769 and JMJ770 contained an amyE::Pspac-sad67-D56N cat allele identical to that in Ireton et al.,

1993, and a ∆spo0A::spec identical to that in Grossman et al., 1992 with cat swapped for spec.

The amyE::PspoIIA-lacZ cat allele was derived from KI938 (Chung et al., 1994). Essentially identical alleles with PspoIIE and PspoIIG were also used.

Construction of selective markers for mating and competition experiments. ICEBs1 was marked with the cat gene (conferring chloramphenicol resistance) between the divergently transcribed genes yddJ and spbK (yddK). Markers used to select ICEBs1-containing and ICEBs10 cells were all integrated at lacA and contained spec (spectinomycin resistance), mls (macrolide- lincosamide-streptogramin resistance), or kan (kanamycin resistance). The mls and kan constructs also contained constitutively expressed fluorescent proteins BFP and RFP,

68 respectively. The plating efficiency if all markers was verified, and control competitions

(described below) were performed to measure marker-associated fitness effects.

Construction of ICEBs1 mutants. Fitness measurements of ICEBs1 mutants were performed in a version of ICEBs1 unable to excise and replicate (locked-in ICEBs1). All mutants were isogenic to JMJ646 (ICEBs1 oriT* ∆attR::tet), which is unable to excise due to the attR::tet deletion (Lee and Grossman, 2007). The origin of transfer was mutated (oriT*) to prevent

ICEBs1 replication while integrated, which is detrimental (Lee and Grossman, 2007; Menard and

Grossman, 2013). The markerless oriT* mutation was constructed by cloning nicK(oriT*) from pJT245 and ~1 kb of upstream sequence into pCAL1422 (a plasmid that contains E. coli lacZ) by isothermal assembly (Gibson et al., 2009), essentially as previously described (Thomas et al.,

2013). The resulting plasmid, pJMJ196, was integrated into the chromosome by single-crossover recombination. Transformants were screened for loss of lacZ, indicating loss of the integrated plasmid, and PCR was used to identify a clone containing the oriT* allele. Markerless deletions of ICEBs1 sequence were also generated using pCAL1422-derived plasmids. The rapI-phrI deletion was generated using pJMJ430 and removes the rapI and phrI ORFs. The Pxis deletion was generated using pJMJ199 and removes sequence from 149 bp to 27 bp upstream of the xis

ORF. This removes the presumed -35 and -10 of the promoter but does not remove the known regulatory sites at the neighboring immR promoter (Auchtung et al., 2007). The ydcO deletion was generated using pELS1 and fuses the first four and last two codons of ydcO.

Construction of ectopic rapI-phrI construct. The rapI-phrI ORFs plus the promoter region

(352 bp upstream of rapI) and 112 bp of downstream sequence were cloned into pMMH253

(vector for integration at bcaP). The resulting plasmid (pJMJ354) was linearized and introduced

69 to B. subtilis by transformation and selection for kanamycin resistance. The corresponding empty control construct was generated by transforming linearized pMMH253.

Construction of lacA::Pxis-ydcO. We expressed ydcO from the Pxis promoter by cloning existing elements from a Pxis gene expression construct marked with mls at lacA and inserting the ydcO ORF by isothermal assembly. The resulting product was introduced to the chromosome by transformation and selection for MLS resistance. The Pxis-empty control strain contains an identical construct lacking an ORF fused to Pxis.

Biofilm mating experiments

Cultures were started from resuspended light lawns (described above) in S750 minimal medium at an initial OD600 of 0.05. Cultures were grown to mid-exponential phase (OD600

~0.5) at 37ºC with shaking. Cells were pelleted, resuspended in 1x Spizizen’s salts, and diluted to an OD600 of 0.01. Donor and recipient strains were mixed at the indicated frequencies and 50 µl of the mixture was spotted onto the center of MSgg agar plates. Spots were allowed to dry at

30ºC before flipping the plates. Plates were incubated at 30ºC for 4 days. At the time of inoculation, the strain mixes were diluted and plated on LB agar plates containing the appropriate antibiotics to determine the initial CFU/ml of the donor and recipient strains. After 4 days, the mature biofilms were scraped from the agar surface with sterile wooden sticks and resuspended in 5 ml 1x Spizizen’s salts, followed by mild sonication to disperse the cells. Cells were diluted and selectively plated to determine the final CFU/ml of transconjugants.

Competition experiments

Cells were prepared for competition experiments as described above for biofilm mating experiments. Strain mixtures at the indicated frequencies were spotted onto MSgg agar plates for

70 biofilm competitions and DSM agar plates for sporulation competitions. Plates were incubated at

30ºC for 4 days unless otherwise indicated. Biofilms/colonies were collected, disrupted, and plated as described above. For time-course competitions, two replicate biofilms/colonies were collected at each of the indicated times. Sporulation frequency was determined by selective plating before and after a heat treatment at 85ºC for 20 minutes to enumerate total CFUs and

CFUs derived from heat-resistant spores. Relative fitness of ICEBs1-containing cells over

ICEBs10 cells was determined as (pe/(1-pe))/(pi/(1-pi)), where pe,pi are the frequencies of

ICEBs1-containing cells and the end and beginning of the competition, respectively. Control competitions between ICEBs1-cured cells were performed to determine the fitness associated with the lacA::{Pveg-mTagBFP mls} marker (JMJ574) used in ICEBs1-containing cells relative to the lacA::{Ppen-mApple2 kan} marker used in ICEBs1-null cells (JMJ550). When the mls- marked cells were started at a frequency of 0.01, their relative fitness was 0.7 ± 0.09 (average and standard deviation from 3 independent experiments and a total of 9 biofilms).

