Sortase Enzymes and Their Integral Role in the Development of Streptomyces Coelicolor

Sortase enzymes and their integral role in the development of Streptomyces coelicolor

Andrew Duong

A Thesis Submitted to the School of Graduate Studies In Partial Fulfillment of the Requirements of the Degree of Master of Science

McMaster University Copyright by Andrew Duong, December, 2014

Master of Science (2014) McMaster University (Biology) Hamilton, Ontario

TITLE: Sortase enzymes and their integral role in the development of Streptomyces coelicolor

AUTHOR: Andrew Duong, B.Sc. (H) (McMaster University)

SUPERVISOR: Dr. Marie A. Elliot

NUMBER OF PAGES: VII, 77

Abstract

Sortase enzymes are cell wall-associated transpeptidases that facilitate the attachment of proteins to the peptidoglycan. Exclusive to Gram positive bacteria, sortase enzymes contribute to many processes, including virulence and pilus attachment, but their role in Streptomyces coelicolor biology remained elusive. Previous work suggested that the sortases anchored a subset of a group of hydrophobic proteins known as the long chaplins. The chaplins are important in aerial hyphae development, where they are secreted from the cells and coat the emerging aerial hyphae to reduce the surface tension at the air-aqueous interface. Two sortases (SrtE1 and SrtE2) were predicted to anchor these long chaplins to the cell wall of S. coelicolor. Deletion of both sortases or long chaplins revealed that although the long chaplins were dispensable for wild type-like aerial hyphae formation, the sortase mutant had a severe defect in growth. These two sortases were found to be nearly redundant, as deletion of individual enzymes led to only a modest change in phenotype. In vitro analysis of sortase cleavage activity showed that both sortases recognized the unique LAXTG pentapeptide sequence found in the long chaplins, and 11 other putative substrate proteins. Transcriptional analysis revealed that a number of genes typically expressed during aerial hyphae development were not expressed in a sortase deletion mutant. This suggests that the sortases have a role in transcriptional regulation, a phenomenon that has not been described previously. Current work is focused on addressing the mechanism(s) by which sortases affect transcriptional regulation, with a specific focus on the role of the proteins that they anchor to the cell wall (sortase substrates) in aerial growth.

Acknowledgements

First, and foremost, I’d like to thank Dr. Marie A. Elliot for all of her efforts in supporting my academic career. She’s been a supportive and kind mentor that has encouraged me to perform my best and is someone who I greatly admire for her thirst for knowledge and compassion for others.

I’d like to thank my committee members, Dr. Turlough Finan and Dr. Lori Burrows for their honest and helpful feedback for these past few years. The input that I’ve received has helped both my research and my evolution as a critical person. I’d also like to thank Dr. Alba Guarné for introducing me to the bench, which sparked my interests in primary research. I would also like to extend special thanks for Dr. Juliet Daniel for her continuous support and guidance. These professors have taught me that mentorship is too often an ignored, but invaluable component of life; of which, the successes of millions are dependent.

I have been lucky to work with some fantastic people during my time in the lab. All have had an impact, but in particular, I’d like to thank: Dr. David Capstick for his friendship and support, while being an encyclopedia of science and pop culture; Dr. Chan Gao for being that weird, cat-loving, big sister I never had; Dr. Henry Haiser for the intellectual conversations about science in pubs; Dr. Julia Swiercz for being my annoying ‘lab little sister’ with a huge heart; Dr. Hindra and Dr. Mary Yousef for being two of the nicest people I have ever worked with, and for teaching me the value of family; Dr. Emma Sherwood for her optimism and wonderful lab quirks that I never get tired of discovering; Matthew Moody for never being afraid to share his weirdness and whose integrity I greatly admire; Danielle Sexton for being one of the most caring people I’ve ever met, and despite that, also being my lab twin (CATCATCAT); Rachel Young for being so kind, brilliant, selfless, and badass at the same time.

Beyond school, I’d like to thank my family, and especially my sister Anita, for reminding me of life beyond work. I also want to thank the people who have become like family over these past few years: Catherine Nguyen for being so wonderful, uncool, and generous with her kindness. Her friendship is one that I will always cherish for the impact she has had on me; Nolan D’Souza for being a confidant, a brother, and whose tenacity is something of legends; and to Sanjan George for reminding me to remember the important things in life. I’d also like to extend my gratitude towards the Kwan family for their kindness these last few years.

Special thanks goes to Michelle J. Kwan, who has contributed so much of the positivity in my life, and without whom, I would not have made it to this point. Her patience, compassion, and encouragement have done so much in providing everlasting support through this journey.

Table of Contents Abstract ...... I Acknowledgements ...... II List of Figures ...... V List of Tables ...... VI List of Abbreviations ...... VII Chapter 1: Introduction ...... 1 1.1 The Actinomycetes ...... 1 1.2 Streptomyces coelicolor ...... 1 1.3 The developmental life cycle of S. coelicolor ...... 2 1.4 Regulation of development ...... 2 1.4.1 Vegetative growth ...... 3 1.4.2 Aerial hyphae formation ...... 3 1.4.3 Sporulation ...... 3 1.5 Surfactants in development ...... 4 1.5.1 SapB ...... 4 1.5.2 Chaplins ...... 5 1.6 Sortases ...... 6 1.6.1 Sortase A ...... 7 1.6.2 Sortase B ...... 7 1.6.3 Sortase C ...... 8 1.6.4 Sortase D ...... 9 1.6.5 Sortases in the actinomycetes: SrtE and SrtF ...... 9 1.7 Sortases and the chaplins ...... 9 1.8 Aims of this thesis ...... 10 1.9 Figures ...... 11

Chapter 2: Materials and methods ...... 14 2.1 Bacterial strains and plasmids ...... 14 2.2 Bacterial culturing ...... 14 2.3 Bacterial and molecular genetic techniques ...... 15 2.3.1 Plasmid isolation from Escherichia coli ...... 15 2.3.2 Agarose gel electrophoresis ...... 15 2.3.3 PCR amplification ...... 15 2.3.4 Purification of PCR products ...... 16 2.3.5 Purification of DNA fragments from agarose gels ...... 16 2.3.6 Restriction digest of DNA ...... 16 2.3.7 Phosphorylation and dephosphorylation of DNA ...... 16 2.3.8 DNA ligation ...... 16 2.3.9 Chemical transformation of DNA into E. coli ...... 17 2.3.10 Electroporation of DNA into E. coli ...... 17 2.3.11 Conjugation of plasmids and cosmids from E. coli into S. coelicolor ...... 18 2.3.12 Gene deletion by PCR-targeting mutagenesis ...... 18 2.3.13 Isolation of genomic DNA from S. coelicolor ...... 20 2.3.14 Isolation of RNA from S. coelicolor ...... 20 2.3.15 Reverse transcription PCR ...... 20

III

2.4 Biochemical Techniques ...... 21 2.4.1 Purification of His-tagged proteins ...... 21 2.4.2 Sortase cleavage assay ...... 22 2.4.3 Mass spectrometry analysis of cleavage products ...... 22 2.5 Tables ...... 23

Chapter 3: Sortases in S. coelicolor development ...... 33 3.1 Introduction ...... 33 3.2 Results ...... 34 3.2.1 Bioinformatic prediction of sortases and their substrates ...... 34 3.2.2 Deletion of the sortases leads to a defect in development ...... 34 3.2.3 Scanning electron microscopy analysis of sortase mutants ...... 35 3.2.4 Redundancy of srtE1 and srtE2 in morphological development ...... 35 3.2.4 Role of the sortase-dependent long chaplins in spore maturation ...... 36 3.2.5 Expression of the sortase genes ...... 36 3.2.6 In vitro analysis of sortase cleavage analysis ...... 37 3.2.7 Deletion of sortases changes in expression of aerial hyphae-specific genes 38 3.3 Discussion ...... 39 3.3.1 SrtE1 and SrtE2 recognize a unique substrate motif ...... 39 3.3.2 Housekeeping sortases modulate development ...... 41 3.4 Tables ...... 43 3.5 Figures ...... 45

Chapter 4: Probing the role of sortase substrates in aerial development ...... 54 4.1 Introduction ...... 54 4.2 Results and Discussion ...... 54 4.2.1 Creation of sortase substrates mutants ...... 54 4.2.2 Transcriptional analysis of sortase substrate genes ...... 55 4.2.3 Accumulation of proteins in the cell membrane of a sortase deletion mutant 56 4.3 Future directions ...... 57 4.3.1 Genetic analysis of the roles of sortase substrates in development ...... 57 4.3.1.1 Hypothesis 1: Sortase substrates are integral to aerial development 557 4.3.1.2 Hypothesis 2 Induced accumulation of sortase substrates delays development ...... 58 4.3.3 Identification of the regulatory networks through transcriptional analysis ... 59 4.4 Towards a comprehensive model for the role of sortase substrates in ...... 59 morphological development 4.5 Figures ...... 61

Chapter 5 Conclusions and Future directions ...... 63 5.1 Summary of Research ...... 63 5.2 A new working model of aerial development facilitated by the sortases ..... 63 5.3 Long term goals ...... 64

Appendix ……………………………………………………………………………………….67 6.1 Analysis of the covalently attached cell wall proteome ………………………67 6.1.2 Identification of sortase substrates ………………………………………….. 67 6.1.3 Determining substrate specificity of the sortases in vivo ………………..... 68 Works Cited ...... 69 IV

List of Figures

Chapter 1 Figure 1.1 Developmental life cycle of Streptomyces coelicolor Figure 1.2 Scanning electron micrograph of an S. coelicolor spore Figure 1.3 The protein domain architecture of the long chaplins and predicted model of sortase-dependent long chaplin anchoring Chapter 3 Figure 3.1 Plate phenotype of sortase mutants in S. coelicolor Figure 3.2 Scanning electron micrographs of wild type and ∆srtE1/E2 mutants Figure 3.3 Plate phenotype of sortase, chaplin and long chaplin mutants. Figure 3.4 A – Scanning electron micrograph of wild type and ∆chpABC spore B – Distribution of rodlet length in wild type and ∆chpABC strains Figure 3.5 A – Genetic organization of housekeeping sortases and upstream genes in S. coelicolor B – Semi-quantitative RT PCR analysis of sortase gene expression Figure 3.6 In vitro sortase cleavage assay showing cleavage of ChpC and ChpB peptides Figure 3.7 LC/MS analysis of the cleavage products in the sortase in vitro cleavage assay Figure 3.8 Semi-quantitative RT PCR analysis of genes expressed during aerial growth in wild type, long chaplin (∆chpABC) and a double sortase mutant (∆srtE1/E2) Figure 3.9 Alignment of SrtE1, SrtE2 and SrtA

Chapter 4 Figure 4.1 Plate phenotype of sortase substrate deletion mutants Figure 4.2 Semi-quantitative RT PCR results of sortase substrate expression

List of Tables

Chapter 2 Table 2.1 Streptomyces strains Table 2.2 Escherichia coli strains Table 2.3 Plasmids and cosmids Table 2.4 Oligonucleotides Table 2.5 Taq polymerase (GeneDirex) PCR reaction protocol Table 2.6 Standard PCR cycling conditions

Chapter 3 Table 3.1 List of predicted sortases in S. coelicolor Table 3.2 List of predicted sortase substrates

List of Abbreviations

aa Amino acid Ala, A Alanine bp Base pair C Celsius C Carboxy CWSS Cell wall sorting signal Cys Cysteine dH2O Distilled water DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate DTT Dithiothreitol Glu, E Glutamic acid EDTA Ethylenediaminetetracetic acid Fwd Forward FRT Flippase recognition target g Grams Gly, G Glycine His, H Histidine IPTG Isopropyl β-D-1-thiogalactopyranoside l Litre LB Luria Bertani M Molar MALDI ToF Matrix-assisted laser desorption/ionization time of flight mg Milligram Min Minute ml Millilitre mM Millimolar N Amino nm Nanometer nt Nucleotide OD Optical density PAGE Polyacrylimide gel electrophoresis PCR Polymerase chain reaction Rev Reverse rpm Revolutions per minute s Seconds SDS Sodium dodecyl sulfate SEM Scanning electron microscopy SOB Super optimal broth TE Tris EDTA Thr, T Threonine Tat Twin-arginine transport TBE Tris Boric Acid EDTA buffer tipA Thiostrepton-inducible promoter Tris Tris(hydroxymethyl)aminomethane TSB Tryptone soya broth VII

U Units X-gal 5-bromo-4-chloro-3-indoyl-D-galactopyranoside YEME Yeast extract-malt extract YT Yeast tryptone broth µg Microgram µl Microliter

VIII

Chapter 1: Introduction

Preface: The names of sortases have been modified to reflect the annotation assigned by Dramsi et al. (2005). Historically, sortases were assigned letters in the chronological order they were identified, but due to the vast number of sortases discovered in the past 10 years, sortases with similar characteristics or sequence similarity have been grouped for consistency.

1.1 The Actinomycetes Gram positive bacteria are subdivided into low GC content (firmicutes) and high GC content (the actinobacteria) bacteria (Goodfellow and Williams, 1983). The actinobacteria have diverse ecological niches and morphology. Pathogenic members of this phylum contribute to human disease, and include Mycobacterium (tuberculosis and leprosy) and Corynebacterium (diphtheria) (Burkovski, 2013). However, the actinobacteria are also beneficial to human health. The secondary metabolites produced by many of these organisms likely have environmental/ecological significance, and humans have co-opted a large number of these molecules for use as antibiotics to treat bacterial infections, amongst other ailments (Waksman et al., 2010). Of these, the streptomycetes are considered to be one of the largest reservoirs of secondary metabolites having medical benefit (de Lima Procópio et al., 2012; Liu et al., 2013).

1.2 Streptomyces coelicolor The streptomycetes are one of the most studied actinomycetes, and are the source of over half of all naturally-derived antibiotics (Bentley et al, 2002). Many members of the streptomycetes encode more than 20 secondary metabolite biosynthetic gene clusters, but the associated products have not been detected in many instances (Medema et al., 2011; Zhong et al., 2013). One of the first characterized streptomycetes was Streptomyces coelicolor. Initially selected as a model organism for its production of a blue secondary metabolite, this species has revealed a wealth of information not only regarding antibiotics, but also the complexities of multicellular development in bacteria (Hopwood, 1999). The genome of S. coelicolor is considered to be large for a bacterium, at 8.6 Mbp, with a high GC content (72%) (Bentley et al., 2002). This large genome encodes approximately 7,800

protein-coding genes, nearly double that of the pathogenic actinomycete, Mycobacterium tuberculosis (Cole et al., 1998; Bentley et al., 2002). Given the large number of genes encoded by S. coelicolor, precise control of their expression is no trivial task. S. coelicolor devotes over 12 percent of its genome to regulation (Bentley et al., 2002). Not only is production of antibiotics tightly regulated, but S. coelicolor also has a complex multi-stage life cycle that requires specific regulation.

1.3 The developmental life cycle of S. coelicolor The incredibly large genome of S. coelicolor is justified when you compare the development of S. coelicolor to most other bacteria, which typically reproduce through binary division (Blattner et al., 1997). S. coelicolor exhibits a multicellular life cycle with three distinct stages. Beginning with germination of a spore, germ tubes emerge and grow via hyphal tip extension to form a vegetative mycelium. Aerial hyphae then extend from the vegetative mycelium, growing out of the culture medium. This formation of aerial hyphae often coincides with the production of secondary metabolites. The aerial hyphae go on to develop into spore chains, and mature into dormant exospores (Figure 1.1) (Flärdh and Buttner, 2009).

