Characterization of an exclusion mechanism in an integrative and conjugative element in Bacillus subtilis

Characterization of an exclusion mechanism in an integrative and conjugative element in Bacillus subtilis

Monika M. M. Avello B.A. Chemistry

Florida International University 2010

Submitted to the Department of Biology in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Biology Massachusetts Institute of Technology

September 2018

C 2018 Monika M. M. Avello. All rights reserved. The author hereby grants to MIT permission to reproduce

and to distribute publicly paper and electronic copies of this thesis document in whole or in part

in any medium known or hereafter created.

Signature redacted

Signature redacted

Signature redacted

MASSACHUSETTS INSTITUTE A''y E. Keating

OF TECHNOLOGY Professor of Biology and Biological Engineering

Characterization of an exclusion mechanism in an integrative and conjugative element in Bacillus subtilis

by

Monika M. M. Avello

Submitted to the Department of Biology on August 2018 in partial fulfillment of the requirements for the degree of

Doctor of Philosophy in Biology Abstract

Horizontal gene transfer is the acquisition of new genetic material that can confer novel

phenotypes to bacteria and contribute to their evolution. Conjugation is an important mechanism of horizontal gene transfer that involves the direct transfer of DNA between two cells and is mediated by mobile genetic elements encoding type IV secretion systems. Conjugative elements prevent redundant transfer by a mechanism known as exclusion that inhibits their cognate

secretion systems. Exclusion is prevalent among conjugative elements, suggesting it is advantageous and potentially essential. Yet very few exclusion mechanisms are characterized, and the advantages they provide are not well understood. My work characterizes the exclusion mechanism of an integrative and conjugative element found in a Gram-positive bacterium. In combination with several other studies, my results point to a potentially conserved mechanism and novel benefits of this phenomenon, furthering our understanding of how mobile genetic elements regulate their transfer, impact their bacterial hosts, and mediate horizontal gene transfer.

Thesis Supervisor: Alan D. Grossman Title: Professor of Biology

Acknowledgements

I'm truly grateful to Alan Grossman, a phenomenal mentor who's mastered the art of directness and clear feedback (no punches pulled) balanced with kindness, understanding, and tact. The members of the Grossman lab have all been wonderful and supportive colleagues, beside and away from the bench. I will miss them very much.

I would not have dreamed of setting foot into MIT, if it were not for my peer and mentor, Leilani Chirino. Her enthusiasm for science inspired me to conduct biomedical research during our undergraduate years at Florida International University; it was her direct influence that led me to MIT -first as a post-baccalaureate student, then as a graduate student. I would also like to acknowledge Dr. DeEtta Mills, who welcomed me with open arms into her lab, as well as Dr. Charles Bigger & Aileen Landry from the MBRS-RISE program for championing students and encouraging their scientific pursuits.

My family has always been fiercely supportive of my academic pursuits, in particular my mother Mieko, father Dario, and younger sister Aika.

Last (but not least!) I'd like to thank my partner, Justin Wright, for being a fan of dinosaurs, mountains, and cuddles, becoming a top notch brewer of espresso (despite not drinking coffee at all) and making me coffee every morning, and graciously excusing my stressed-out self (especially during thesis writing).

Table of Contents

Chapter 2 -Identification, characterization, and benefits of an exclusion mechanism in an integrative and conjugative element of Bacillus subtilis -36

List of Tables

Chapter Table Title Page

1 1 ICEBs] genes and functions 19

List of Figures

Chapter Figure Title Page

1 ICE lifecycle 13

Models of T4SSs from Gram-Negative and

2 Gram-Positive bacteria 16

3 Genetic map of ICEBs] 18

4 The T4SS of ICEBs] 21

5 Regulation of ICEBs] 23

Topologies of F/Rl00, SXT/R391, and

6 R64/R621a exclusion proteins and targets 25

2 1 Genetic map of ICEBsJ 68

ICEBs] in recipient cells inhibits acquisition of

pC194 mobilized by ICEBs], but not Tn916,

2 conjugation machinery 69

In recipient cells, ICEBs] gene yddJ is necessary

3 and sufficient for exclusion 70

Isolation of exclusion-resistant conG mutations

4 in ICEBs] 71

ICEBs] and ICEBat] homology and exclusion

5 specificity 73

Exclusion is beneficial to ICEBsJ and its host cells by preventing loss of viability due to

Chapter 1

Overview

Horizontal gene transfer describes the acquisition of new genetic material by various processes and is prevalent in microbes. Horizontal gene transfer can confer novel and advantageous phenotypes to bacteria and accounts for their rapid adaptability and evolution. Conjugation is an important mechanism of horizontal gene transfer, involving the direct transfer of DNA between two cells, mediated by mobile genetic elements encoding type IV secretion systems. Exclusion describes mechanisms by which conjugative elements prevent redundant transfer, typically by blocking their cognate secretion systems. Most conjugative elements encode an exclusion mechanism, suggesting it is advantageous and important. My work characterizes the exclusion mechanism of an integrative and conjugative element found in a Gram-positive bacterium and highlights potentially conserved strategies and benefits of this phenomenon, furthering our understanding of how mobile genetic elements regulate their transfer, impact their bacterial hosts, and mediate horizontal gene transfer.