Gene expression assays

Cultures were grown from single colonies in liquid DSM at 37º with shaking. Cells were harvested at the indicated timepoints. For β-galactosidase assays, growth was stopped by the addition of toluene (~1.5% final concentration). β-galactosidase specific activity ({∆A420 per minute per ml of culture per OD600} x 1000) was measured as described (Miller, 1972) after pelleting cell debris. For RT-qPCR assays, cells were harvested directly into ice-cold methanol

(1:1 methanol to culture volume) and pelleted. RNA was isolated using Qiagen RNeasy PLUS kit with 10 mg/ml lysozyme. iScript Supermix (Bio-Rad) was used for reactions to generate cDNA. Control reactions without reverse transcriptase (-RT) were

71 performed to assess the amount of DNA present in the RNA samples. RNA was degraded by adding 75% volume of 0.1 M NaOH and incubating at 70ºC for 10 minutes, followed by neutralization with an equal volume of 0.1 M HCl. qPCR was done using SSoAdvanced SYBR master mix and CFX96 Touch Real-Time PCR system (Bio-Rad). Primers used to measure epsB were oJJ363 (5’-CGGAACAATATCGCACCATTC-3’) and oJJ364 (5’-

CGCTGCACTGAACGATTTAC-3’). Primers used to quantify tasA were oJJ367 (5’-

GGATCACTTGCGATCAAAGAAG-3’) and oJJ368 (5’-

CTTCAAACTGGCTGAGGAAATC-3’). Primers used to quantify abrB were oJJ339 (5’-

CAAGTAACTGGTGAAGTTTCTGATG-3’) and oJJ340 (5’-

GGATTTCGCTGATGATTTGCTC-3’). Primers used to measure the control locus gyrA were oMEA128 (5’-TGGAGCATTACCTTGACCATC-3’) and oMEA129 (5’-

AGCTCTCGCTTCTGCTTTAC-3’). The relative transcript copy numbers (as indicated by the

Cp values measured by qPCR) of epsB, tasA, and abrB were normalized to gyrA after subtracting the signal from -RT qPCR reactions.

Sporulation assays

Cultures were grown from single colonies in liquid DSM at 37ºC with shaking. IPTG was added at a final concentration of 1 mM to induce sad67-D56N at the end of exponential growth.

Spore titers were measured 24 hours later by plating after heat-treatment at 85ºC for 20 minutes.

Spore frequencies were normalized to the total CFUs at the time of IPTG induction, due to viability differences of the strains at T24.

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79 Table 1: Frequency of transconjugants generated in biofilm matings

Initial frequency ICEBs1 donors1 Final frequency transconjugants2 0.008 ± 0.002 0.415 ± 0.131 0.099 ± 0.027 0.635 ± 0.15 0.470 ± 0.051 0.444 ± 0.070 0.891 ± 0.033 0.063 ± 0.008

1 ICEBs1-containing cells (JMJ592) were mixed with ICEBs1-cured cells (JMJ550). The initial frequencies reported are the average ± standard deviation from three independent experiments. 2 The final frequencies of transconjugants reported are the average ± standard deviation from a total of 9 biofilms from 3 independent experiments.

80 A. B.

10 10

1 1 Relative fitness Relative fitness 0.1 0.1 0.01 0.1 0.5 0.9 0.01 0.1 0.5 0.9 Initial frequency ICEBs1 Initial frequency ICEBs1 C. D. 1010 1010

109 109 Total CFU yield CFU Total Total CFU yield CFU Total 108 108 0.01 0.1 0.5 0.9 0.01 0.1 0.5 0.9 Initial frequency ICEBs1 Initial frequency ICEBs1

Figure 1. The fitness of ICEBs1-containing cells during development depends on their initial frequency in the population. The fitness of ICEBs1-containing cells (JMJ593) relative to ICEBs1-cured cells (JMJ550) was measured by competition during biofilm growth (A) or growth on sporulation medium without biofilm formation (B). The growth yields of the populations were determined by counting all CFUs derived from cells and spores. Data presented are the pooled measurements from 3 independent experiments. A total of 9 populations were analyzed per initial frequency. Boxes extend from the lower to upper quartiles of the data, and the line inside the box indicates the median fitness. Whiskers indicate the range of the measurements.

81 A. B. 100 100 80 80 60 60 40

% Spores 40 % Spores 20 20 0 0 1 freq. 0.1 freq. Bs1 0.01 Bs1 0.5 ICE ICE 0 20 40 60 0 20 40 60 C. Hours D. Hours 100 100 80 80 60 60 40 40 % Spores % Spores 20 20 0 0 1 freq. 0.1 freq. Bs1 0.01 Bs1 0.5 ICE 0 20 40 60 ICE 0 20 40 60 Hours Hours

Figure 2. ICEBs1-containing cells delay sporulation in a frequency-dependent manner. ICEBs1-containing cells (JMJ593, black circles) delayed sporulation compared to ICEBs1- cured cells (JMJ550, open squares) when they were started at a frequency of 0.01 during competition in biofilms (A) and on sporulation medium (C). When ICEBs1-containing cells were started at a frequency of 0.9 in biofilms (B) sporulation was not delayed, and on sporulation medium it was slightly delayed (D). Boxes below each graph show the frequency of ICEBs1-containing cells at each timepoint. Data shown are the average from two populations per timepoint with error bars indicating the standard deviation.