1.4 Regulation of development Progression through the life cycle occurs through a series of many smaller, complex processes. Many of the components required for germination, vegetative and aerial growth, and sporulation are life stage-specific, requiring extensive control of associated gene expression. S. coelicolor has two broad classes of genes that are important for the progression from one stage to the next. These are the bld and whi genes. Interestingly, the different stages of development are phenotypically distinct, and can be observed with the naked eye. Disruption of genes that are involved in the progression between different life stages leads to a distinctive vegetative (bald or bld) or aerial (white or whi) phenotype. The bald colonies appear slightly translucent, whereas the colonies arrested at the aerial hyphae stage have a white and ‘fluffy’ appearance. Notably, sporulating colonies have a characteristic grey colour, courtesy of a polyketide spore pigment (Elliot et al., 2007).

1.4.1 Vegetative growth S. coelicolor growth occurs through hyphal tip extension. This growth model is in contrast to that of many rod-shaped bacteria where new cell wall material is incorporated into the lateral cell wall (Carballido-López and Errington, 2003). Instead, S. coelicolor growth occurs at the poles, and depends on the polar tip localization of DivIVA, which is proposed to recruit the cell wall biosynthetic machinery needed for cell growth (Flärdh, 2003a; Flärdh, 2003b). The assembly of DivIVA foci on lateral walls of the growing mycelia results in the growth of branches (Hempel et al., 2008), and ultimately this gives rise to the unique branching phenotype of vegetative mycelia.

1.4.2 Aerial hyphae formation The erection of aerial hyphae from the vegetative mycelium coincides with the production of secondary metabolites (Nguyen et al., 2002). It is currently hypothesized that the initiation of aerial hyphae development (and onset of secondary metabolism) is triggered by a lack of nutrients, as aerial hyphae develop into the major reproductive structures (spores) of these organisms (Flärdh and Buttner, 2009). The activity of the bld genes is required for aerial hyphae formation; many bld gene products also contribute to antibiotic production. There are 12 annotated bld genes, (Merrick, 1976; Elliot et al., 2007; Chater et al., 2010, Pope et al., 1997; Den Hengst et al., 2010), and most of these encode regulatory proteins.

1.4.3 Sporulation The aerial hyphae are initially multigenomic, before septation divides them into unigenomic compartments that mature into dormant spores, thus completing the life cycle. The final stage in sporulation involves the deposition of a grey polyketide into the spore coat. This pigment is encoded by the whiE gene cluster (Yu and Hopwood, 1995; Ryding et al., 1999). Beyond whiE, 11 additional whi genes have been identified, and these have been categorized as either early or late sporulation genes. The ‘early’ sporulation genes include whiA, B, G, H, I, and J, and these are collectively responsible for chromosome segregation and septation. Late sporulation genes (including whiE and whiD) are involved in the maturation of the spores, including spore wall thickening (Flärdh et al., 1999). 3

Sporulation is not required for the growth of Streptomyces in the lab. The vegetative mycelia grow through hyphal tip extension, and although cross walls occasionally separate segments of the mycelia, classical cell division does not occur. Consequently, proteins such as FtsZ are not essential for growth, but are required for sporulation (McCormick et al., 1994; Wasserstrom et al., 2013). ftsZ expression is upregulated in the aerial hyphae, where the protein then polymerizes, initially in a helical pattern in the prespore chain, before collapsing into multiple rings within the spore chain, delineating the sites of septation (Grantcharova et al., 2005; Loose and Mitchison, 2013).

1.5 Surfactants in development During aerial development and sporulation on an agar plate, the aerial hyphae have to extend from the aqueous culture medium, into the air. At this liquid-air interface, surface tension provides a barrier to aerial growth, and this must be bypassed in order to facilitate upward growth (Willey et al., 1991). Surfactant molecules coat the developing bacteria, and aid in crossing the liquid-air interface by reducing surface tension. Both bacteria and fungi secrete surfactants to reduce the surface tension, allowing these organisms to extend out of the liquid interface (Willey et al., 1991). S. coelicolor uses the chaplins and SapB to perform this function (Tillotson et al., 1998; Claessen et al., 2003; Elliot et al., 2003).

1.5.1 SapB SapB is a secreted lantibiotic produced by the ram (rapid aerial mycelicum) gene cluster (Gaskell et al., 2012). Lantibiotics are ribosomally encoded molecules that undergo post translation modification to create a characteristic lanthionine bridge (Kodani et al., 2004). SapB is secreted and coats the emerging aerial hyphae during growth on glucose-containing media. The SapB propeptide is encoded by ramS. ramC encodes a lantibiotic synthetase, whose expression (and that of the entire ramCSAB gene cluster) is activated by the orphaned response regulator, RamR (Gaskell et al., 2012). RamC dehydrates the serine and threonine residues in RamS, allowing for cyclization with specific cysteine residues to create lanthionine rings (Kodani et al., 2004; Willey and Gaskell, 2011). The molecule is secreted by two transport proteins, RamA and RamB. Interestingly, exogenous addition of SapB to bld mutants can restore aerial

growth to these mutants (Willey et al., 1993). As SapB is not produced on media lacking glucose, reduction of surface tension is facilitated by the chaplins instead.

1.5.2 Chaplins Unlike SapB, the chaplins are expressed during growth on both rich and minimal media (Capstick et al., 2007). bldN encodes a sigma factor that is expressed during late vegetative growth and is responsible for directing the expression of the chaplins, in association with an as yet unknown transcription factor(s) (Elliot et al., 2003; Bibb et al., 2012). There are eight chaplin proteins produced by S. coelicolor, and these all share a well-conserved “chaplin domain”. The chaplin domain comprises ~50 amino acids, and is highly hydrophobic: approximately 50-60% of the residues in the chaplin domain are hydrophobic (Elliot et al., 2003). The chaplins can be subdivided into two groups based on the number of chaplin domains present. ChpABC are termed the ‘long’ chaplins, containing two chaplin domains, whereas ChpDEFGH are considered to be the ‘short’ chaplins, containing a single chaplin domain. All eight chaplin proteins further possess an N-terminal secretion signal that is cleaved following secretion,(Elliot et al., 2003). The chaplins share some functional redundancy, as not all chaplins are required for the development of aerial hyphae, and different Streptomyces species encode different chaplin complements (although all encode chpC, chpE and chpH) (Di Berardo et al., 2008). To facilitate functional analyses, a ‘minimal chaplin strain’ was created, that included chpCEH, encoding the long chaplin ChpC, the short chaplin ChpH, and the conditionally essential short chaplin ChpE. In the presence of other chaplin genes, attempts to delete chpE have been unsuccessful in the absence of additional suppressor mutations (Di Berardo et al., 2008). Unlike most of the other chaplins, ChpE (and one chaplin domain of ChpB) lacks the cysteine residue required for intramolecular disulphide bond formation (Elliot et al., 2003; Sawyer et al., 2011). The chaplins are secreted to the surface of the growing aerial hyphae and spores, where they polymerize into long fibres having amyloidogenic properties (Capstick et al., 2011). In conjunction with the rodlin proteins, the chaplins are organized into a paired rodlet ultrastructure that is visible on the spore surface using high resolution electron microscopy (Figure 1.1) (Di Berardo et al., 2008). The rodlins appear to assist in connecting chaplin fibres, but their deletion only affects fibre association on the surface, and seems to have no effect on development (Claessen et al., 2002). 5

The long chaplins are unique in that in addition to having two chaplin domains, their C-terminus also contains a sortase recognition motif.

1.6 Sortases Sortases are transpeptidase enzymes found exclusively within Gram positive bacteria (Comfort and Clubb, 2004). Sortases are the only enzymes known to covalently link proteins to the cell wall and therefore play an important role in determining the surface architecture of Gram positive bacteria (Hendrickx et al., 2011). Secretion, recognition, cleavage, and transpeptidation of the sortase substrates are all coordinated to ensure appropriate localization of these proteins. The sortase recognition motif, also known as the cell wall sorting signal (CWSS) is typically a ~35 residue long region at the C-terminus of secreted proteins. It contains a highly conserved pentapeptide sequence, followed by a stretch of hydrophobic residues, and a short positively charged tail (Schneewind et al., 1992; Dramsi et al., 2008). In Staphylococcus aureus, where sortases were first characterized, the pentapeptide sequence recognized and cleaved by the major housekeeping sortase SrtA, is LPXTG, where the third residue, X, is a variable amino acid (Schneewind et al., 1992). Following secretion of a substrate protein, sortases recognize this pentapeptide sequence, cleaving it between the threonine and glycine residues. Sortases are cysteine transpeptidases, and they therefore use an active site cysteine residue to perform a nucleophilic attack on the bond between the threonine and glycine residues, forming a thioester with the threonine residue (Ton-That et al., 1999). The sortase enzyme transfers the N-terminal product to the lipid II component of the cell wall, creating a covalent bond between lipid II and the substrate (Ton-That and Schneewind, 1999; Ton- That et al., 2002; Perry et al., 2002). In S. aureus, sortases anchor proteins to the pentaglycine bridge of the peptidoglycan (Zong, Mazmanian, et al., 2004; Zong, Bice, et al., 2004). Sortase-dependent anchoring to the crosswalls of the peptidoglycan requires newly formed or nascent peptidoglycan to serve as an anchoring site. This is typically found at the site of cell elongation and growth (e.g. cross wall or a hyphal tip) (Spirig et al., 2011). To date, the majority of characterized sortase substrates are targeted for secretion via the Sec secretion pathway, and are secreted from areas of new cell wall growth (Kline et al., 2009; Schneewind and Missiakas, 2013). 6

Sortase substrates are extremely diverse in their function, and a combination of substrate function and specific sorting signal (pentapeptide motif) define the sortase class. There are currently six categories of sortases (Sortase A-F), and these anchor substrates involved in sporulation, pilus assembly, iron acquisition, and general housekeeping functions of the cell.

1.6.1 Sortase A First described in 1992 by Schneewind et al, sortase A was identified as the enzyme responsible for anchoring protein A to the cell wall of S. aureus. Protein A is a virulence factor that inhibits recognition of the bacterial cell by host cells during infections (Navarre and Schneewind, 1994; Maresso and Schneewind, 2008). Since the initial identification of sortase A, it has been shown that SrtA anchors a number of different proteins (not all involved in pathogenesis) that share the same CWSS containing a conserved LPXTG pentapeptide motif (Ton-That et al., 1999; Ton-That et al., 2000; Zong, Bice, et al., 2004; Marraffini et al., 2004; Ton-That et al., 2004). SrtA is considered to be a housekeeping sortase, and is encoded independently of its substrates (Bolken et al., 2001; Weiss et al., 2004; Swaminathan et al., 2007). Housekeeping sortases are expressed constitutively throughout the life cycle of the bacterium, and contribute to the general surface architecture of many Gram positive bacteria. The cognate substrates of the housekeeping sortases are often located in a different genetic locus relative to their processing sortase (Comfort and Clubb, 2004). Identifying housekeeping sortase substrates in new genomes can be difficult, and relies on prior knowledge (or educated guesses) of the CWSS’s pentapeptide recognition sequence. Housekeeping sortases and their substrates have been best studied in S. aureus, but their role in the unique lifestyles of other firmicutes is expanding their functional repertoire. To date, housekeeping sortases have been shown to be involved in not only pathogenesis, but also pilin attachment, and biofilm formation (Budzik et al., 2008; Donahue et al., 2014; Shaik et al., 2014).

1.6.1 Sortase B Unlike housekeeping sortases, Class B sortases are not constitutively expressed (Mazmanian et al., 2003), and they are typically encoded from the same “iron-regulated surface determinant (isd)” locus as their substrates. The isd locus in S. aureus is

associated with soluble iron or heme acquisition. SrtB recognizes, and binds the heme- binding protein, IsdC, which facilitates the translocation of the heme ligand into the cell (Mazmanian et al., 2003). IsdC contains an alternative CWSS from the SrtA substrates. While still maintaining the general architecture of the CWSS (pentapeptide sequence - hydrophobic region - positively-charged tail), SrtB recognizes an NPQTN pentapeptide sequence instead (Mazmanian et al., 2003; Schneewind and Missiakas, 2012). The sortase- dependent anchoring of IsdC to the cell wall occurs via a mechanism analogous to that of SrtA (Marraffini and Schneewind, 2005). Interestingly, two other proteins involved in heme binding, IsdA and IsdB are constitutively present on the cell surface, and are anchored to the cell wall by SrtA (Mazmanian et al., 2003). During growth in iron-limiting environments, IsdC is secreted and anchored to the cell wall by SrtB. The anchored IsdC transfers heme from IsdAB to the membrane translocation factors IsdDEF for import into the cell (Mazmanian et al., 2003; Marraffini and Schneewind, 2005). Heme acquisition in iron rich environments is exploited by the SrtA-dependent anchoring of IsdAB, which is retained until environmental iron becomes scarce, at which point SrtB aids in importing heme into the cell.

1.6.2 Sortase C The third class of sortases (Sortase C) is involved in the anchoring of pilin proteins to the cell wall. Unlike in Gram negative bacteria, where pilin subunits are non- covalently associated with the inner or outer membrane, pilin subunits in Gram positive bacteria are anchored to the cell wall by sortases (Kline et al., 2010; Hendrickx et al., 2011). In Corynebacterium diptheriae, SpaA forms the majority of the pilin shaft. SpaA contains an LPLTG pentapeptide signal and is initially recognized by SrtC. The SpaA protein contains a lysine that functions as an amino acceptor for newly cleaved SpaA proteins, serving as an attachment site for the new substrate. This lysine acceptor acts much like the pentaglycine bridge in SrtA-catalyzed reactions, and allows for polymerization of SpaA subunits to form the pilus. The incorporation of another pilin protein, SpaB arrests pilus polymerization, and promotes the transfer and anchoring of the entire pilus to the cell wall by the housekeeping sortase, SrtA (Ton-That and Schneewind, 2004; Spirig et al., 2011). The assembly of multiple sortase substrate

subunits to form a complex has only been observed for class C sortases (Nobbs et al., 2008)

1.6.3 Sortase D Class D sortases also have an extremely specialized function: promoting sporulation. Class D sortases have been characterized in Bacillus anthracis, where two sortase D substrates have been described. BasH and BasI are expressed during endospore formation and both contain an LPNTA pentapeptide sequence in their C- terminus. Analogous to the SrtA situation, cleavage occurs between the threonine and alanine residues (Marraffini and Schneewind, 2006). SrtD is expressed prior to and during sporulation in both the mother cell and the forespore, while its substrates basI and basH are expressed solely in the envelope of the pre-divisional cell, and in the forespore, respectively (Marraffini and Schneewind, 2006; Marraffini and Schneewind, 2007). Bioinformatic analyses have shown that homologues of Class D sortases are highly conserved in many genomes within the firmicutes, alongside genes encoding similar SrtD substrates possessing LPNTA pentapeptide CWSS; although there is limited information about this class of protein and its substrates outside of the firmicutes (Aucher et al., 2011).

1.6.4 Sortases in the actinomycetes: SrtE and SrtF The sortases have been studied extensively in the low G+C bacteria. In high G+C bacteria, most research has been confined to Corynebacterium. The SrtE and SrtF designations were assigned to actinomycete-specific sortases not previously described. The class F designation for actinomycete-specific sortases has been reserved for sortases having unknown functions (Dramsi et al., 2005). SrtE enzymes are functionally analogous to the housekeeping sortase, SrtA, in the firmicutes, In S. coelicolor, these sortases are not predicted to be encoded adjacent to any of their cognate substrates, but in silico work by Elliot et al, (2003) predicted that the previously described long chaplins would be targets for these sortase enzymes. The long chaplins have a prototypical CWSS, only with an LAXTG pentapeptide sequence instead of the S. aureus LPXTG recognition motif (Elliot et al., 2003).

1.7 Sortases and the chaplins The presence of a C-terminal sorting signal provides a putative mechanism for chaplin fibre formation on the cell surface. Anchoring of the long chaplins on the surface of the aerial structures by a sortase enzyme may nucleate chaplin polymerization and ultimately promote fibre formation. As regular anchoring to the peptidoglycan may not necessarily be required for fibre formation, the short chaplins could exploit these long chaplin-mediated nucleation sites, in assembling into long amyloid fibres on the surface, which in turn aid in the formation of aerial structures.

1.8 Aims of this thesis The class E sortases recognize and cleave a cell wall sorting signal found within a number of proteins in S. coelicolor. When this work was initiated, the role of the sortases in S. coelicolor morphological development was still elusive. In chapter three, this work examines of the role of these sortases in morphological development using phenotypic analyses of sortase mutants, biochemical characterization of sortase activity, identification of substrate proteins in vivo, and determining the surface architecture of the spore. An outline of future work aimed at probing the function of the sortase substrates during development is described in chapter four.

1.9 Figures

Figure 1.1: Developmental life cycle of S. coelicolor. A free spore germinates, extending a germ tube, which grows and develops into the vegetative mycelium. Aerial hyphae are erected from the vegetative mycelia, growing upwards from the culture medium. The aerial hyphae develop into the spore chains, which mature and septate into mature, individual spores. Image modified from Swiercz and Elliot, 2012.

Figure 1.2: Scanning electron micrograph of the surface of an S. coelicolor spore. The surface is coated in paired fibres composed of chaplin and rodlin proteins. Image modified from Marie Elliot.

Figure 1.3: The protein domain architecture of the long chaplins and predicted model of sortase-dependent long chaplin anchoring. Following cleavage of the N- terminal secretion signal (not shown), the long chaplins (substrate) are secreted, and recognized at their C-terminus by a sortase enzyme. The C-termini of sortase substrates contain a pentapeptide sequence (LAXTG) followed by a string of hydrophobic residues, and a positively charged tail. Following recognition of the sortase cell wall sorting signal (CWSS), the pentapeptide sequence is cleaved, and the N-terminal fragment is covalently bound to the peptidoglycan. In the predicted model of sortase-dependent long chaplin anchoring, anchored long chaplin proteins facilitate short chaplin (individual purple oval without CWSS) polymerization to form the fibres that coat the surface of aerial hyphae and spores in S. coelicolor.

Chapter 2: Materials and Methods

2.1 Bacterial strains and plasmids All Streptomyces strains used in this work are listed in Table 2.1, and all Escherichia coli strains are listed in Table 2.2. Plasmids are described in Table 2.3. Streptomyces stocks were stored as spores when appropriate. To create spore stocks, a single colony was divided and streaked as lawns on three MS agar plates overlaid with cellophane discs and grown for five days. Spores were harvested by scraping the lawns with a flat edge of a metal spatula, and the resulting biomass was resuspended in 12.5 ml of distilled water in a glass universal vial. The samples were sonicated in a water bath to liberate individual spores. The spore suspension was passed through a 10 ml syringe containing a cotton filter to remove large debris and the filtrate was pelleted at 1248 x g for 5 minutes at 4°C. The supernatant was removed, and the spore pellet was resuspended in 1-2 ml of 40% glycerol and stored at -20°C. When mycelia stocks were appropriate, a single colony was divided and streaked as lawns on three MS agar plates overlaid with cellophane discs and grown for 5 days. Vegetative or aerial mycelia were harvested by scraping the lawns with a flat edge of a metal spatula, and the biomass was resuspended in 12.5 ml of distilled water. The samples were then transferred to a 15 ml Pyrex® homogenizer (Corning). The samples were homogenized and the homogenate was pelleted at 1248 x g for 5 mintues at 4°C. The supernatant was removed, and the pellet was resuspended in 2 ml of 40% glycerol and stored at -20°C. Stocks of E. coli cultures were made by mixing equal volumes of overnight cultures and 40% glycerol. E. coli stocks were stored at -80°C.

2.2 Bacterial culturing S. coelicolor strains were grown on MS agar, rich R5 agar, Difco nutrient agar, or in a 1:1 mixture of yeast extract–malt extract and tryptic soy broth (YEME-TSB) liquid medium (Kieser et al., 2000). Liquid cultures were grown in glass universal bottles containing a stainless steel spring, or baffled flasks at 30°C in a shaking incubator at 200 rpm. All E. coli strains were cultured in liquid or agar Luria Bertani medium (LB) (Miller, 1972). Strains that were grown in the presence of the salt sensitive antibiotic

hygromycin, were cultured in SOB medium or grown on Difco nutrient agar (Hanahan, 1983; Kieser et al., 2000). Antibiotics, when appropriate, were added to the media in the following final concentrations (µg/ml): ampicillin, 100; apramycin, 50; chloramphenicol, 50; hygromycin, 50; kanamycin, 50; and nalidixic acid, 25.

2.3 Bacterial and molecular genetic techniques 2.3.1 Plasmid isolation from E. coli Plasmid and cosmid DNA were isolated from E. coli cultures using previously described protocols (Sambrook and Russell, 2001). Briefly, up to 5 ml of E. coli cells were collected by centrifugation and resuspended in 100 µl of TEG solution (50 mM Tris, 20 mM EDTA, 1% glucose, pH 8). Cells were lysed by the addition of 250 µl of freshly prepared alkaline lysis buffer (0.1 M NaOH, 1% SDS), and incubated at room temperature for 5 min. The mixture was precipitated with the addition of 350 µl of precipitation buffer (3M NaOAc, pH 4.8). The insoluble sample was isolated by centrifugation, and the aqueous phase was transferred to a new tube for phenol:chloroform:isoamylalcohol (25:24:1) extraction. Two volumes of 95% ethanol were added to the resulting aqueous phase, and incubated on ice for 10 minutes. The DNA pellet was collected by centrifugation for 10 minutes, and was resuspended in 50 µl of TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0).

2.3.2 Agarose gel electrophoresis Agarose gels were prepared as a 1%-2% agarose solution in TBE buffer (89 mM Tris-HCl, 89 mM boric acid, 2 mM EDTA, pH 8.3) (Sambrook and Russell, 2001). Twenty-five micrograms ethidium bromide solution (10 mg/ml) (Bioshop) was added to each 50 ml solution prior to pouring the gel. One hundred base pair and 1 kb ladders (GeneDirex) were used to approximate DNA sizes.

2.3.3 PCR amplification Amplification of genes was performed using Phusion polymerase (New England Biolabs) according to manufacturer directions in 50 µl reaction volumes. Primers used for PCR amplification are listed in Table 2.4. Reaction conditions and thermal cycling parameters are summarized in Tables 2.5 and 2.6. Confirmation of genetic clones were 15

tested by PCR using Taq DNA polymerase (GeneDirex) in 25 µl volumes using the parameters outlined in Table 2.6.

2.3.4 Purification of PCR products Purification of PCR products involved a phenol:chloroform:isoamyl alcohol extraction previously described by Sambrook and Russell (2001). Following phenol:chloroform extraction and ethanol precipitation, samples were resuspended in 25 µl of water.

2.3.5 Purification of DNA fragments from agarose gels To isolate distinct DNA products following PCR amplification or DNA digestion, samples were purified using the PureLink Quick Gel extraction kit (Invitrogen). DNA bands were separated by size by agarose gel chromatography on a 1% agarose gel before excision of the desired bands. Samples were then purified according to the manufacturer’s instructions

2.3.6 Restriction digest of DNA DNA digestions were performed in 25 µl or 50 µl reaction volumes as per the manufacturer’s directions. The enzymes, EcoRI, SacI, NdeI, HindIII, SmaI, and EcoRV were purchased from Thermo Fisher Scientific, whereas, the enzymes, BamHI, XhoI, XbaI, KpnI, and BglII were purchased from New England Biolabs.

2.3.7 Phosphorylation and dephosphorylation of DNA Phosphorylation and dephosphorylation of DNA fragments were performed using T4 kinase and calf intestine alkaline phosphatase (Invitrogen) according to the manufacturer’s instructions.

2.3.8 DNA ligation Ligation of DNA fragments was performed as per the manufacturer’s instructions using T4 DNA ligase (Invitrogen). One unit of enzyme was added to each 20 µl reaction and incubated overnight at 16°C prior to transformation.

2.3.9 Chemical transformation of DNA into E. coli Preparation of competent E. coli and transformation into E. coli cells was conducted using the methods outlined in Sambrook and Russell (2001). Three hundred microlitres of an overnight E. coli culture were subcultured into 30 ml of fresh LB at a inoculum concentration of 1:100. Cultures were grown at 37°C in a shaking incubator at

200 rpm, until they reached a cell density (OD600) of 0.4-0.6. Cells were collected by centrifugation and resuspended in 20 ml of ice cold 25 mM MgCl2. Cells were then collected again, and washed twice with 15 ml of ice cold 20 mM CaCl2. Cells were then resuspended in 500 µl of CaCl2, and stored on ice for immediate transformation. For transformation of plasmid DNA, 2 – 4 µl of plasmid DNA were added to 100 µl aliquots of freshly prepared competent cells. Cell mixtures were incubated on ice for 60 minutes prior to being heat-shocked at 42°C in a water bath for 60 seconds, which was followed by a 60 second incubation on ice. One ml of LB broth was added to the tube, and the sample was incubated at 37°C in a shaking incubator at 200 rpm for 45 minutes. The entire mixture was then plated on LB agar with appropriate antibiotics for plasmid/cosmid selection, and grown overnight at 37°C.

2.3.10 Electroporation of DNA into E. coli Preparation of electrocompetent cells and electroporation of DNA into cells was performed using the methods outlined in Sambrook and Russell (2001). Briefly, 300 µl of overnight cultures were inoculated into 30 ml of fresh SOB media at an inoculum concentration of 1:100 and grown at 37°C at 200 rpm in a shaking incubator until they reached a cell density (OD600) of 0.4-0.6. Cells were collected by centrifugation, and washed three times with 20 ml of ice cold 10% glycerol (Hanahan, 1983). Cells were then collected by centrifugation, and the cell pellet was resuspended in 500 µl of ice cold 10% glycerol. Introduction of DNA into E. coli cells by electroporation began with the addition of 2 µl of plasmid DNA into 100 µl of freshly prepared electrocompetent cells. Following a 30 min incubation on ice, the cell-DNA mixture was transferred to an electroporation cuvette (2 mm, VWR). The cuvette was placed into a BioRad MicropulserTM, and subjected to a 2.5 kV pulse. Immediately following the electric pulse, 1 ml of LB was added to the cell mixture, and after which everything was transferred to a new tube. The electroporated cells were incubated at 37°C at 200 rpm for 60 minutes, before being 17

plated on LB agar with the appropriate antibiotics for plasmid/cosmid selection. Transformed cells were typically grown at 37°C overnight, apart from during cosmid preparation for gene deletion, as described below.

2.3.11 Conjugation of plasmids and cosmids from E. coli to S. coelicolor Conjugations were performed as previously described by Kieser et al. (2000). Plasmids and cosmids were conjugated from E. coli ET12567 containing plasmid pIJ790, to ensure that DNA was not methylated prior to conjugation, and to provide the necessary conjugation machinery, respectively. Overnight E. coli cultures were subcultured 1:100 into 20 ml of LB, and grown at 37°C in a shaking incubator until reaching an OD600 of 0.4. Cells were then pelleted and washed twice with 10 ml fresh LB medium. Cells were resuspended in 1 ml of LB medium. To germinate spores, 5 µl of a spore stock (106 spores/µl) were added to 500 µl of 2× YT medium, and germinated at 50°C for 10 min. Vegetative and aerial hyphae stocks were grown by adding 5 µl of a concentrated mycelial stock to 500 µl of 2× YT, and incubating at 30° for 3.5 h. Following incubation, the germinated spores or growing mycelia were mixed with the washed E. coli cells and plated on MS agar medium. These plates were then incubated at 30°C overnight , after which plates were overlayed with 25 µl of apramycin (50 mg/ml) and 20 µl nalidixic acid (25 mg/ml) in one ml of water. Plates were grown for an additional 4- 5 days at 30°C.

2.3.12 Gene deletion by PCR-targeting mutagenesis Generation of gene deletions in S. coelicolor was performed using the protocol outlined by Gust et al (2003). Genes were replaced using antibiotic resistance cassettes (apramycin-resistance) containing an oriT sequence and flanked by FRT (flippase recognition target) sites. S. coelicolor cosmids were used for construction of gene deletions. Genes to be deleted were replaced with apramycin-resistance gene and introduced into S. coelicolor (Redenbach et al., 1996). When an appropriate cosmid was not available, the genes selected for deletion were PCR amplified, along with 3-4 kb of flanking sequences, using Phusion polymerase (Thermo Fisher Scientific). These DNA fragments were then ligated into a modified Topo 2.1 vector (Invitrogen) having both ampicillin and kanamycin resistance genes.

Initially, the cosmids or ‘knockout plasmids’ were electroporated into electrocompetent BW25113/pIJ790 E. coli cells for further genetic manipulation. Following amplification of the apramycin resistance cassette using primers with 5' extensions containing 39 bp of gene-specific DNA flanking the sequence to be deleted, and PCR or gel-purification, the resulting PCR products were electroporated into electrocompetent cosmid/plasmid-containing BW25113/pIJ790 strains that had been cultured in the presence of 10 mM arabinose. Recombinant cosmids/plasmids were selected for by growth of the transformed cells on LB agar plates supplemented with apramycin. These knockout constructs were then confirmed by both PCR (using primers listed in Table 2.4) and restriction digestion using diagnostic restriction enzymes (so as to allow differentiation between wild type and ‘mutant’ cosmids/plasmids) prior to electroporation into E. coli strain ET12567/pUZ8002 to facilitate conjugation into S. coelicolor. Transconjugants were screened for double recombination (and replacement of the wild type gene) based on their apramycin resistance and kanamycin sensitivity (where the kanamycin resistance gene is located on the plasmid/cosmid vector backbone). Gene deletion clones were confirmed by PCR following isolation of chromosomal DNA from the putative mutant strain. Removal of the antibiotic resistance cassette was required in some cases, so as to eliminate any possible polar effects caused by the presence of the antibiotic resistance gene. To do this, a conjugatable plasmid (pUWL-Flp) containing the Flp recombinase gene, an oriT and a hygromycin-resistance selection marker, was conjugated into the knockout mutant (Fedoryshyn et al., 2008). Following selection of transconjugants containing the Flp plasmid, the strain was screened for loss of apramycin resistance, signifying removal of the antibiotic resistance gene. These strains were confirmed by PCR using primers flanking the deleted sequence. Creation of the srtE1 complementation construct required PCR amplification (See 2.3.3.) of the srtE1 gene using the H69 cosmid as template. Creation of the srtE2 complementation construct utilized the H69 cosmid in which srtE1 was replaced by an apramycin resistance cassette and then excised as a template.

2.3.13 Isolation of genomic DNA from S. coelicolor Genomic DNA was isolated from 2 day old, 10 ml cultures of S. coelicolor grown in 1:1 YEME:TSB liquid medium. Following collection of the vegetative mycelial by centrifugation, DNA was isolated using the Norgen Genomic DNA Isolation Kit. Samples were stored in TE buffer at -20˚C.