82 A. Biofilm B. Biofilm Sporulation

10 10

1 1 Relative fitness Relative fitness 0.1 0.1

Bs1 0 0

rapI-phrI rapI-phrIBs1 rapI-phrIBs1 rapI-phrI ∆ :: :: :: ICE rapI-phrI rapI-phrI ∆ ICE ∆ bcaP bcaP::rapI-phrI bcaP bcaP locked-in ICE Bs1 Bs1 ICE ICE

Figure 3. The ICEBs1 cell-cell signaling genes, rapI-phrI, are necessary but not sufficient to confer a selective advantage. (A) rapI-phrI are required for high fitness. Fitness of cells containing locked-in ICEBs1 (JMJ646) or an isogenic rapI-phrI mutant (JMJ686) was measured relative to ICEBs1 cured cells (JMJ550) when the ICEBs1-containing cells were started at a frequency of 0.01. (B) rapI-phrI alone are not sufficient, and other ICEBs1 gene(s) are necessary for high fitness. Cells containing rapI-phrI alone (JMJ576) or also containing ICEBs1∆rapI-phrI (JMJ785) were started at a frequency of 0.01 in competitions against ICEBs1-cured cells (JMJ714) in biofilms or on sporulation medium. Data shown are from three independent experiments with a total of 9 populations analyzed per condition. Boxes extend from the lower to upper quartiles, and the middle line indicates the median fitness. Whiskers indicate the range of the fitness measurements.

83

10

1 Relative fitness 0.1

xis Bs1 P ydcO ∆ ∆

locked-in ICE

Figure 4. The Pxis promoter and ydcO are required for the fitness benefit of ICEBs1. Fitness of cells containing locked-in ICEBs1 (JMJ646) or isogenic mutants of Pxis (JMJ662) or ydcO (JMJ703) was measured relative to ICEBs1-cured cells (JMJ550) during competition in biofilms. ICEBs1-containing cells were started at a frequency of 0.01. Data shown are from 3 independent experiments with a total of 9 populations per condition. Boxes extend from the lower to upper quartiles of the data, and the middle line indicates the median fitness. Whiskers indicate the range of the fitness measurements.

84 A. B. 100 100 80 80 60 60 40 40 % Spores % Spores 20 20 0 0 0.1 0.1 freq. freq.

0.01 0.01 ydcO ydcO 0 20 40 60 80 0 20 40 60 80 Hours Hours

Figure 5. ydcO alone is sufficient to inhibit sporulation and provide a selective advantage. Cells that constitutively express ydcO (JMJ725, black circles) were inhibited in sporulation compared to wild-type (JMJ727, open squares) during competition in biofilms (A) and on sporulation medium (B). Cells containing ydcO were started at a frequency of 0.01. Boxes below each graph show the frequency of ydcO-containing cells at each timepoint. Data shown are the average from two populations per timepoint with error bars indicating the standard deviation.

85 A. PspoIIA-lacZ PspoIIE-lacZ PspoIIG-lacZ 150 150 80 60 100 100 40 50 50 20 -gal specific activity -gal specific activity -gal specific activity β β 0 0 β 0 -2 0 2 4 6 -2 0 2 4 6 -2 0 2 4 6 Hours post-exponential Hours post-exponential Hours post-exponential B. epsB tasA abrB 4 8 20

3 6 15 2 4 10 1 2 5 Arbitrary Units Arbitrary Units Arbitrary Units 0 0 0 -2 -1 0 1 2 3 -2 -1 0 1 2 3 -2 -1 0 1 2 3 Hours post-exponential Hours post-exponential Hours post-exponential

Figure 6. ydcO inhibits expression of genes associated with sporulation initiation and biofilm formation. (A) Cells with ydcO (black circles) and wild-type cells (open squares) were grown in DSM. Cells were harvested at the indicated times and β-galactosidase specific activity was measured. Strains: PspoIIA-lacZ Pxis-ydcO (JMJ732), PspoIIA-lacZ WT (JMJ735), PspoIIE-lacZ Pxis-ydcO (JMJ731), PspoIIE-lacZ WT (JMJ734), PspoIIG-lacZ Pxis-ydcO (JMJ733), PspoIIG-lacZ WT (JMJ736). A representative experiment is shown. (B) Cells with ydcO (JMJ749, black circles) and wild-type cells (JMJ750, open squares) were grown in DSM, and cells were harvested at the indicated times. cDNA was synthesized using reverse transcriptase and RT-qPCR was used to measure expression of biofilm-associated genes epsB and tasA as well as a regulatory gene abrB. The transcript copy numbers of these genes were measured relative to a housekeeping gene gyrA. The data reported are the average of three technical replicates from one experiment. The relative expression levels are shown normalized to WT at T-1.

86 Table 2: B. subtilis strains used1 Strain Relevant genotype JMJ550 ICEBs10 lacA::{Ppen-mApple2 kan} JMJ574 ICEBs10 lacA::{Pveg-mTagBFP mls} JMJ576 ICEBs10 bcaP::{PrapI-rapIphrI kan} lacA::{Pveg-mTagBFP mls} JMJ592 ICEBs1 yddJ-cat-yddK lacA::{Pveg-mTagBFP mls} JMJ593 ICEBs1 conEK476E yddJ-cat-yddK lacA::{Pveg-mTagBFP mls} JMJ646 ICEBs1 oriT* attR::tet lacA::{Pveg-mTagBFP mls} JMJ662 ICEBs1 ∆Pxis oriT* attR::tet lacA::{Pveg-mTagBFP mls} JMJ686 ICEBs1 oriT* ∆rapIphrI attR::tet lacA::{Pveg-mTagBFP mls} JMJ703 ICEBs1 oriT* ΔydcO attR::tet lacA::{Pveg-mTagBFP mls} JMJ714 ICEBs10 lacA::spec bcaP::kan JMJ725 ICEBs10 lacA::{Pxis-ydcO mls} JMJ727 ICEBs10 lacA::{Pxis-empty mls} JMJ731 ICEBs10 lacA::{Pxis-ydcO mls} amyE::{PspoIIE-lacZ cat} JMJ732 ICEBs10 lacA::{Pxis-ydcO mls} amyE::{PspoIIA-lacZ cat} JMJ733 ICEBs10 lacA::{Pxis-ydcO mls} amyE::{PspoIIG-lacZ cat} JMJ734 ICEBs10 lacA::{Pxis-empty mls} amyE::{PspoIIE-lacZ cat} JMJ735 ICEBs10 lacA::{Pxis-empty mls} amyE::{PspoIIA-lacZ cat} JMJ736 ICEBs10 lacA::{Pxis-empty mls} amyE::{PspoIIG-lacZ cat} JMJ749 JH642 ICEBs10 lacA::{Pxis-ydcO mls} JMJ750 JH642 ICEBs10 lacA::{Pxis-empty mls} JMJ769 JH642 ICEBs10 lacA::{Pxis-ydcO mls} amyE::{Pspac-sad67D56N cat} spo0A::spec JMJ770 JH642 ICEBs10 lacA::{Pxis-empty mls} amyE::{Pspac-sad67D56N cat} spo0A::spec JMJ785 ICEBs1 oriT* ∆rapIphrI attR::tet bcaP::{PrapI-rapIphrI kan} lacA::{Pveg- mTagBFP mls} JMJ786 ICEBs10 spo0A∆Ps lacA::{Ppen-mApple2 kan} JMJ788 ICEBs1 conEK476E yddJ-cat-yddK spo0A∆Ps lacA::{Pveg-mTagBFP mls}

1All strains derived from NCIB3610 plasmid-free unless otherwise indicated.

87 Appendix A

Genetic screen to isolate ydcO suppressor mutants

88 As part of our efforts to identify the target of YdcO, I performed a screen to isolate mutants that were resistant to YdcO-mediated sporulation inhibition. Mutations in host genes that improve sporulation despite overproduction of YdcO could indicate what host protein or pathway YdcO targets. To reduce the likelihood of isolating mutants of ydcO, the screen was performed with a strain containing two copies of ydcO constitutively expressed from the Pxis promoter (JMJ772: JH642 ICEBs10 lacA::{Pxis-ydcO mls} amyE::{Pxis-ydcO kan}). This strain produces spores at a frequency of ~0.05% of total CFUs after 24 hours in stationary phase in liquid sporulation medium (DSM). Spontaneous mutants that sporulate at a higher frequency will be enriched in the final population of heat-resistant spores. Multiple rounds of spore germination, growth, and sporulation were performed until such mutants made up the majority of the population and could easily be identified.

The first cycle of enrichment consisted of growing JMJ772 in DSM until approximately 24 hours of stationary phase. The resulting population was heat-treated for 20 minutes at 85ºC to kill vegetative cells and plated on LB agar. Plates were incubated until the germinated spores had formed pinpoint-sized colonies. At least 10,000 pinpoint colonies were resuspended from the plates and diluted by a factor of 100 into fresh DSM to start the next cycle of enrichment. The frequency of spores in the culture was measured after each cycle. When enrichment of mutants was obvious (the sporulation frequency of the population greatly exceeded that of the parent strain JMJ772), individual isolates from the population were characterized. In a pilot attempt at the screen, 4 rounds of enrichment resulted in a sporulation frequency of 4%, and a mutant with a different colony morphology was isolated (JMJ795). The screen was repeated with 8 independent cultures. After 5 rounds of enrichment, several candidate mutants were isolated. In

89 some cases, multiple candidates from the same culture were isolated if they were phenotypically different. The sporulation frequency of each isolate was measured at T24 in DSM, and both copies of Pxis-ydcO were sequenced to make sure the increased sporulation was not the result of mutations in ydcO or Pxis. I identified 6 suppressor mutants with sporulation frequencies ranging from ~3 - 60%. The characterizations of these strains are described in Table 1. Other candidates were missing one copy of Pxis-ydcO.