2.3.14 Isolation of RNA from S. coelicolor Streptomyces strains were cultured on MS agar or solid R5 medium and biomass was harvested and stored at -80˚C prior to RNA isolation. RNA was extracted using the protocol outlined in Moody et al. (2013). Briefly, cells were lysed by extensive vortexing with glass beads in a guanidium thiocyanate solution (4 M guanidium thiocyanate, 25 mM trisodium citrate dihydrate, 0.5% w/v sodium N-lauroylsarcosinate, 0.8% 2- mercaptoethanol). Nucleic acids were isolated following two phenol:chloroform extractions, and precipitation in isopropanol at -80˚C overnight. Precipitated nucleic acids were pelleted, and washed with 70% ethanol before resuspension in nuclease free water. Turbo DNase (Ambion) was added to the samples to remove contaminating DNA.

2.3.15 Reverse transcription PCR Analysis of gene expression was performed by semi-quantitative reverse transcription PCR. Three micrograms of purified RNA isolated from S. coelicolor were reverse transcribed using internal gene primers (listed in Table 2.4). The RNA/primer was mixed with four microliters of 5× First Strand buffer (Invitrogen) in a final volume of 20 µl and was initially treated with 40 U of RNaseOUT™ Recombinant Ribonuclease Inhibitor (Invitrogen), and incubated at room temperature for five minutes. This was followed by the addition of 200 units (u) of SuperScriptIII® Reverse transcriptase (Invitrogen) and incubation at 50°C for 50 minutes. The enzymes were inactivated by heat, at 72°C for 5 minutes. The PCR reaction was performed using Taq DNA polymerase (GeneDirex) in 25 µl reaction volumes, using the protocol described in section 2.3.3 for an optimized number of amplification cycles (Tables 2.5 and 2.6). Experiments were repeated using two additional biological replicates to confirm results.

2.4. Biochemical techniques 2.4.1 Purification of His-tagged proteins Plasmids (derived from pET15b, Novagen) encoding N-terminally histidine tagged protein-encoding gene fusions were transformed into E. coli BL21 Rosetta (Novagen) strains for the purpose of overexpression and purification of proteins. Two housekeeping sortases, srtE1 and srtE2 were cloned into the NdeI and BamHI sites of the pET15b vector. Additionally, as controls, the active-site cysteine mutants and srtA from S. aureus were cloned into the same site of pET15b for purification. Overnight cultures of all five BL21 strains carrying the pET15b plasmid containing one of the five sortase genes or gene variants were inoculated into 1 l of LB medium to an inoculum concentration of 1:100 with ampicillin and chloramphenicol. Cultures were incubated at 37˚C in a shaking incubator (200 rpm) until they reached an optical density at 600 nm ranging from 0.4-0.6. Protein overexpression was induced by the addition of 1 mM IPTG, and cultured at 30˚C for an additional 5 hours before the biomass was harvested.

Cell pellets were resuspended in 5 ml protein lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8), with one half of a Complete, EDTA-free, protease inhibitor tablet (Roche) and lysozyme (final concentration 1 mg/ml), prior to incubation on ice for 30 minutes. The cells were then sonicated (output 4, duty 40%) for six intervals for 6 sec with 10 sec incubations on ice between sonication bursts. Cell lysates were then separated via centrifugation at 11,955 × g for 30 minutes at 4°C. The resulting supernatant was then mixed with 1 ml Ni-NTA resin (Qiagen) for one hour at 4°C on a shaking platform. The resin-associated proteins were then washed with 4 ml wash buffer 1 (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8), and 1 ml wash buffer

2 (50 mM NaH2PO4, 300 mM NaCl, 100 mM imidazole, pH 8). Samples were eluted with

5 ml elution buffer (50 mM NaH2PO4, 300 mM NaCl, 500 mM imidazole, pH 8). Eluted samples were buffer exchanged using Amicon-ultra centrifugal filter units (Millipore) into sortase cleavage buffer (50 mM Tris-Cl pH 7.5 ,150 mM NaCl, 1 mM DTT) at 1248× g and 4°C to a final volume of approximately 0.5 ml. Purity of samples was determined using SDS PAGE.

2.4.2 Sortase cleavage assay Purified protein concentrationw were determined using the Bradford protein assay (Biorad) (Bradford, 1976). The cleavage of synthetic peptides was achieved by incubating 100 ml reactions containing purified wild-type and mutant sortase proteins (10 mM or 50 mM final concentration), together with 50 mM of each modified peptide substrate in cleavage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT and 5 mM CaCl2) for 18 h at 30°C. Peptide substrates (Biopep-tide) included: AALAETGSD (ChpC sorting signal; putative SrtE1/E2 substrate), VSLAHTGTD (ChpB sorting signal; putative SrtE1/E2 substrate), QALPETGEE (S. aureus SrtA substrate) and VENPQTNAG (SrtB substrate). All peptides had a 2-aminobenzoyl (Abz) fluorophore conjugated to their N-terminus, and a 2,4- dinitrophenyl (Dpn) quencher affixed to their C-terminus. Fluorescence was measured at 320 nm (excitation) and 420 nm (emission) for both the cleavage reactions and the peptides alone (negative control), with peptide alone fluorescence being subtracted from the overall reaction fluorescence.

2.4.3 Mass spectrometry analysis of cleavage products Mass spectrometry analysis was performed at the Center for Microbial Chemical Biology at McMaster University. LC-ESI-MS data were obtained using an Agilent 1100 Series LC system (Agilent Technologies Canada, Inc.) and a QTRAP LC/MS/MS System (ABSciex). One hundred µl of the sortase cleavage reaction was subjected to reverse phase HPLC using a C18 column (3 μm, 120 Å, 4.6×100 mm; Dionex Corporation) at a flow rate of 1 ml/min, under the following conditions: isocratic 5% solvent B (0.05% formic acid in acetonitrile), 95% solvent A (0.05% formic acid in water) followed by a linear gradient to 97% B over 20 min.

Table 2.1: Streptomyces strains used in this work Streptomyces strain Genotype Reference M600 SCP1- SCP2- (Chakraburtty and Bibb, 1997) E200 M600 ∆sco3850::apr This work (∆srtE1::apr) E200A M600 ∆sco3850 (∆srtE1) This work E201 M600 ∆sco3849::apr (∆srtE2::apr) E201A M600 ∆sco3849 (∆srtE2) This work E202 M600 ∆sco3849-3850::apr This work (∆srtE1/E2::apr) E202A M600 ∆sco3849-3850 This work (∆srtE1/E2) E206 M600 ∆chp::apr ∆chpB Duong et al, 2012 ∆chpC::vio J3150 M600 ΔchpAD Duong et al, 2012 ΔchpCH::aadA ΔchpB ΔchpF ΔchpG ΔchpE (Δchp) E207 M600 ∆sco2971::apr This work E208 M600 ∆sco1734::apr This work E212 M600 ∆sco0644::apr This work E210 M600 ∆sco5650::apr This work E211 M600 ∆srtE1/E2 This work ∆sco5650::apr

Table 2.2: E. coli and S. aureus strains used in this work E. coli strains Use Reference DH5α Plasmid construction and Invitrogen subcloning ET12567/pUZ8002 Generation of methylation MacNeil et al, 1992 free DNA BW25113 Construction of cosmid- Gust et al, 2003 based knockouts BL21 DE3 pLysS Rosetta Protein overexpression Novagen S. aureus strain S. aureus wild type Cloning of srtA Holden et al, 2004

Table 2.3: Plasmids and cosmids used in this work

Plasmids Use or genotype Reference pIJ2925 General cloning vector Janssen and Bibb, 1993 pMC131 pIJ2925 + srtE1 Duong et al, 2012 pMC131a pIJ2925 + srtE1 (C320A) Duong et al, 2012 pMC132 pIJ2925 + srtE2 Duong et al, 2012 pMC132a pIJ2925 + srtE2 (C220A) Duong et al, 2012 pMC133 pIJ2925 + srtE1/E2 Duong et al, 2012 pMS82 Integrating plasmid vector Gregory et al., 2003 (hygr) pMC134 pMS82 + srtE1 Duong et al, 2012 pMC134a pMS82 + srtE1 (C320A) Duong et al, 2012 pMC135 pMS82 + srtE2 Duong et al, 2012 pMC135a pMS82 + srtE2 (C220A) Duong et al, 2012 pMC136 pMS82 + srtE1/E2 Duong et al, 2012 pET15b Overexpression of 6x His Novagen tagged proteins pMC139 pet15B + srtE1 Duong et al, 2012 pMC139a pet15B + srtE1 (C320A) Duong et al, 2012 pMC140 pet15B + srtE2 Duong et al, 2012 pMC140a pet15B + srtE2 (C220A) Duong et al, 2012 pMC141 pet15B + srtA Duong et al, 2012 pSET152 Integrating plasmid vector Gregory et al, 2003 (Aprar) pIj773 Plasmid containing Gust, et al, 2003 apramycin resistance cassette StH69 Cosmid used for srtE1 and Redenbach et al, 1996 srtE2 knockout StI11 Cosmid used for sco1734 Redenbach et al, 1996 knockout StE59 Cosmid used for sco2971 Redenbach et al, 1996 knockout pUWL hyg FLP Replicative plasmid (pIJ101 Fedoryshyn et al, 2008 origin) with constitutively expressed Flp recombinase TOPO 2.1 Cloning vector used for Invitrogen plasmid creation pMC235 TOPO vector, self ligated This work pMC236 TOPO vector::sco0644 + This work 3kb flanking sequence pMC237 TOPO vector:sco0644::apr This work + 3 kb flanking sequence St1G2 Cosmid for deletion of Redenbach et al, 1996 sco2270

pMC239 St1G2, sco2270::apr This work StC82 Cosmid for deletion of Redenbach et al, 1996 sco2492 StC61A Cosmid for deletion of Redenbach et al, 1996 sco2682 pMC240 StC61A, sco2682::apr This work pMC241 TOPO vector::sco3176 + 3 This work kb flanking sequence pMC242 TOPO vector::sco3176::apr This work + 3 kb flanking sequence pMC243 TOPO vector::sco4782 + 3 This work kb flanking sequence St6A9 Cosmid for deletion of Redenbach et al, 1996 sco5650 pMC244 St6A9, sco5650::apr This work pMC245 TOPO vector:: sco6904 + 3 This work kb flanking sequence

Table 2.4 Oligonucleotides used in this work Italicized sequences correspond to nucleotide extensions on knockout primers that anneal to antibiotic resistance cassettes. Bolded nucleotides indicate sites of mutagenesis. Underlined sequences indicate inserted restriction enzyme recognition sites.

Primer Name Sequence (5' to 3') Function Knockout and complementation of srtE1 and srtE2 H69-20c KO I GGGGCTCGCCCCGGTGGTGGGCAGGGCCACGGCGTGACCATTCC Knockout of srtE1 GGGGATCCGTCGACC H69-20c KO II TCCGTGTCGGTGGTCGCTGCCACTGGTCATCCGCCCTTATGTAGGC Knockout and knockout check for srtE1 TGGAGCTGCTTC H69-19c KO I AGCCGGATGCGCTCGTCAGTTAAGGGCGGATGACCAGTGATTCCG Knockout of srtE2 GGGATCCGTCGACC H69-19c KO II CTGAGGGACCGGGGCTCCGTCGTGTACGTCAGGAGGTCATGTAGG Knockout and knockout check for srtE2 CTGGAGCTGCTTC H69-19c TEST I TTCACCAGCAAGTACCGCAT Knockout check for srtE2 H69-19c TEST II TTCCCGGCTTCGGCGACTGA Knockout check for srtE2 H69-19c TEST III CGACATCCTGGTGGAGAAGG Knockout check for srtE2 SCO3850p I TGCAGTTCCACCCGGAGTCGGTGCT Knockout check for srtE1 and srtE1/E2; cloning srtE1, srtE2, and srtE1/E2 SCO3850 end CCACGTTCGTCCACCACAGCG Cloning srtE1 SCO3849 end CCTCGAACCCGCAGCCCGGCAGC Cloning srtE2 and srtE1/E2 New 3850 KO III GAGGCCGAGCAGGCCCCGAAGGCGCCCCTGTCGCGCGTG Cloning srtE2 H69-20c KO II TCCGTGTCGGTGGTCGCTGCCACTGGTCATCCGCCCTTATGTAGGC Cloning srtE2 TGGAGCTGCTTC SCO3850p II ATCCGCAGCGCCACCGTCTCGTCGT Knockout check for srtE1 and srtE1/E2 Mutagenesis of srtE1 3850 C to S a CCCTCACCACCTCTACGCCGGAGTTC Site directed mutagenesis of srtE1 active site Cys to Ser 3850 C to S b GAACTCCGGCGTAGAGGTGGTGAGGG Site directed mutagenesis of srtE1 active site Cys to Ser 3850 C to A a CCCTCACCACCGCTACGCCGGAGTTC Site directed mutagenesis of srtE1 active site Cys to Ala 3850 C to A b GAACTCCGGCGTAGCGGTGGTGAGGG Site directed mutagenesis of srtE1 active site Cys to Ala Overexpression of sortases 3850 NdeI GGGTCGCATATGACGAACGTGCGGGCGACG SrtE1 overexpression and purification 3850 BamHI GGGTGCGGATCCTTAACTGACGAGCGCATCC SrtE1 overexpression and purification 3849 Nde GGTGCCATATGACGAACGTGGTCGCCGACC SrtE2 overexpression and purification 3849 BamHI GGGTGCGGATCCTCAGCGCAGCTCCTTCGG SrtE2 overexpression and purification SrtA NdeI AACATATGAAACCACATATCGATAATTATC Overexpression and purification of SrtA SrtA BamHI AAGGATCCTTATTTGACTTCTGTAGCTACAA Overexpression and purification of SrtA Knockout and complementation of sortase substrates 1734 Fwd CGCCGCATCCCCAGTGACCGTTTCGAGGAGTTCCCCATGATTCCGG Knockout of SCO1734

GGATCCGTCGACC 1734 Rev CGTTCCCCGTTCCCCCGTCGTCCCCCCGCCTCCGCGTCATGTAGG Knockout of SCO1734 CTGGAGCTGCTTC 1734 up GGGAGGTACGCGGCGTCG Knockout check and cloning SCO1734 1734 down CCGTCCTCGCCACGAAGTACGACG Knockout check and cloning SCO1734 1734-gene-R CGACCGCCGCCCTGCACG Knockout check for SCO1734 1734-gene-F GGACAGCACCACGCCCTACATCGC Knockout check for SCO1734 2971 Fwd TCGACGACTAAATCGGCTACTGGGGGTAGCAACTACATGATTCCGG Knockout of SCO2971 GGATCCGTCGACC 2971 Rev GTTCTGCGTGCGCGGTGCGTACGCGGACGGCTACCGTCATGTAGG Knockout of SCO2971 CTGGAGCTGCTTC 2971 up CGTTCGCCGCTGATGTCTGG Knockout check for SCO2971 2971-gene-R CGTCGGGTCCGTCGGGTCCG Knockout check for SCO2971 2971 down GCTGCGCCCGTGAGTGAGGGCAGG Knockout check for SCO2971 2971-gene-F CGGTGAGGAGCTCGCCGAGACCGG Knockout check for SCO2971 2971 upstream TTCAACGGCGAGATCGACG Cloning of SCO2971 2971 downstream ATGACGTACGACCGGTTGGT Cloning of SCO2971 RT-PCR rdlA-forward GGATGGGGATCCCGTAGGACAGTGCGTCGCTACG Transcription profiling for rdlA SCC46.03C down CCCGTTGTCGTCCCCGATGG Transcription profiling for rdlA chpD-F GCGCTGCTGTCGTCGCGGG Transcription profiling for chpD SCC46.02c down GGGAACGTGCACGGGAACC Transcription profiling for chpD chpG-F CCATGTCGCGTATCGCGAAGGG Transcription profiling for chpG 26c down CCGATGACGTCGATGGTGTTGC Transcription profiling for chpG rRNA-1 AGAGTTTGATCCTGGCTCAG Transcription profiling for 16S rRNA rRNA-2 CGAACCTCGCAGATGCCTG Transcription profiling for 16S rRNA nepA-up GGCAAGCATCCGTACCGCCCG Transcription profiling for nepA 2SC10A7.06c GCTGCGTGGTGGACGAGTTGC Transcription profiling for nepA General use primers M13 F CGCCAGGGTTTTCCCAGTCACG Sequencing M13 R GCGGATAACAATTTCACACAGG Sequencing T7 Fwd TAATACGACTCACTATAGGG Sequencing T7 term GCTAGTTATTGCTCAGCGG Sequencing Sortase RT PCR primers 3850in fwd2 CCACTCCTGGACGACGAGACGG transcription profiling for srtE1 3850in fwd2 TCCACCACAGCTGGTAGGTGACGA transcription profiling for srtE1 3849in fwd GGTGGACGAACGTGGTCGCCGA transcription profiling for srtE2 3849in rev CGTTGAGGATCTTCATCGACGTGCCCT transcription profiling for srtE2 3851in fwd CACGCCCAGGACGGTTTCGACGG transcription profiling for sco3851