I also PCR amplified and sequenced loci where we expected we might find suppressor mutations (spo0A and spo0H) in 4 of the 6 mutants isolated (JMJ795, JMJ833, JMJ834,

JMJ837). I did not find mutations at either locus. Further screening of individual loci or genome sequencing will be necessary to identify the suppressor mutations. Our inability to identify mutations in spo0A or spo0H does not necessarily rule them out as targets of YdcO. Mutations in either genes that suppress YdcO-mediated sporulation inhibition may not exist or might impair growth/viability such that they do not become enriched.

90 Table 1: ydcO suppressor mutants

Strain Sporulation Morphology on LB Notes frequency at T24 (compared to JMJ772)

JMJ795 3% More opaque, sharper/ 3-fold fewer total CFUs at T24 defined colony edges than JMJ772 JMJ833 18% Identical JMJ834 59% Colony center more opaque JMJ837 3% Identical JMJ843 11% Identical From same culture as JMJ833, but not identical. JMJ843 lost mlsR but has lacA::{Pxis-ydcO}. JMJ846 32% Identical

91 Appendix B

yddI is also important for ICEBs1 host fitness

92 During preliminary experiments to identify the ICEBs1 gene(s) responsible for the fitness benefit of ICEBs1-containing cells, I found that deleting yddI caused a significant drop in fitness.

Expressing yddI at an ectopic locus from an IPTG-inducible promoter restored wild-type fitness in the presence of IPTG, indicating that the fitness drop was due to loss of yddI rather than unintended consequences of the deletion.

I found that in the absence of yddI, cells with ICEBs1 had a fitness cost. I made a clean deletion of yddI in the locked-in ICEBs1 background (JMJ664) and measured fitness of the mutant relative to ICEBs1-cured cells (JMJ550). In the same experiments, I measured fitness of an isogenic strain containing yddI (wild-type ICEBs1, JMJ646) as well as an identically marked

ICEBs1-cured strain (JMJ574) to determine neutral fitness. During biofilm growth with the

ICEBs1-containing strains started at a frequency of ~0.01, the fitness of the yddI mutant was below neutral (median 0.39 ± 0.23 for ∆yddI compared to 0.70 ± 0.09 for neutral). The median fitness of the isogenic wild-type ICEBs1-containing strain was 14.2 ± 2.9. These results are from at least 3 independent experiments with 3 replicate biofilms in each experiment (n=9 biofilms for yddI and the neutral control, n=12 biofilms for wild-type). In a different experiment, I verified that ectopic expression of yddI restored ~wild-type fitness to the yddI mutant.

I also found that yddI alone was not sufficient to provide a selective advantage to cells. I cloned yddI and inserted it at an ectopic locus in an ICEBs1-cured strain under the control of the

IPTG-inducible promoter Pspank. Expression of yddI alone, or in combination with rapI-phrI did not provide a selective advantage. Expression of yddI did not confer any additional benefit to cells expressing ydcO, suggesting that yddI is not involved in the developmental inhibition

93 mechanism of selection mediated by ydcO. Instead, we hypothesize that yddI is needed to mitigate a cost of ICEBs1 to the host.

yddI is predicted to encode a 168 amino acid protein with one transmembrane helix predicted at the N-terminus. YddI has predicted structural similarity (Phyre2, Kelley et al., 2015) to a conjugation protein, TcpC, encoded by the plasmid pCW3. In this system, TcpC is required for conjugation and interacts with 3 other conjugation genes including the hydrolase (Porter et al., 2012). In ICEBs1, yddI is located immediately downstream of cwlT, which encodes a cell wall hydrolase. YddI is not essential for ICEBs1 conjugation, but could be needed to regulate activity of CwlT or other components of the conjugation machinery.

Exposure to purified CwlT kills B. subtilis cells, suggesting that regulation of cell wall hydrolase activity during mating is critical (DeWitt and Grossman, 2014).

Table 1. B. subtilis strains used.

Strain Relevant genotype

JMJ550 ICEBs10 lacA::{Ppen-mApple2 kan} JMJ574 ICEBs10 lacA::{Pveg-mTagBFP mls} JMJ646 ICEBs1 oriT* ∆attR::tet lacA::{Pveg-mTagBFP mls} JMJ664 ICEBs1 oriT* ∆yddI ∆attR::tet lacA::{Pveg-mTagBFP mls}

All strains are derived from NCIB3610 plasmid-free.

94 References

DeWitt, T., and Grossman, A.D. (2014) The Bifunctional Cell Wall Hydrolase CwlT Is Needed for Conjugation of the Integrative and Conjugative Element ICEBs1 in Bacillus subtilis and B. anthracis. Journal of Bacteriology 196: 1588–1596.

Kelley, L.A., Mezulis, S., Yates, C.M., Wass, M.N., and Sternberg, M.J.E. (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10: 845–858.

Porter, C.J., Bantwal, R., Bannam, T.L., Rosado, C.J., Pearce, M.C., Adams, V., et al. (2012) The conjugation protein TcpC from Clostridium perfringens is structurally related to the type IV secretion system protein VirB8 from Gram-negative bacteria. Molecular Microbiology 83: 275– 288.

95 Appendix C

Prolonged ICEBs1 induction is detrimental to host cells

96 Overview

Prior to investigating the fitness effects of ICEBs1 in the context of biofilms, I performed batch-culture competitions to measure the fitness of ICEBs1-containing cells. Under these conditions, we found that cells containing ICEBs1 had a strong frequency-dependent fitness cost that was dependent on induction of ICEBs1 by the cell-cell signaling genes rapI-phrI. The cost was primarily due to stationary phase defects; ICEBs1-containing cells entered stationary phase early and plateaued at a lower density. Different aspects of the ICEBs1 life cycle contributed to the fitness cost. Blocking excision and subsequent replication (locked-in ICEBs1), as well as deletion of the conjugation genes, each alleviated the stationary phase defects.

ICEBs1-containing cells have a frequency-dependent fitness cost

We found that ICEBs1-containing cells had a strong fitness cost when they were the minority in the population during batch-culture competition. We co-cultured ICEBs1-containing and

ICEBs1-cured cells in defined minimal medium (S750 1% w/v glucose, 0.1% w/v monopotassium glutamate) at 37ºC with shaking. Every 12 hours (approximately 10 doublings), cultures were diluted by a factor of 1000 into fresh medium. Every 24 hours, cultures were selectively plated to enumerate ICEBs1-containing and ICEBs1-cured cells. When the ICEBs1-containing cells were started at a low frequency (~0.1), their abundance decreased sharply (Fig. 1A). When ICEBs1- containing and ICEBs1-cured cells were started at an equal frequency (~0.5), the amount of

ICEBs1-containing cells only decreased slightly (Fig. 1A). When cells contained a version of

ICEBs1 lacking rapI-phrI, which are needed for efficient induction, there was minimal change in the relative abundance of ICEBs1-containing cells at either initial frequency (Fig. 1B). We suspect that the fitness cost of ICEBs1-containing cells is due to the inability to turn off induction

97 of ICEBs1 under these conditions. When ICEBs1-containing cells are the minority in the population, they are unlikely to sense the secreted inhibitory peptide, PhrI, for 3 reasons: 1) relatively few cells contain ICEBs1 and therefore secrete PhrI, 2) the liquid cultures are well- mixed so PhrI is likely diluted efficiently, and 3) all cells in the culture can internalize Phr peptides, soaking up the inhibitory signal. Additionally, there is no appreciable change in the total frequency of ICEBs1-containing cells due to mating in these conditions. The maximum frequency of transconjugants was detected in the 1:1 mixture at ~60 generations (7.7 x 10-4 transconjugants/donor).

ICEBs1 induction is detrimental during stationary phase

We found that the fitness cost associated with ICEBs1 was primarily due to early entry into stationary phase and a plateau at lower density. We monitored growth of a mixed culture containing roughly equal initial numbers of ICEBs1-containing and ICEBs1-cured cells. A version of ICEBs1 that was unable to excise from the chromosome was used to prevent curing of

ICEBs1. Induction of ICEBs1 was stimulated by over-expression of rapI throughout growth.

RapI was also over-produced in the ICEBs1-cured cells to control for effects of RapI. Separate cultures were grown in LB, and IPTG (final concentration 1 mM) was added to stimulate rapI expression 1 hour prior to mixing the strains. Cultures were diluted into fresh LB (with 1 mM

IPTG) and mixed such that the strains were present at a 1:1 ratio and an initial total OD600 of

0.002. Samples of the culture were selectively plated at the indicated times. Cells containing

ICEBs1 entered stationary phase earlier than the ICEBs1-cured cells by about 60 minutes (~3 doublings) (Fig. 2). The density of the ICEBs1-containing cells plateaued at ~1 x 108 CFU/ml, while the density of ICEBs1-cured cells eventually reached ~1 x 109 CFU/ml. There was no

98 obvious defect in growth during exponential phase, but these growth curves were not sensitive enough to measure growth rate. This suggests that constitutive induction of ICEBs1 during stationary phase is particularly detrimental to host cells.

ICEBs1 replication and conjugation genes contribute to cost

We found that preventing excision and subsequent replication of ICEBs1 as well as removal of the conjugation genes both alleviated the growth and stationary phase defects of ICEBs1- containing cells. We used a plate reader to monitor growth of cells containing variants of

ICEBs1. All strains contained the IPTG-inducible Pspank(hy)-rapI allele to stimulate ICEBs1 induction. Cultures of each strain were grown in S750 minimal medium (1% w/v glucose, 0.1% w/v monopotassium glutamate), and IPTG (1 mM final concentration) was added to induce rapI for two hours prior to starting growth curves. Cultures were diluted into fresh minimal medium

(with 1 mM IPTG) to an OD of 0.01 and 200 µl volumes were added to the wells of a 96-well plate (10 replicate wells per strain). Growth curves were performed at 37ºC with shaking at 1200 rpm, and the OD600 was read every 4 minutes. Cells containing an un-inducible ICEBs1 (∆Pxis) grew essentially identically to ICEBs1-cured cells (not shown) and were used as a control in this experiment. Cells containing wild-type ICEBs1 exited exponential growth early and plateaued at a lower density in stationary phase (Fig. 3A). A version of ICEBs1 unable to excise and replicate

(locked-in) grew essentially identical to the ∆Pxis strain (Fig. 3B). This suggests that replication of ICEBs1 is a major contributor to the growth and stationary phase defects. This could be due to use of host resources during replication or the increase in overall ICEBs1 gene expression due to increased copy number. Cells containing ICEBs1 without any of its conjugation genes grew essentially identically to the ∆Pxis strain, but plateaued at an intermediate density between the

99 wild-type and ∆Pxis variants (Fig. 3C). This suggests that expression of the conjugation genes and/or assembly of conjugation machinery is detrimental to host cells.