3851in rev CGGCGCGCGGTCCACCACAC transcription profiling for sco3851 3850up start GTGGTGGGCAGGGCCACGG transcription profiling for sco3851-srtE1 3851in down NEW CCGGCCGAGCTGGAGGTGAC transcription profiling for srtE1 3850in CtermFWD ACATCACCCTCACCACCTGTACGCCG transcription profiling for srtE1-srtE2 3849in NtermREV ACAGCGAGTACACGACGAACAGGCCC transcription profiling for srtE1-srtE2 Sortase substrate deletion primers 0644 3kb up GGCGAACGCCTTCTCGTACT Knockout plasmid creation for knockout of sco0644 0644 3kb down GCCTTCATCCGGGTCGTCAA Knockout plasmid creation for knockout of sco0644 sco0644 KO fwd CATTTCATGCCCGAGTCCGTCACGACACAGGAGCACGCCATTCCGG Knockout of sco0644 GGATCCGTCGACC sco0644 KO rev CGGCCCCGCCCACCCGCGCTCAGGCGGCTCCGGCAGCCG TGT Knockout of sco0644 AGG CTG GAG CTG CTT C sco0644 up CGG GGT GCT GCC GCT Knockout check of sco0644 sco0644 down CAAGTCATGAGGCATCCTTCTTCCGCG Knockout check of sco0644 0644 in rev CCCGGTCGATGGCGGC Knockout check of sco0644 2270 KO fwd CACCCCCGACACCCCCCGACACCCCAGGAGAACCGAGCCATTCCG Knockout of sco2270 GGGATCCGTCGACC 2270 KO rev CGGACAGTATCGGGCGCGGACCGCCCCGGCCCGCAACGGATGTA Knockout of sco2270 GGCTGGAGCTGCTTC 2270 up2 CTGCCCGACCTCGGCAGCG Knockout check of sco2270 2270 down2 CGGGCCCGTACGAAGCGCAG Knockout check of sco2270 2270 in rev GGCACCGTTGCCGGCGG Knockout check of sco2270 2492 KO fwd CCTCATATCCGCCCATCCACACCGTTCCCGGGACTCTCGATTCCGG Knockout of sco2492 GGATCCGTCGACC 2492 KO rev ACGCGGGCCGGGCGCGGGGCGGTTCGGGCCGCGCCGCGGTTGTA Knockout of sco2492 GGCTGGAGCTGCTTC 2492 up 2 CCCGTCTCACCGTACGCGCC Knockout check of sco2492 2492 down2 CAGCGCCGCGTTCAGCCTCA Knockout check of sco2492 2492 in TCGTACGCCTCGGAGCAGTTCTTGA Knockout check of sco2492 2682 KO F GTGTTTTCTTCGCACGCTGCGCCGTCTCTGCCCGGTCGAATTCCGG Knockout of sco2682 GGATCCGTCGACC 2682 KO R TCACTGCTCGTTCCTCCTGCGGACGAACAGCAGGACCGCTGTAGGC Knockout of sco2682 TGGAGCTGCTTC 2682 up CGGGACGAACGGGATGGATT Knockout check of sco2682 2682 down ACCACCAGCTCACCAAGGAC Knockout check of sco2682 2682 midR GCCGCCCCGTCCTGGGC Knockout check of sco2682 2971 3kb up TCCAGGAATGGATCGGCACC Knockout plasmid creation for knockout of sco2971 2971 3kb down GCGCAGTATGGAGACGACCT Knockout plasmid creation for knockout of sco2971 3176 3kb up GTGTCTACGAGGACAGGGGC Knockout plasmid creation for knockout of sco3176 3176 3kb down GTTGGGGTGCTTGGTGAACG Knockout plasmid creation for knockout of sco3176 29

3176 KO F ATGAGACCTAGCTCCCTGCGCTCGCCGTGGACGCGTGCGATTCCG Knockout of sco3176 GGGATCCGTCGACC 3176 KO R TCAGGCCGCGGTGCCGCCCGTCCTGCGGCGGCGTGAGACTGTAGG Knockout of sco3176 CTGGAGCTGCTTC 3176 up AGGTCCACGCACATTGACGA Knockout check of sco3176 3176 down CTGTACACGTCCAGCGGCTA Knockout check of sco3176 3176 midR GACCGGTGATCGTGACGGGCAG Knockout check of sco3176 4782 3kb up GTCCGTGACCTCGTTGGTGA Knockout plasmid creation of sco4782 4782 3kb down CACGCCGACTACGTCTCCTT Knockout plasmid creation of sco4782 4782 KOF3 ACCACGTCCGCCCCCGCGAACCCCTCGCCGAGCACCTCCATTCCG Knockout of sco4782 GGGATCCGTCGACC 4782 KOR3 GAAGAGGACCCGCACCCCCCTCCGAGCGCGGGTCCCCCTTGTAGG Knockout of sco4782 CTGGAGCTGCTTC 4782 up GTCCTCGAGTTCCTCCGCC Knockout check of sco4782 4782 down TCGTACTGGCCCAACAAGGG Knockout check of sco4782 4782 midR CGCCGGCCAGGTACTCGAGGTT Knockout check of sco4782 sco5650 KO Fwd TCTTCACCCCCCGCATCCGCGCTCCCGTAAAGGACAAACATTCCGG Knockout of sco5650 GGATCCGTCGACC Sco5650 KO rev ACGGGACGGGCCACCGCCGTGAACGGGTCGGCGCGGGCGTGTAG Knockout of sco5650 GCTGGAGCTGCTTC sco5650 up 2 CGCCGGCACTGGACCGAGAC Knockout check of sco5650 sco5650 down 2 CACGGACGTGCTGCTGGCCT Knockout check of sco5650 sco5650 in rev TGCACGGCGTCGACGGG Knockout check of sco5650 6904 3kb up AACACCACCCCTTGACACCC Knockout plasmid creation of sco6904 6904 3kb down CAATGCAACACGGCCGTCAG Knockout plasmid creation of sco6904 6904 KOF2 CGCTGACCTCCCCCGACCGATCAAGGAGTCCTCTTCCTCATTCCGG Knockout of sco6904 GGATCCGTCGACC 6904 KOR2 GCTGCGGGGGCTCCGTGTGTTGAAGAAGGGTGGGTGCGGTGTAGG Knockout of sco6904 CTGGAGCTGCTTC 6904 Up CTGATCTCCACCGAACCGCT Knockout check of sco6904 6904 down GATCCACCGGTCTAGCTGCC Knockout check of sco6904 6904 midR CGCGGCCGGAGTCTCGGC Knockout check of sco6904 Sortase substrate RT PCR 0644 RT F CGACGGTGGACACCGAACTC Transcription profiling of sco0644 0644 RT R CCTTCTTGAGCGGGTCGAGG Transcription profiling of sco0644 1734 RT F AGGCGCTCTACGACTGGAAC Transcription profiling of sco1734 1734 RT R GCCAGGTCCAGACCCTTGAA Transcription profiling of sco1734 sco2270 RT F CTACGAGTTGACGTGGGGCA Transcription profiling of sco2270 sco2270 RT R CTCCGGTGAAGGTGAAGGCA Transcription profiling of sco2270 sco2492 RT F AGAAGGGCGACGAGCACTAC Transcription profiling of sco2492 30

sco2492 RT R CTCGCTCCCGGAGTCCTTC Transcription profiling of sco2492 2682 RT F TCGACATCCAGAACCCGACG Transcription profiling of sco2682 2682 RT R GACCTGCGGGTAGCTGTTCT Transcription profiling of sco2682 sco2971 RT F CCGGTGCCTCCCTGACC Transcription profiling of sco2971 sco2971 RT R AGGACCTGTACCGATGTCGC Transcription profiling of sco2971 3176 RT F ATCCCCAACAGCGGCTACAA Transcription profiling of sco3176 3176 RT R GAACGGGTTGTGGATGTCGC Transcription profiling of sco3176 4782 RT F GCACGAGTTCTCGCTGAACG Transcription profiling of sco4782 4782 RT R TCCTCCCAGGTCCTGTCCTC Transcription profiling of sco4782 sco5650 RT F ACATCAGCGAGTTCTCCGCC Transcription profiling of sco5650 sco5650 RT R CAGTCCACGGGGAAGGAGAC Transcription profiling of sco5650 6904 RT F AGCGTCGAGATCACCACCAG Transcription profiling of sco6904 6904 RT R CCTCCTTGAGCCGGTAGACG Transcription profiling of sco6904 sco5650mutagenesis 5650 mut overlap 1 GCACCGGTCTCGGGGAGGTTGGAGTCA Site directed mutagenesis of SCO5650 LAXTG to LPXTG 5650 mut overlap 2 TGACTCCAACCTCCCCGAGACCGGTGC Site directed mutagenesis of SCO5650 LAXTG to LPXTG

Table 2.5 Taq PCR reaction conditions (GeneDirex).

Component Final concentration Primer 1 0.2 µM Primer 2 0.2 µM dNTP 200 µM Taq polymerase buffer (10x) 1x DMSO 5% Taq polymerase 1.5 units

dH20 To 25 µl

Table 2.6 Standard PCR cycling conditions for Phusion polymerase (Thermo Fisher Scientific).

Temperature Time

1) 98˚C 4 minutes 2) 98˚C 20 seconds Repeat 30 times 3) 60˚C 20 seconds 4) 72˚C 30 seconds/kb 5) 72˚C 5 minutes 6) 18˚C Hold

Chapter 3: Sortases in S. coelicolor development

Preface: This results of 3.2.1 -3.2.7 have been published in the journal, Molecular Microbiology (Duong et al., 2012). This work was performed in conjunction with Dr. David Capstick and Christina DiBerardo, and their relevant work has been cited. All newly constructed Streptomyces coelicolor strains were constructed by Christina DiBerardo. Dr. David Capstick performed a subset of the phenotypic analyses, and performed RT PCR analysis of chpH, chpD, chpF, rdlA and nepA in the ∆chpABC, ∆srtE1/E2 and wild type strains. Template RNA used for RT PCR analysis was isolated by both Dr. David Capstick and Andrew Duong. Maureen Bibb performed sample preparation for high resolution SEM, and SEM was performed by Kim Findlay.

3.1.1 Introduction Sortases are a family of enzymes that facilitate the anchoring of proteins to the cell wall of Gram positive bacteria (Ton-That et al., 1999). This group of proteins is further subdivided into classes, based on their functional role. SrtA, the first characterized sortase, was first described as a housekeeping sortase, responsible for the attachment of multitude of secreted proteins to the developing cell wall of Staphylococcus aureus (Navarre and Schneewind, 1999). Class B sortases anchor substrates involved in iron acquisition, whereas class C and D sortases are involved in anchoring proteins associated with pili formation and sporulation, respectively (Pallen et al., 2001; Ton-That and Schneewind, 2004; Ton-That et al., 2004; Dramsi et al., 2005; Litou et al., 2008). Sortases are predicted to exist in most actinomycete species but their specific roles in the biology of these bacteria remain largely elusive (Pallen et al., 2001). The actinomycete-specific sortases have been assigned to the ‘sortase E’ (SrtE) and ‘sortase F’ classes. Similar to the SrtA enzymes studied to date, the SrtE enzymes are housekeeping sortases, but are found in the actinomycetes rather than the firmicutes, and appear to recognize a different sorting signal. Sortase F are sortases of unknown function that are specific to the actinomycetes.

3.2 Results 3.2.1 Bioinformatic prediction of sortases and their substrates S. coelicolor is predicted to be a rich reservoir of sortase enzymes. Previous work has identified seven sortase like proteins encoded by this species, based on their similarities to SrtA in S. aureus (Pallen et al., 2001; Comfort and Clubb, 2004). Two of these sortases, SCO3849 and SCO3850 were classified as SrtE enzymes, for their similarity to SrtA (and thus their likely housekeeping properties), as well as their presence in a high G+C organism (Spirig et al., 2011). Sortase substrates are predicted based on the presence of a C-terminal “sorting signal”, in addition to an N-terminal secretion signal. This sorting signal was identified in 14 S. coelicolor proteins, all containing the same LAXTG pentapeptide sequence in their CWSS. To date, only four of these substrates have been studied, including CbpA and the three long chaplin proteins (ChpABC) (Claessen et al., 2003; Elliot et al., 2003; Walter and Schrempf, 2008). Housekeeping sortases typically anchor substrates encoded in disparate loci from the sortase gene. These 14 putative substrates are predicted to be substrates of the housekeeping sortases as their corresponding genes are not found adjacent to any sortase-encoding gene.

3.2.2 Deletion of the sortases leads to a defect in development S. coelicolor contains two predicted housekeeping sortases, SrtE1 and SrtE2 (SCO3850 and SCO3849, respectively). We generated both individual and double deletions mutants of srtE1 and srtE2 using the ReDirect Knockout protocol. As both genes are adjacent, to reduce polar effects of the deletion, we created a markerless, in frame deletion mutant of srtE1 containing a 39 bp “scar” sequence as well (Gust et al., 2006). These strains were used for further experimental and phenotypic analysis and were subjected to complementation of the mutant phenotypes. To test the effects of deleting one or both sortase genes, sortase deletion mutants were cultured on MS agar for five days, and the strains were examined for developmental defects, relative to a wild type strain. The wild type strain, the double sortase mutant (∆srtE1/E2), as well as the double sortase mutant complemented with either srtE1 or srtE2 on an integrative plasmid (pMS82) were all grown for up to 5 days for phenotypic analysis.

In comparison to the wild type strain, the double sortase mutant had an extremely delayed progression through its life cycle. Following 2 days of growth on MS agar, the wild type strain had established a robust vegetative mycelium, and had begun to form aerial hyphae, as evidenced by the white hyphae on the plate (not shown). The mutant required 4 days of growth before aerial hyphae were evident. Furthermore, after 5 days, the wild type strain sporulated, as evidenced by the expression of the spore-associated, grey-pigmented polyketide, WhiE (Ryding et al., 1999). In contrast, ∆srtE1/E2 remained arrested at the aerial hyphae stage, and did not progress to sporulation, even after extended incubation (14 days). Restoration of the wild type phenotype in the ∆srtE1/E2 was achieved by the introducing wild type versions of one or both sortase genes on an integrative plasmid (see Figure 3.1).

3.2.3 Scanning electron microscopy analysis of sortase mutants To further examine the developmental defects observed for the double sortase mutant, we used SEM to probe colony morphology. Wild type and ∆srtE1/E2 strains were streaked onto MS agar medium and grown for 5 days prior to sample preparation. As seen in Figure 3.2a, the wild type strain formed long, mostly uniform spore chains. The sortase mutant; however, did not form spore chains, but was arrested during what appeared to be aerial hyphae formation. Interestingly, the ∆srtE1/E2 aerial hyphae appeared to be branching structures. This is atypical for aerial hyphae, as the branching phenotype is only associated with vegetative mycelia in wild type cells (Figure 3.2a). High resolution scanning electron micrographs of the same strains showed a complex paired rodlet ultrastructure on the surface of the wild type spore, which was not visible on the aerial hyphae of the ∆srtE1/E2 strain (Figure 3.2b).

3.2.4 Redundancy of srtE1 and srtE2 in morphological development To investigate the role of the individual sortases in development, we introduced srtE1 or srtE2 cloned into an integrating vector (pMS82) into the double sortase mutant (∆srtE1/E2). The introduction of srtE1 into ∆srtE1/E2 fully rescued the mutant phenotype, where growth and development were indistinguishable from that of the wild type strain. The introduction of srtE2 into ∆srtE1/E2 could restore sporulation, but development was modestly delayed compared to the wild type strain: after 2 days growth, it remained ‘bald’, but after 5 days, it was virtually indistinguishable from wild type strain (Figure 3.1). 35

The ability of either sortase to rescue the ∆srtE1/E2 phenotype suggests that srtE1 and srtE2 are functionally redundant. The slight delay in the ∆srtE1 strain may also suggest that SrtE1 is the primary sortase.

3.2.4 Role of the sortase dependent long chaplins in spore maturation The three long chaplins (ChpA, B and C) are all predicted sortase substrates, and the chaplins as a whole have a demonstrated role in promoting aerial development. A ‘long chaplin’ mutant strain was constructed (Duong et al, 2012), and strikingly, the phenotype of the ∆srtE1/E2 mutant was far more severe than that of the long chaplin mutant (Figure 3.3). It had been originally hypothesized that the sortase-dependent anchoring of the long chaplins would facilitate the raising of aerial hyphae. Instead, ∆chpABC exhibited only a slight delay in aerial development compared to the wild type strain at 2 days, but after 6 days growth on MS agar, the mutant colony phenotype was similar to the wild type strain, and the strain appeared to sporulate. The chaplin mutant (∆chp) had the greatest defect in development, relative to the long chaplin and double sortase mutant (Figure 3.3) (Duong et al, 2012). High resolution SEM analysis was employed to further investigate the ∆chpABC spores (Figure 3.4a). While the spores themselves appeared wild type, the organization of the rodlet ultrastructure appeared to be less uniform in comparison to the wild type strain. Measurement of the rodlet length of the wild type and ∆chpABC strains revealed that average rod length in both strains was similar, between 120-140 nm (Figure 3.4b). However, further analysis of the rodlet lengths revealed a wider distrubtion in the long chaplin mutant (from 20 nm to 440 nm, N=369) compared to the wild type strain (40 nm to 240 nm, N=207) (Figure 3.4b).

3.2.5 Expression of the sortase genes The deletion of both sortases lead to a severe defect in aerial development. We sought to test if the expression of both genes was limited to the transition from vegetative to aerial growth, as no phenotypic defects were noted in vegetative growth. Analysis of sortase expression was performed using RNA isolated from a 96 hour time course of wild type S. coelicolor grown on MS agar. Both sortases genes were highly expressed during vegetative growth (24-48 hours), but transcripts were not detected at

96 hours of growth (Figure 3.5b, fragments iv and vi), a time-point that corresponds to late sporulation in S. coelicolor. The srtE1 and srtE2 genes are only separated by 13 bp, and given this short distance, we hypothesized that the genes were co-transcribed. Semi-quantitative RT PCR was performed using primers spanning these two genes. RT PCR revealed that both sortases were indeed cotranscribed (Figure 3.4b, fragment v). No transcript was detected using primers that amplified a region extending from the upstream gene into srtE1 (Figure 3.5b, fragment ii), indicating that srtE1 and srtE2 were co-transcribed independently of the upstream gene, sco3851.

3.2.6 In vitro analysis of sortase cleavage activity Sortases act by recognizing and cleaving a specific pentapeptide motif found in the C-terminus of some secreted proteins. SrtA, the most extensively characterized sortase to date, recognizes an LPXTG motif, and cleaves between the threonine and glycine residues (Mazmanian et al., 1999; Ton-That et al., 1999). In S. coelicolor, as well as other actinomycetes, the predicted sortase substrates possess a C-terminal LAXTG sequence, instead of the canonical LPXTG motif (Pallen et al., 2001; Elliot et al., 2003). These putative substrates also possess a hydrophobic domain and positively charged tail following the pentapeptide sequence, all of which are positioned at the extreme C- terminal end of the protein. Investigation of each sortases’ affinity for the LAXTG pentapeptide was probed utilizing an in vitro substrate cleavage assay. Briefly, the extracellular domains of SrtE1 and SrtE2 were overexpressed in E. coli as N-terminally His-tagged fusions, and were purified by nickel affinity chromatography. Active site mutant variants (containing cysteine to alanine mutations) were also overexpressed and purified, and served as negative controls. Purified proteins were incubated with synthetic peptides containing the LAXTG pentapeptide sequence hypothesized to be recognized by the sortases. The peptides further possessed an N-terminal 2-aminobenzoyl (Abz) fluorophore, as well as a C-terminal dinitrophenyl (Dnp) molecule that quenches fluorescence of the Abz, when in close proximity. Fluorescence is observed when the peptide is cleaved, and the fluorophore is liberated from the quenching properties of Dpn. A number of peptides were tested: including LAETG and LAHTG sequence variants, which were found in predicted S. coelicolor sortase substrates; LPETG, the canonical pentapeptide sequence 37

from the the SrtA (from S. aureus) substrate ‘Spa’; and NPQTN, the pentapeptide sequence in IsdC, a class B sortase substrate (Mazmanian et al., 2000). Unlike Spa, IsdC is not recognized by the S. aureus, SrtA, and thus, was not expected to be recognized by either of the SrtE1 or SrtE2 S. coelicolor housekeeping sortases (Bentley et al., 2006). As seen in Figure 3.6, both SrtE1 and SrtE2 were capable of cleaving the two S. coelicolor-associated peptide sequences (LAETG and LAHTG). In contrast, very little fluorescence was observed when SrtE1 and SrtE2 were incubated with the Spa (LPETG) or IsdC (NPQTN)-derived peptides. The active site mutants (cysteine to alanine) showed little cleavage activity with any of the peptides relative to the wild type sortases. Our positive control, SrtA from S. aureus, was able to cleave Spa, but not IsdC or either of the S. coelicolor-specific peptides (Mazmanian et al., 1999; Ton-That et al., 1999). The amount of cleavage/fluorescence seen by the cysteine mutants was comparable to the SrtA protein, whose sortase activity was specific to the LPXTG motif (Figure 3.6) (Mazmanian et al., 1999; Ton-That et al., 1999). Further analysis of sortase activity was performed by analyzing the cleavage products following in vitro cleavage. Liquid chromatography, followed by mass spectrometry was performed to determine the sites at which each sortase cleaved the pentapeptide sequence. Similar to SrtA from S. aureus, both SrtE1 and SrtE2 cleaved the LAXTG pentapeptide sequence between the threonine and glycine residues (Figure 3.7, i, ii, and iv)(Mazmanian et al., 1999). Cleavage of SrtA substrates was exclusive to this one site (Figure 3.6b, vi), but our data revealed that there was an additional cleavage between the second and variable third residues (Figure 3.7, i, ii, and iv). Both SrtE1 and SrtE2 exhibited cleavage at this additional site in the LAETG and LAHTG pentapeptide motif in our in vitro cleavage assay.

3.2.7 Deletion of sortases mediates aerial hyphae-specific genes SrtE1 and SrtE2 appear to be important for S. coelicolor development, and this effect is independent of the long chaplins. It was unclear what other genes involved in development were affected by the deletion of both sortases, leading to the developmental defect. Previous studies have unveiled a number of developmental stage- specific genes that we employed to monitor developmental progession at a genetic level (Claessen et al., 2002; Elliot et al., 2003; Dalton et al., 2007).

Analyses of genes typically expressed during aerial development were conducted using semi quantitative RT-PCR. Transcript levels were assayed throughout a 96 hour RNA time course for wild type, ∆chpABC, and ∆srtE1/E2 strains grown on MS agar (Figure 3.8). In the wild type strain, chpH was constitutively expressed at all time points, whereas chpD, chpF, rdlA and nepA, were upregulated during aerial growth (72 – 96 hours) (Figure 3.8). In the ∆chpABC strain, there was a slight reduction in transcript abundance for all of these genes, except for chpH, which was expressed constitutively in both the wild type and mutant strains. Most strikingly, except for chpH, there was a dramatic decrease and/or delay in the expression of these genes in the ∆srtE1/E2 strain. chpD expression was highly expressed in the wild type strain during aerial development, but its expression was delayed until 96 hours in the ∆srtE1/E2 strain, where the other genes were not seen to be expressed at all.

3.3 Discussion This work collectively demonstrates that the housekeeping sortases in S. coelicolor are required for multicellular development, and have a role in regulating gene expression during aerial growth.

3.3.1 SrtE1 and SrtE2 recognize a unique substrate motif Substrate recognition by the sortases is a highly regulated process. Often, sortase substrate-encoding genes are co-localized with their cognate sortase-enoding gene, and the sole function of that sortase is to anchor those substrate proteins (Pallen et al., 2001). However, in most characterized sortase systems to date, the majority of predicted sortase substrates are not encoded near a sortase-encoding gene, and it is housekeeping sortases that anchor these proteins to the cell wall. (Pallen et al., 2001; Comfort and Clubb, 2004; Spirig et al., 2011). Within the streptomycetes, the sortase recognition pentapeptide sequence for SrtE1 and SrtE2 was confirmed to be LAXTG (Figure 3.6). In all, there are fourteen proteins in S. coelicolor that have this motif followed by a stretch of hydrophobic residues and a positively charged tail at the extreme C-terminal end. This pentapeptide motif is expected to be recognized and cleaved by these two sortase enzymes in vivo, after which they anchor the N-terminal end of the cleaved product to the nascent peptidoglycan (Comfort and Clubb, 2004). Of all the sortase substrates identified in 39

Gram positive bacteria to date, the majority (>82%,) of substrates possess an LPXTG pentapeptide sequence rather than an LAXTG motif (Comfort and Clubb, 2004). Co- crystallization of SrtA from S. aureus with a ‘substrate peptide’ bearing the LPETG motif, (Suree et al,2009) showed that the proline in the sortase recognition motif is a key residue for interaction with Ile182 and Ala118, which reside in the catalytic domain of SrtA. The equivalent Ala118 residue is conserved within both SrtE1 and SrtE2, but these enzymes lack the equivalent isoleucine, and in its place is a threonine (Figure 3.9). It is conceivable that this modification of the catalytic domain sequence alters the specificity for substrate pentapeptides, allowing for recognition of those possessing an alanine in the second position. This convergent evolution of sortase and substrate is evident in most actinomycetes (Comfort and Clubb, 2004). Active site sortase mutants were expected to show no cleavage when incubated with the LAXTG pentapeptides. However, reduced cleavage of the fluorescent peptides relative to the wild type proteins was observed. It is possible that the cysteine to alanine mutations caused a change in protein conformation that allowed for some cleavage activity to occur. This compensatory mechanism of cleavage is predominantly an observation of the in vitro cleavage assay, as cysteine to alanine mutants of both sortases are unable to complement ∆srtE1/E2 mutants in vivo (Duong et al, 2012). SrtE1/SrtE2 recognition of an unusual pentapeptide sequence is accompanied by a possible unique cleavage site. The canonical SrtA enzymes exclusively cleave between the threonine and glycine residues of the LPXTG motif. Here, we show that both SrtE1 and SrtE2 cleave at this site, but in addition, also cleave between the alanine and the variable residue (i.e. between the second and third amino acids). Interestingly, this alternative cleavage site appears to be the dominant one - at least in vitro, as evidenced by the mass spectrometry analysis of the cleavage products following the in vitro sortase cleavage assay (Figure 3.5b). The additional cleavage site could also represent an alternative attachment site for sortase substrates. Cleavage at the alternative site could facilitate anchoring of sortase substrate sites to other proteins, instead of the nascent peptidoglycan. It is possible that this dominant cleavage site is not applicable in vivo, but further analyses of the covalent bonding sites of the sortase substrates could identify its relevance (Mazmanian et al., 1999, Ton-That and Schneewind, 1999; Marraffini and Schneewind, 2005). An in vivo analysis of sortase substrates anchored to the peptidoglycan would allow us to determine if the additional 40

cleavage site is an artifact of the in vitro assay, or if SrtE1 and SrtE2 represent a subset of sortase enyzmes with a dynamic ability to identify and cleave different sites of the same substrate proteins. A secondary cleavage site has never been reported previously for any characterized sortase enzyme, and as such it represents a unique opportunity to probe the cleavage specificity of sortase enzymes.

3.3.2 Housekeeping sortases modulate development SrtE1 and SrtE2 are essential for the normal development of S. coelicolor. It was previously hypothesized that the housekeeping sortases would aid in aerial development by facilitating attachment of the long chaplins to the peptidoglycan, which in turn were proposed to anchor the short chaplins. Deletion of the three long chaplins (chpABC) only resulted in a slight delay in aerial development; in contrast to the effect of deleting the housekeeping sortases, whose deletions dramatically altered aerial development and prevented sporulation. This suggests that the sortases mediate development in a long chaplin-independent manner. The dramatic defect of the sortase mutant was obvious at the level of colony morphology, but scanning electron microscopy of the hyphae revealed that the deformity extended to a microscopic level. Previous studies have shown that the expression of a number of genes are upregulated during the formation of aerial hyphae (Claessen et al., 2002; Elliot et al., 2003; Dalton et al., 2007). Here, transcript analyses revealed that expression of many of these genes (chpD, chpF, rdlA, nepA) was extremely delayed and/or non-existant in the sortase mutant compared to the wild type. Thus, the deletion of cell wall anchoring enzymes thus appears to affect gene expression and in turn, influences development of the entire organism. Regulation of development through sortase enzymes is a process that is currently unique to S. coelicolor. In S. aureus, deletion of srtA modifies its complement of proteins at the cell surface, but cellular morphology and division remain the same (Mazmanian et al., 2000). To our knowledge, SrtE1 and SrtE2 provide a unique opportunity to study the function of cell wall modifying enzymes and their abilities to regulate transcription within a cell. There are many possible explanations as to how the sortases function to regulate development. It is possible that the absence of sortase activity changes the extracellular topology, triggering a signal cascade, delaying aerial development within the cell. S. coelicolor is predicted to encode 85 histidine kinases and 41

79 response regulators that are capable of responding to a number of environmental factors (Barakat et al., 2010; Rodriguez et al., 2013). There is great interest in understanding the roles of the many two component systems in S. coelicolor, but the majority of signals and functions for these two component systems are still unknown. S. coelicolor encodes 14 predicted sortase substrates, including the three long chaplins. Deletion of the three long chaplins did not result in a defect in development equivalent to that of the sortase mutant, suggesting that the long chaplins are dispensable for normal aerial development. The remaining 11 sortase substrates are not predicted to have a role in the development of S. coelicolor. Most of the substrates of are categorized as proteins of unknown function, and only a subset of these have predicted functional domains. The substrate proteins have not been further characterized, and without additional information, it is difficult to deduce their role in S. coelicolor development. Further work into the role of these substrates or the two component systems is needed to determine the mechanism of sortase function in the aerial development of S. coelicolor.