Conclusions

These experiments, which show that ICEBs1 is detrimental to host cells, were performed prior to recognizing the fitness benefit provided by ICEBs1 by inhibiting development. The conditions used here (rich medium or minimal medium with high glucose) inhibit development, and it is unlikely the developmental inhibition played a role in the fitness cost due to ICEBs1. In these conditions, cultures remain in stationary phase for a prolonged period of time without the option of sporulation. When ICEBs1 is induced constantly (naturally by cell-cell signaling or by rapI over-expression), ICEBs1 replication and conjugation gene expression are detrimental to host cells. The cost may be greater in stationary phase than during growth because increasingly limited cellular resources are being diverted to ICEBs1. Additionally, conjugation requires a cell- wall degrading , CwlT, which may partially degrade donor cell wall to allow assembly of the conjugation machinery on the cell surface. Cells in stationary phase may have a limited capacity to repair and maintain the cell wall.

100 A. Initial frequency 0.1 Initial frequency 0.5

107 107 ICEBs1 106 106 ICEBs10 105 105 104 104 3 3 CFU/ml CFU/ml 10 10 102 102 101 101 0 20 40 60 0 20 40 60 Generations Generations

B. Initial frequency 0.1 Initial frequency 0.5

107 107 ICEBs1(∆rapI-phrI) 106 106 ICEBs10 105 105 104 104

CFU/ml 3 3 10 CFU/ml 10 102 102 101 101 0 20 40 60 0 20 40 60 Generations Generations

Figure 1. ICEBs1 induction incurs a frequency-dependent fitness cost to host cells. A) ICEBs1-containing cells (JMJ89, black circles) decrease in abundance in a frequency- dependent manner when co-cultured with ICEBs1-cured cells (JMJ63, open squares). B) Deletion of the cell-cell signaling genes rapI-phrI (JMJ86, black circles) alleviates the cost to ICEBs1-containing cells.

101

10 10 locked-in ICEBs1 109 ICEBs10 108 107

CFU/ml 106 105 104 0 100 200 300 400 Time (minutes)

Figure 2. ICEBs1 induction is detrimental during stationary phase. In a mixed culture, cells containing locked-in ICEBs1 (JMJ211, black circles) enter stationary phase earlier and plateau at a lower density than cells without ICEBs1 (JMJ184, open squares).

102

A.

B.

C.

Figure 3. ICEBs1 replication and conjugation gene expression contribute to growth and stationary phase defects. A) Induction of wild-type ICEBs1 (JMJ192, blue lines) by over- expression of rapI causes pre-mature slowing of growth and plateau at a lower density compared to an un-inducible version of ICEBs1 (∆Pxis, JMJ216, green lines). B) Cells with locked-in ICEBs1 (JMJ211, magenta lines) have no growth or stationary phase defects. C) Deletion of the conjugation genes (JMJ185, black lines) relieves the growth inhibition and partially relieves the stationary phase defect. The growth curves of each of the ten replicate wells are shown as individual lines.

103 Table 1. B. subtilis strains used

Strain Relevant genotype JMJ63 ∆comC::cat JMJ86 ∆comC::mls ICEBs1 ∆rapI-phrI::kan JMJ89 ∆comC::mls ICEBs1 yddM-spec-attR (non-disruptive insertion) JMJ184 amyE::{Pspank(hy)-rapI cat} JMJ185 amyE::{Pspank(hy)-rapI spec} ICEBs1 ∆conQ ∆conB-cwlT ∆rapI-phrI::kan JMJ192 amyE::{Pspank(hy)-rapI spec} ICEBs1 ∆rapI-phrI::kan JMJ211 amyE::{Pspank(hy)-rapI spec} ICEBs1 oriT* ∆rapI-phrI::kan ∆attR::mls JMJ216 amyE::{Pspank(hy)-rapI spec} ICEBs1 ∆Pxis ∆rapI-phrI::kan

All strains are derived from PY79, which naturally lacks ICEBs1 unless otherwise indicated.

104 Chapter 3

Conclusions and Perspectives

105 Mobile genetic elements (MGEs) are important agents of horizontal gene transfer in bacteria

(Frost et al., 2005). They also have profound effects on the physiology and fitness of the cells that contain them, largely due to cargo genes they carry. Prior to widespread sequencing of bacterial genomes, mobile genetic elements and their associated cargo genes were typically discovered because of phenotypes conferred by cargo genes, notably antibiotic resistance. Now, numerous putative MGEs, especially ICEs, have been identified based on sequence similarity to known MGEs and many do not contain cargo genes with obvious functions (Cury et al., 2017;

Guglielmini et al., 2011).

ICEBs1 from B. subtilis was discovered both bioinformatically by similarity to another ICE

(Burrus et al., 2002) and by studying its regulatory genes (Auchtung et al., 2005). Despite being one of the most well-studied ICEs, no cargo genes within ICEBs1 were known until recently.

ICEBs1 benefits or protects its host cells in at least three ways. Exclusion, mediated by yddJ, protects the host from lethal excessive conjugation (Avello et al., 2019). Abortive infection, mediated by spbK (yddK) protects populations of ICEBs1-containing cells from a bacteriophage,

SPβ (Johnson et al., 2020, manuscript in preparation). In this work, I characterized a third cargo gene that provides a selective advantage to ICEBs1 host cells by modulating development. When

ICEBs1-containing cells are the minority in a dense population, the conjugative life cycle is induced. We found that when ICEBs1 is induced during growth in a biofilm or conditions that promote sporulation, ICEBs1-containing cells delayed sporulation and grew more on average than cells without ICEBs1. We found that one ICEBs1 gene, ydcO, was necessary and sufficient to inhibit both biofilm and sporulation gene expression, likely by targeting the master

106 developmental regulator Spo0A. We suspect that other cargo genes in MGEs alter aspects of host physiology, such as development, rather than providing entirely new traits.

Mechanism of developmental inhibition

We found that ydcO inhibited early sporulation and biofilm-associated gene expression, suggesting that Spo0A or the phosphorelay was the target of YdcO. We ruled out the phosphorelay as a target, as YdcO still inhibited sporulation when the phosphorelay was bypassed with a constitutively active form of Spo0A. We propose that YdcO directly targets

Spo0A~P, preventing it from regulating expression of target genes. Spo0A is activated by phosphorylation, which stimulates Spo0A dimerization and DNA binding (Lewis et al., 2002).

Spo0A~P is de-activated by multiple phosphatases (Spo0E, YisI and YnzD) (Ohlsen et al., 1994;

Perego, 2001). Since YdcO could still inhibit sporulation when we used a form of Spo0A that did not depend on phosphorylation for its activity, YdcO most likely targets Spo0A dimerization,

DNA binding, or interaction with RNA polymerase. In addition to inactivation by phosphatases,

Spo0A~P activity is also regulated by inhibition of its association with RNA polymerase. A complex of MecA and ClpC bind to Spo0A, inhibiting its effects on Spo0A-activated genes but not Spo0A-repressed genes (Prepiak et al., 2011; Tanner et al., 2018). This suggested that Spo0A was still able to bind to DNA in order to repress gene expression but could not interact with RNA polymerase to stimulate expression (Tanner et al., 2018). We showed that ydcO affected expression of Spo0A-activated genes; spoIIA, spoIIE, spoIIG are directly activated by Spo0A, and biofilm gene expression is stimulated indirectly by Spo0A activation of sinI expression

(Kearns et al., 2004; Sonenshein, 2000). We also found that ydcO affected expression of abrB,

107 which is repressed by Spo0A (Perego et al., 1988). This suggests that YdcO likely prevents DNA binding by Spo0A.

Regulation of development by cargo genes

Bacteria use developmental transitions to adapt to changes in their environment. Biofilm formation is a near universal behavior in bacteria. Sporulation is critical for survival and the lifestyle of many bacteria, including important pathogens. We suspect that altering the regulation of development is a common strategy employed by mobile genetic elements to benefit host cells.

Multiple MGEs in Bacillus anthracis contribute to the regulation of biofilm production and sporulation in ways thought to benefit the host during infection of and during growth in the soil (Dale et al., 2018; Schuch and Fischetti, 2009; White et al., 2006). Our work demonstrated that a MGE provides an advantage by enabling the host to exploit other cells during development. Development also has implications for the cell-to-cell spread of MGEs, which may be another reason for targeting development with cargo genes. Biofilm formation promotes spread of MGEs but also creates limitations for transfer in some cases (Madsen et al.,

2012; Stalder and Top, 2016). MGEs that promote or inhibit development might do so to increase their transfer.

Impacts of mobile genetic elements on their hosts

Mobile genetic elements have inherent costs, as they rely on host cells for their vertical inheritance and horizontal spread between cells (Baltrus, 2013). MGEs can also carry cargo genes that provide a fitness benefit to the host under some conditions. Many MGEs, especially

ICEs, have been discovered bioinformatically and do not contain cargo genes with obvious functions (Cury et al., 2017; Guglielmini et al., 2011). Genes of unknown function or without an

108 annotated function account for ~60% of all ICE genes (Cury et al., 2017). This is a large reservoir of potential cargo genes with novel functions. In the case of ICEBs1, we were only able to ascribe a function to an unknown gene, ydcO, by performing competitions of ICEBs1- containing and ICEBs1-cured cells under biofilm- and sporulation-stimulating conditions.

Interestingly, in other conditions that did not promote development, we found that induction of

ICEBs1 imposed a significant cost to cells. This highlights that cargo gene function and fitness of host cells is highly dependent on growth conditions. In another study, a lysogenic phage was found to provide a selective advantage during growth in medium derived from the host’s natural environment, but not in standard laboratory medium (Tree et al., 2014). Many MGEs without obvious cargo genes may in fact provide benefits in the right conditions. Characterizing benefits provided by MGEs to host cells is likely to reveal new roles for cargo genes and new types of interactions between MGEs and their host cells.

109 References

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