3.4 Tables

Table 3.1 List of predicted sortases in S. coelicolor. Sortase F denotes actinomycete-specific sortases of unknown function, where SrtE denote housekeeping sortases

Sortase SCO number SrtF1 SCO0935 SrtF2 SCO2480 SrtF3 SCO2841 SrtF4 SCO3737 SrtE2 SCO3849 SrtE1 SCO3850 SrtF5 SCO7450

Table 3.2 List of predicted sortase substrates, and their anchoring sortase

Sortase Protein or predicted Processing Sortase recognition substrate function sortase motif SCO0644 Hypothetical protein SrtE1/SrtE2 LAETG SCO0934 Hypothetical protein SrtF1 (SCO0935) Unknown SCO1674 ChpC – long chaplin SrtE1/SrtE2 LAETG SCO1734 Cbp – Cellulose binding SrtE1/SrtE2 LAETG protein SCO1860 Putative secreted protein SrtE1/SrtE2 LAETG SCO2270 Putative protein with role SrtE1/SrtE2 LASTG in iron acquisition SCO2479 Hypothetical protein SrtF2 (SCO2480) Unknown SCO2492 Hypothetical protein SrtE1/SrtE2 LAETG SCO2682 Putative secreted protein SrtE1/SrtE2 LAETG SCO2716 ChpA – long chaplin SrtE1/SrtE2 LAETG SCO2842 Hypothetical protein SrtF3 (SCO2841) Unknown SCO2971 Putative secreted protein SrtE1/SrtE2 LAETG SCO3176 Putative secreted protein SrtE1/SrtE2 LASTG SCO3738 Putative integral SrtF4 (SCO3737) Unknown membrane protein SCO4782 Hypothetical protein SrtE1/SrtE2 LAATG SCO 5650 Hypothetical protein SrtE1/SrtE2 LAETG SCO6904 Putative secreted protein SrtE1/SrtE2 LAHTG SCO7257 ChpB SrtE1/SrtE2 LAHTG SCO7449 Hypothetical protein SrtF5 (SCO7450) Unknown

3.5 Figures

Figure 3.1 Phenotype of sortase mutants on MS agar after 7 days growth. Deletion of one housekeeping sortase (∆srtE1/E2 + srtE1 pMS82 and ∆srtE1/E2 + srtE2 pMS82) results in a modest delay in development, but after 7 days growth, the phenotype resembles the wild type strain (M600). Deletion of both sortases delays aerial hyphae formation and inhibits spore formation. Image modified from Duong et al, 2012.

Figure 3.2 Scanning electron micrographs of wild type (M600) and double sortase mutant (∆srtE1/E2) strains after growth on MS agar for 5 days. A) Wild type spores in spore chains are more uniform, whereas ∆srtE1/E2 does not develop into spores, and forms branched aerial hyphae. White bars = 10 µm. B) The paired rodlet ultrastructure is visible on the spores of M600 spores, whereas the surface of the ∆srtE1/E2 strain appears smooth and lacks the rodlet ultastructure. White bars = 200 nm. Scanning electron micrographs provided by Kim Findlay.

Figure 3.3 Phenotypic analysis of the wild type (M600), long chaplin mutant (∆chpABC), chaplin mutant (∆chp), and the double sortase mutant (∆srtE1/E2) grown on MS agar for 2 and 6 days. The wild type and long chaplin mutant strains are able to sporulate, as evidenced by the grey pigmentation, whereas the double sortase mutant strain is arrested at the aerial hyphae stage. The chaplin mutant, lacking all eight chaplins, had the greatest defect in development in growth, and only grows vegetatively.

Figure 3.4 The rodlet ultrastructure of M600 and ∆chpABC strains A) Scanning electron micrograph showing the paired rodlet ultrastructure of spores in the wild type (M600) and long chaplin mutant (∆chpABC). White bars = 200 nm. B) A distribution of rodlet lengths in the wild type (N = 207) and the long chaplin mutant (N=307).

Figure 3.5 Transcript analysis of the srtE1/E2 locus A) Diagram of genetic organization of housekeeping sortases and upstream gene. The numbered fragments correspond to regions used for RT-PCR analysis in B. B) Semi-quantitative PCR of sortase genes (iv and vi) and upstream genes (i). Fragments used to test for co- transcription include (ii), (iii), and (v).

Figure 3.6 Sortase cleavage assay showing in vitro cleavage of fluorescent peptides by sortases and active site mutants. SrtE1 and SrtE2 cleaved both the ChpC and ChpB peptides. Active site mutants SrtE1 C320A and SrtE2 C220A showed considerably less cleavage of all peptides. SrtA was most effective at cleaving the Spa (LPETG) peptide.

Figure 3.7: LC/MS analysis of the cleavage products in the in vitro cleavage assay. Four cleavage products are evident when ChpC (LAETG) or ChpB (LAHTG) peptides are incubated with SrtE1 or SrtE2 (i, ii, and iv). Four cleavage products were seen, suggesting that cleavage of the peptide was occurring between the threonine (T) and the glycine (G) residues as well as between the alanine (A) and the variable third residue of the pentapeptide (glutamic acid, E, or histidine, H) SrtE1 and SrtE2 active site mutants showed no cleavage, and only the full length peptide was observed (iii and v). Cleavage of the Spa peptide (LPETG) was only evident when incubated with SrtA (vi).

Figure 3.8: Semi-quantitative RT PCR analysis of genes expressed during aerial growth. RT PCR was performed using RNA isolated from wild type, long chaplin (∆chpABC) and a double sortase mutant (∆srtE1/E2) over a four day time course grown on MS agar. Amplification of 16S rRNA was used as a positive loading control for RT PCR.

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CLUSTAL O(1.2.1) multiple sequence alignment

SrtA ------0 SrtE1 VTALRPERDSGTAGDQGSSYGQPYGDSGAFGGGRYEESAAGEENRPPLLDDETVALRIPE 60 SrtE2 ------0

SrtA ------0 SrtE1 PPAPRTAAGTGPIGGGPDGGGRAARRKAAKRRHGRRGAPRDQAPEEEAEQAPKAPLSRVE 120 SrtE2 ------VAATTD---TEHQEQA------G 14

SrtA ------MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKIEQY 45 SrtE1 ARRQARARKPGAAVVASRAIGEIFITTGVLMLLFVTYQLWWTNV---RAHAQANQA---- 173 SrtE2 TGGRGRRRPGRIAAQAVSVLGELLITAGLVMGLFVVYSLWWTNV---VADRAADKQ---- 67 .:: :*::: *...* : .: . ::

SrtA DKNVKEQASKDNK---QQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVS 102 SrtE1 ASNLQDDWAN------GKRSP-GSFEPGQGFALLHIPKLDVVVPIAEGISSKKVLDRGMV 226 SrtE2 AEKVRDDWAQDRVGGSGQDGP-GALDTKAGIGFLHVPAMSEGDILVEKGTSMKILNDGVA 126 .::::: :: : * : . :.:* . : :: : *: *:

SrtA FAEEN------ESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKM 155 SrtE1 GHYAEDGLKTAMPDAKAGNFGLAG HRNTH--GEPFRYINKLEPGDPIVVETQDKYFVYKM 284 SrtE2 GYYTDPVKATLPTSDEKGNFSLAAHRDGH--GARFHNIDKIEKGDPIVFETKDTWYVYKT 184 : . . *:.:*.* . * :. : *. : .:. : **

SrtA TSIRD-VKPTDVEVLDE------QKGKDKQLTLITCDDYNE---KTGVWEKRKIFVATEV 205 SrtE1 ASILPVTSPSNVSVLDPVPKQSGFKGPGRYITLTT CTPEFTSKYRMIVWGKM---VEERP 341 SrtE2 YAVLPETSKYNVEVLGGIPKESGKKKAGHYITLTTCTPVYTSRYRYVVWGEL---VRTEK 241 :: .. :*.** * : :** ** : ** : * .

SrtA K------206 SrtE1 RSKG--KPDALVS* 352 SrtE2 VDGDRTPPKELR-- 253

Figure 3.9 Alignment of the two S. coelicolor housekeeping sortases with the S. aureus sortase, SrtA. The conserved alanine residue is highlighted in blue, and the isoleucine in place of the threonine residue is highlighted in red. The extended N-terminus of SrtE1 is highlighted in green.

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Chapter 4: Probing the role of sortase substrates in aerial development

Preface: The work presented in this chapter is ongoing and conclusions drawn are from the results presented. Future results may alter the presented hypotheses. Two deletion mutants referred to in this chapter, ∆sco1734 and ∆sco2971 were created by C. DiBerardo (Duong et al., 2012).

4.1 Introduction Our previous work had demonstrated that the sortases are important for the development of aerial hyphae in S. coelicolor (Duong et al., 2012). The deletion of the long chaplins, which comprise three of the predicted 14 sortase substrates (Table 3.2), had little effect on aerial hyphae formation. Here, we investigate several previously uncharacterized sortase substrates, and work towards identifying genes misregulated in the sortase mutant to determine if they have roles in morphological development requiring the function of sortases.

4.2 Results and Discussion 4.2.1 Creation of sortase substrate mutants The sortases anchor proteins to the cell wall, and we have shown that their deletion affects gene expression and aerial development. Sortase-dependent anchoring of one or more of the 14 substrate proteins may influence development. Deletion of the three long chaplin genes did not result in a drastic change of phenotype, and their deletion did not affect the expression of aerial hyphae-specific genes, suggesting that the absence of the long chaplins is not the cause of the altered transcription of developmental genes (Figure 3.8). One or more of the remaining 11 substrates (i.e. excluding the long chaplins) may promote aerial development following their attachment to the cell wall, possibly by triggering a signaling cascade that leads to aerial hyphae formation. It is not obvious which of the substrates have a role in development, as the remaining 11 sortase substrates have no homology to other proteins involved in development. Our first approach was to delete each sortase substrate, and monitor the phenotypic changes associated with this loss. If a substrate is involved in facilitating

normal aerial development, its deletion should recapitulate the developmental phenotype seen for the ∆srtE1/E2 mutant. Sortase substrates studied were prioritized based on the presence of regions of interest, conservation of genes, and if the genes were previously studied. Currently, four sortase substrate mutants have been created. Null mutations of sco0644, sco1734, sco2971 and sco5650 have been constructed using the ReDirect protocol (Gust et al., 2006). Deletion of these genes did not result in a change in morphological phenotype with the wild type strain on MS agar (Figure 4.1), suggesting that these substrates, at least individually, do not have a role in aerial development under these conditions. The deletion of the remaining sortase substrates is underway.

4.2.2 Transcript analysis of sortase substrate genes In some pathways, the expression of substrate genes can depend on the activity of the protein required for their function. For example, in E. coli and Salmonella typhimurium, the secretion of flagellar structural proteins (FlgM) affects the expression of downstream genes (fliA and fliC) that are involved in flagellar movement (Karlinsey et al., 2000). This hierarchy of modified gene expression following secretion could also be occurring during aerial development. Sortase secretion and localization might activate expression of its substrate genes. To test this hypothesis, we assessed the transcription of sortase substrates in wild type and the double sortase mutant strain using RT PCR analysis. Briefly, RNA was isolated over a 4 day time course from both the wild type and ∆srtE1/E2 strains. The sortase substrate transcripts were reverse transcribed, and PCR was performed to monitor relative transcript levels for these genes in both strains. Comparing the expression profiles of 11 sortase substrate genes revealed that most of the substrate genes exhibited a modest decrease in expression in the ∆srtE1/E2 strain relative to wild type (Figure 4.2). One exception to this expression reduction was sco2270. In the wild type strain, expression was highest after 24 and 72 hours of growth on MS agar (Figure 4.2). However, in the ∆srtE1/E2 strain, there was only one peak in expression at 48 hours. This suggested that the expression of sco2270 may be indirectly dependent on the expression of both sortases. For the remaining sortase substrates, expression of the

sortases does not appear to have a profound effect on the expression of the substrate genes. These results revealed that expression of most of the predicted sortase substrates did not require the presence of the sortases. However, due to the observed decrease in expression of sco2270 in the sortase mutant strain, it is possible that misregulation of sco2270 transcription delays aerial hyphae formation and sporulation. SCO2270 has a putative HtaA domain, which is involved in heme or iron acquisition and transport. Iron limitation delays aerial hyphae formation, but the underlying mechanism remains to be determined (Traxler et al., 2012). If SCO2270 plays a role in promoting aerial hyphae formation, it can be hypothesized that the deletion of sco2270 may affect aerial hyphae formation, and that the delayed aerial growth and absence of sporulation seen in the double sortase mutant may be associated with delayed expression of sco2270 (Figure 4.2).

4.2.3 Accumulation of proteins in the cell membrane as a result of the sortase deletion An alternative hypothesis to explain the ∆srtE1/E2 mutant phenotype is that the sortase substrates are normally transcribed (Figure 4.2) and are (presumably) translated and secreted as normal; however, without a processing sortase at the surface, the secreted substrates accumulated in the membrane, causing membrane stress and delays proper aerial growth. Precedence for this phenomenon has been observed in Actinomyces oris. SrtA is an essential protein and suppressor mutants could only be isolated when either lcp (a transglycosylase) or acaC (a glycoprotein) were deleted (Wu et al., 2014). The authors demonstrated that both AcaC and Lcp were secreted (but not covalently attached to the cell wall) in the ∆srtA mutant. Deletion of either gene suppressed the lethal ∆srtA mutant phenotype because the accumulation of glycosylated AcaC (requiring both AcaC and Lcp) lead to cell death due to ‘membrane-jamming’ (Wu et al., 2014). In an analogous way, deletion of both Streptomyces housekeeping sortases could lead to an accumulation of sortase substrates (or modified derivatives) in the membrane, resulting in a membrane stress response, altered gene expression, and in defective development. To test this hypothesis, we are currently deleting the remaining 11 sortase substrates (individually, at least initially) in a ∆srtE1/E2 mutant strain. If an accumulation of one of these substrates causes the aerial growth defect, then deletion of the 56

corresponding substrate gene in the ∆srtE1/E2 strain should rescue the mutant phenotype. Currently, the deletion mutant of sco5650, one of the most highly expressed substrate genes, as shown by RT-PCR and RNA-seq, appeared to relieve the developmental defect seen in the ∆srtE1/E2 strain (Figure 4.2) (Moody et al., 2013). Attempts to complement this phenotype by introducing sco5650 on an integrative plasmid have however, been unsuccessful. Current efforts are directed towards recreating the ∆sco5650 mutant in the ∆srtE1/E2 strain to rule out any technical problems or suppressor mutations that may have occurred when the original ∆sco5650 was constructed. Knockout mutants of the remaining 10 individual sortase substrate mutants are currently being generated in the ∆srtE1/E2 background.

4.3 Future directions 4.3.1 Genetic analysis of the roles of sortase substrates in development In other Gram positive bacteria, the deletion of housekeeping sortases attenuates pathogenesis and pilus formation, but a fundamental defect in morphological development resulting from sortase deletion is unique to S. coelicolor (Gaspar et al., 2005; DeDent et al., 2007). Our previous results demonstrate that the deletion of srtE1/E2 resulted in a change in global gene expression, with a number of genes expressed during aerial development exhibiting delayed or greatly reduced expression (Figure 3.8). It is not obvious how SrtE1 and SrtE2 modulate gene expression. We therefore designed a set of experiments to test different hypotheses to explain this observation.

4.3.1.1 Hypothesis 1: Sortase substrate(s) are integral to aerial development One hypothesis is that sortase deletion results in a mislocalization of the specific sortase substrates, resulting in aberrant sortase substrate function. To date, we have deleted seven sortase substrates (chpA, chpB, chpC, sco0644, sco1734, sco2971 and sco5650) and have observed no defect in development compared to wild type strains (section 4.2.1). The creation of the remaining seven sortase substrate mutants will allow us to address whether a loss of function of one of these sortase substrates results in a phenotypic change similar to that of the double sortase mutant. Following creation of the mutants, the strains will be subjected a comprehensive phenotypic analysis, monitoring development on various media types, as well as in the 57

presence of environmental stress. In addition to their role in development, creation of these mutants will provide allow us to begin characterization of the function of these cell surface proteins.

4.3.1.2 Hypothesis 2: Induced accumulation of sortase substrates delays development As outlined above (section 4.2.3), the accumulation of sortase substrates in the cell membrane may cause a delay in aerial development analogous to the situation previously observed in A. oris (Wu et al., 2014). In addition to individually deleting sortase substrates (as detailed in section 4.2.3), we are also working to create a strain in which a sortase substrate possessing a mutated pentapeptide sorting signal is overexpressed. We hypothesize that such a strain may induce cell membrane stress, analogous to that which may be experienced by the double sortase mutant strain. Taking advantage of the fact that SrtE1 and SrtE2 fail to efficiently cleave the LPXTG pentapeptide motif (Figure 3.6), we are now in the process of mutating the sortase pentapeptide recognition motif in SCO5650, changing it from an LAXTG sequence to LPXTG. We predict that neither SrtE1 nor SrtE2 will recognize and cleave this substrate, resulting in an accumulation of the substrate in the cell membrane. Our rationale for working with sco5650 stems from our RT-PCR results, which showed sco5650 to be highly transcribed (Figure 4.2). If a substrate’s accumulation were to be toxic, it may be reasonable to propose that the highly expressed sortase substrates contribute more to cell membrane stress than the less-expressed sortase substrates. Following mutation of sco5650, it will be cloned downstream of the tipA promoter (in pIJ6902), which can be induced by the addition of thiostrepton to the medium (Huang et al., 2005). Following induction, growth will be monitored relative to a wild type strain grown under the same conditions containing the empty pIJ6902 plasmid. If overexpression of a secreted sortase substrate with a mutant pentapeptide sequence results in cellular membrane stress, we expect to see a delay in aerial development, similar to that seen for the ∆srtE1/E2 strain. It is worth noting that the absence of a change in phenotype following overexpression of a mutant SCO5650 would not invalidate the substrate accumulation hypothesis. It is possible that accumulation of a modified derivative of a sortase substrate or a different sortase substrate is required to cause the phenotype of the

∆srtE1/E2 mutant, whereas the accumulation of the unmodified substrate has no effect on development.

4.3.2 Identification of regulatory networks through transcript analysis (RNA-seq) Of the almost 8000 genes in S. coelicolor, almost 1000 are devoted to regulation (Bentley et al., 2002). It is expected that genes involved in aerial development would exhibit altered expression in the ∆srtE1/E2 mutant due to the delayed aerial development observed for this strain. A small-scale analysis of gene expression in the sortase mutant can be seen in Figures 3.7 and 4.2. Global transcriptome comparisons of the wild type and double sortase mutant throughout development will identify genes that are differentially regulated between these two strains. Genes that exhibit large alterations in gene expression would be predicted to be in the sortase-dependent regulatory network. RNA has been isolated from both wild type and double sortase mutant strains over a 96-hour time course prior to transcript analysis. As the two strains develop at different rates, we will be focusing on aerial hyphae-specific gene expression using the 48 hour sample of the wild type strain, and the 96 hour sample of the double sortase mutant strain: time points representing aerial hyphae growth in either strain. Genes that demonstrate a marked increase or decrease in the double sortase mutant in comparison to the wild type strain will suggest that their expression is sortase-dependent. As the sortases are not predicted to mediate gene expression directly, an intermediary protein or associated regulatory network may control the expression of these differentially expressed genes. Any regulator whose expression is significantly down- or upregulated, in the double sortase mutant, would be of great interest for follow up validation. These regulators may promote or inhibit the expression of genes involved in aerial growth. This would be evident by their change in expression during the developmental time course, and by down-stream mutational analyses.

4.4 Towards a comprehensive model for the role of sortase substrates in morphological development The number of sortase substrates predicted to be anchored by the two housekeeping sortases in S. coelicolor is relatively high, but beyond those of the long chaplins, little is known about their functions. Previously believed to be non-vital for 59

normal cell growth and division, it is becoming more evident that the sortases may be essential for cell growth, through the function of their substrates (Wu et al., 2014). In addition to the results presented thus far, identification and genetic manipulation of the sortase substrates, analysis of gene expression in the double sortase mutant will elucidate the contribution of the sortase substrates to morphological differentiation. Coupled with the identification of genes that are misregulated in the sortase mutant (and subsequent mutational studies), this work will contribute to a model of aerial development that entails sortase function.

4.5 Figure

Figure 4.1 Plate phenotypes of sortase substrate deletion mutants. Strains were cultured on MS agar for 6 days at 30°C. The wild type (M600) strain has undergone sporulation, as exhibited by the grey pigmentation on the surface of the colonies. In contrast to the double sortase mutant (∆srtE1/E2) that arrests at aerial hyphae formation (white), the deletion of the sortase substrates (sco0644, sco1734, sco2971 and sco5650) resulted in no change in phenotype, exhibiting a developmental progression similar to that of the wild type strain.

Figure 4.2: Semi-quantitative RT PCR results of sortase substrate expression RT PCR was performed using RNA isolated from wild type (M600) and double sortase mutant (∆srtE1/E2) strains reveals that the expression of sortase substrate genes is similar in both strains. sco2270 is the only gene that is differentially expressed in the sortase mutant relative to the wild type strain. 16s rRNA was used as a positive RT PCR control. Results were repeated using two biological replicates.

Chapter 5 Conclusions and Future Directions

5.1 Summary of research In the last 20 years of S. coelicolor research, the number of proteins and RNAs implicated in the multicellular development of S. coelicolor has expanded greatly. The complex multi-stage life cycle of S. coelicolor requires coordination of many more genes compared to that of single-celled bacteria. The chaplins are important in facilitating aerial development, but how they are attached to the cell wall had not previously been studied in S. coelicolor (Claessen et al., 2003; Elliot et al., 2003; Di Berardo et al., 2008). Prior to this work, the sortases were thought to anchor the long chaplins, and facilitate the transition from vegetative to aerial development. However this work has revealed that the anchoring of long chaplins is not required for aerial growth. Instead, the work presented in this thesis proposes a novel role for the sortases in development. The first study of sortase function in the actinomycetes is described in Chapter three. Through sortase activity assays, the two housekeeping sortases, SrtE1 and SrtE2, were shown to specifically recognize pentapeptide sequences found within two of the long chaplins (LAETG and LAHTG). The identification of this pentapeptide sequence aided in the subsequent identification of additional sortase substrates of SrtE1 and SrtE2. The presence of an LAXTG pentapeptide motif within a cell wall sorting signal is unique to the actinomycetes, and such a motif is found at the C-terminus of many uncharacterized proteins, which likely represent as-yet uncharacterized sortase substrates. The two sortases in S. coelicolor were also shown to be essential for normal aerial development, expanding the role of sortases from the anchoring of non-essential proteins, to having an essential role in cellular development. Through the deletion of the long chaplins, we determined that the developmental defect in the sortase mutant was not due to the loss of chaplin fibre formation, but through a novel mechanism that remains to be elucidated. Deletion of the sortases results in a severe defect in the expression of many genes that are specifically expressed in aerial hyphae.

5.2 A new working model of aerial development facilitated by the sortases When the chaplins were first identified, it was proposed that the long chaplins had a modified C-terminal cell wall sorting signal that facilitated anchoring to the cell wall

during aerial growth (Claessen et al., 2003; Elliot et al., 2003). The anchoring of the long chaplins was hypothesized to facilitate aerial growth, and catalyze chaplin fibre assembly to assist in reducing surface tension at the soil to air interface. Our work has shown that although the long chaplins are not essential for aerial development, they do play a role in determining the length and organization of chaplin fibres on the spore surface. In contrast, deletion of the two housekeeping sortases, srtE1 and srtE2, resulted in a large defect in aerial growth, suggesting that the sortases play an essential role in the formation of aerial hyphae. There are many possibilities for how sortases affect development, but we hypothesize that the anchoring of one or more non-chaplin sortase substrates triggers a cellular response, affecting the expression of genes required for aerial development. Given that substrates are anchored to the cell wall, anchoring of a sortase substrate may trigger a two-component system to relay a message to initiate aerial development through an alteration in gene expression in response to successful substrate anchoring. The signal(s) for aerial development in S. coelicolor have not been elucidated, but in the presence of an environmental stress, a rapid transition to aerial hyphae and eventually spores (the Streptomyces reproductive structures) could improve the chances of survival. S. coelicolor encodes 85 sensor kinases and 79 response regulators, and of these, only 55 exist as sensor kinase-response regulator pairs (Bentley et al., 2002). Of these pairs, only a handful have been characterized in any capacity, and for the most part, the signal that they respond to is not known (Sheeler et al., 2005; Hutchings et al., 2006; Yepes et al., 2011; Rodriguez et al., 2013). The work in chapter four will address the role of the sortases in aerial development, and identify further targets that are indirectly affected as part of the downstream regulatory cascade. Until now, sortases have never been thought to function in a regulatory capacity. This model of aerial hyphae formation could present the first described instance of sortase-mediated transcriptional regulation.

5.3 Long term goals The short-term future work will be focused on elucidating the role of the sortase substrates in S. coelicolor. By elucidating additional components that play a fundamental role in the development of this multi-cellular bacterium, we hope to contribute to a new model of Streptomyces development from which future hypotheses can be tested. 64

In working towards understanding the role of SrtE1 and SrtE2, the long term direction of this project will be to address the potential redundancy of housekeeping sortases in Streptomyces biology. It is now evident that sortase-containing organisms often harbour more than one sortase enzyme (Maresso and Schneewind, 2008; Spirig et al., 2011). When sortases of multiple classes are present, each sortase recognizes its own unique pentapeptide sequence (and therefore its own substrate), and does not overlap in function (Mazmanian et al., 2003; Marraffini and Schneewind, 2005). However, in many species, multiple sortases of the same class are encoded, and in the case of S. coelicolor, these are co-transcribed and partially functionally redundant. The experiments presented in chapter four will address potential reasons for sortase redundancy, including substrate preference, but not exclusivity. One hypothesis for these “redundant genes” could be that the presence of two housekeeping sortases are a result of gene duplication, which given an appropriate amount of time, could give rise to two non-redundant sortases with highly specialized activities (Serres et al., 2009). Gene duplication, following by specialization is a common phenomenon in bacteria (Francino, 2012; Kondrashov, 2012). The individual sortase mutants have subtly different phenotypes: deletion of srtE1 results in a slightly greater delay in the initiation of aerial hyphae formation compared to the ∆srtE2 and wild type strains. SrtE1 contains an extended N-terminus, relative to SrtE2 and the extensively studied, SrtA (Figure 3.9, green box). The function of this extended N-terminus is unknown, but previous studies have shown that this region is important for SrtE1 function (DiBerardo and Elliot, unpublished). It can be hypothesized that the N-terminus of SrtE1 aids in its localization. As such, the localization signal may facilitate sortase substrate anchoring at the site of cell wall synthesis, expediting aerial hyphae formation, such that even when srtE2 is deleted, the phenotype appears comparable to WT. In contrast, as SrtE2 lacks this localization signal, its localization may be less efficient, so when SrtE2 is the sole housekeeping sortase, there is a subsequent delay before the onset of aerial hyphae formation. Finally, the work here has provided a stepping stone for understanding multicellular development in S. coelicolor. The ultimate goal is to determine the mechanism by which these two housekeeping sortases influence Streptomyces developmental biology. SrtE1 and SrtE2 are predicted to be highly conserved within the actinomycetes,

so the elucidation of their role in S. coelicolor could provide insight into the role of this protein family within diverse high G+C Gram positive bacteria.

Appendix Preface: This section includes ongoing work that is aimed towards the identification of the complete covalently-bound cell wall proteome. The identification and analysis of sortase substrates will be addressed independently from their role in development.

6.1 Analysis of the covalently attached cell wall proteome Bioinformatic analysis has lead to the prediction that S. coelicolor encodes 14 SrtE1 and SrtE2 substrates (Table 3.2), while RT PCR has confirmed the expression of most of these genes during aerial growth (Figure 4.2). We sought to confirm these proteins as sortase substrates by exploiting the fact that sortases provide the only known means of covalently attaching proteins to the peptidoglycan in Gram positive bacteria (Hendrickx et al., 2011). To test this hypothesis, biomass was harvested from strains grown on MS agar at all developmental stages, and we have isolated proteins covalently anchored to the peptidoglycan by taking advantage of the insolubility of peptidoglycan in detergents, like SDS (Calvo et al., 2005). Following the isolation of the insoluble peptidoglycan, the peptides will be liberated from the peptidoglycan by trypsin digestion. The peptides will be identified using tandem mass spectrometry and aligning their sequence with the annotated proteins of S. coelicolor.

6.1.2 Identification of sortase substrates Analysis of the covalently attached cell proteome of wild type S. coelicolor will be used to identify all proteins covalently attached to the cell wall. In addition, a double sortase mutant will be examined to identify proteins attached by the housekeeping sortases. Proteins that are not predicted SrtE1/E2 substrates and are found covalently anchored to the peptidoglycan in the ∆srtE1/E2 mutant may be attached by one of the other five predicted sortases in the S. coelicolor genome (Table 3.1). These five other sortases have sequence similarity to SrtB, SrtC and SrtD, and are not predicted to be housekeeping sortases. For this reason, they have been designated SrtF1 to SrtF5 (actinomycete-specific sortases of unknown function). Since the substrates of non- housekeeping sortase are often located in the same genetic locus as their processing sortases, we expect that predicted SrtF substrates would be present in the covalently- anchored cell wall proteome of the ∆srtE1/E2 mutant. (Table 3.2, SrtF substrates).

The presence of any proteins that are not predicted SrtF substrates in this proteome may represent new sortase substrates not predicted by bioinformatics analysis, or may indicate that there exists an additional, sortase-independent mechanism(s) for covalently attaching proteins to the cell wall.

6.1.3 Determining substrate specificity of the sortases in vivo In addition to experimental validation of the sortase substrates, we also sought to determine if either housekeeping sortase exhibits substrate specificity. Our previous in vivo deletion mutants and in vitro sortase cleavage assays suggested that both SrtE1 and SrtE2 may have both distinct and overlapping functions (Figures 3.1 and 3.6). The deletion mutants suggest that SrtE1 may be the dominant sortase for anchoring proteins to the peptidoglycan in vivo, based on the longer delay in aerial hyphae formation observed for the srtE1 mutant compared to either a srtE2 mutant or the wild type (Figure 3.1). It is not currently known if the third variable amino acid in the LAXTG sequence (glutamine, serine, alanine and histidine) could affect recognition of the pentapeptide by the sortase in vivo. The covalently anchored cell wall proteome from the ∆srtE1 and ∆srtE2 mutants will be compared to determine if either protein displays substrate specificity. The absence of a protein in one sortase mutant (and its presence in the other) would suggest that the substrate is specifically recognized by the sortase deleted in that strain. This would provide an excellent opportunity to functionally dissect the role of two housekeeping sortases in one organism.

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