Horizontal gene transfer

Horizontal gene transfer (HGT) is the acquisition of new genetic material by various mechanisms, contributing to bacterial genomic plasticity and diversity. Several theoretical models argue HGT to be essential for microbial evolution and survival (Koonin 2016). Horizontally acquired DNA is prevalent in bacteria and can range up to ~20-30% of some genomes (Ochman et al. 2000; Narra & Ochman 2006). HGT has been demonstrated to confer bacteria a wide variety of advantageous phenotypes, such as pathogenesis, antibiotic resistance, symbiosis, metabolic capabilities, and biofilm formation.

Tumor inducing (Ti) plasmids allow Agrobacterium tumefaciens to reprogram plant cells, transforming them into gigantic crown galls which produce opines, amino acid-sugar substrates that the bacteria can then use as carbon and nitrogen sources (Pitzschke & Hirt 2010).

The transfer of antibiotic resistance genes has led to the rise of clinically relevant,

antibiotic resistant Gram-negative pathogens such as Escherichia coli, Pseudomonas aeruginosa,

Vibrio cholerae, Proteus mirabilis, Acinetobacter baumannii (Stokes & Gillings 2011) and

Gram-positive pathogens such as Staphylococcus aureus, Clostridium difficile, and various species of Enterococcus and Streptococcus (Roberts & Mullany 2011).

Rhizobia are a genus of Gram-negative soil bacteria that can live symbiotically in

nitrogen fixing nodules on the roots of legume plants (Remigi et al. 2016). Symbiosis requires

nod and nif genes that allow bacteria to respond to plant signals to invade root cells, induce

nodulation, and fix nitrogen. These symbiosis genes are often horizontally acquired via plasmids, ICEs, or genomic islands. Examples include the large (~250-500 kb) plasmids p42d and

pNGR234a/b isolated from Rhizobium etli (Crossman 2005) or the ~500 kb ICEMISymR7a, first

isolated from Mesorhizobium loti and found in related species (Haskett et al. 2017).

Genes encoding resistance to metals such as mercury (Osborn 1997), cadmium, copper, chromium, lead, and nickel (Das et al. 2016) are carried and spread by plasmids and transposons in Gram-negative and Gram-positive bacteria. The TOL plasmid, first isolated from

Pseudomonas putida and found in related species, confers the ability to degrade toluene and

xylene (Burlage et al. 1989). The NAH7 plasmid, also isolated from P. putida, confers the ability to degrade naphthalene (Yen et al. 1988).

Horizontally acquired elements have been shown to affect biofilm formation, mostly in Gram-negative bacteria (Cook & Dunny 2014; Bhatty et al. 2015). Biofilm formation in E.coli K- 12 strains is enhanced when cells contain the F plasmid, and is dependent upon the

conjugative pili (Ghigo 2001; Reisner et al. 2003). The pOLA55 plasmid increases E. coli

biofilm formation by producing type III fimbriae, short pili-like appendages that aid in cell adhesion (Burmolle et al. 2008). The TOL plasmid in Pseudomonas putida enhances biofilm formation by increasing extracellular DNA (D'Alvise et al. 2010).

Mechanisms of HGT

HGT is mediated by a variety of mechanisms encoded by bacteria and/or mobile genetic elements that move within and/or between genomes, including plasmids, integrative and

conjugative elements, integrons, transposons, and bacteriophages. There are three

well-established mechanisms of HGT in bacteria: transformation, transduction, and conjugation (Frost et al. 2005). Conjugation will be described in detail as it is important for this thesis.

Transformation is the uptake of DNA from the environment through bacterially encoded DNA uptake complexes that resemble type II secretion systems and type IV secretion system pili. Over 80 different bacterial species have been shown to be naturally competent (capable of

transformation). Competence is a regulated physiological state many bacteria develop in

response to specific cues, such as entering stationary phase, high cell density, DNA damage, and starvation (Chen & Dubnau 2004; Blokesch 2016).

Transduction is the transfer of bacterial DNA by bacteriophages. General transduction occurs if a phage accidentally packages host cell DNA, creating an infectious particle that delivers bacterial, rather than viral, DNA into a new host cell. Specialized transduction occurs if

a lysogenic phage imprecisely excises flanking chromosomal DNA along with viral DNA and transmits this to new host cells (Canchaya et al. 2003).

Conjugation is the transfer of single stranded DNA (ssDNA) between two directly contacting cells by a type IV secretion system (T4SS) encoded by mobile genetic elements. Conjugation occurs in four different steps (Bhatty et al. 2013; Guddon et al. 2017):

1) The donor and recipient cells contact and form a stable mating pair for the duration of DNA transfer. While not well understood, it is thought that contact between donor and recipient cells signals the start of the DNA transfer process.

2) The DNA to be transferred is processed to ssDNA. This is accomplished by proteins encoded by DNA transfer and replication (DTR) genes. Of these, the relaxase gene is essential. The relaxase nicks the DNA at a specific site within a region known as the origin of transfer (oriT), and covalently binds the resulting ssDNA. The nucleoprotein complex of ssDNA bound to relaxase and auxiliary proteins (which can be host and/or element-encoded) is referred to as the relaxosome.

3) The type IV coupling protein (T4CP) recognizes the relaxosome and couples it to the the T4SS, a multimeric membrane spanning complex encoded by a set of mating pair

formation (MPF) genes.

4) The relaxase-ssDNA complex is transferred from the donor to recipient cell through the T4SS. Transfer is coupled with rolling circle replication of the DNA in the donor. Once transferred into the recipient, the ssDNA is re-circularized and undergoes second strand synthesis to become dsDNA.

Conjugative mobile genetic elements

Conjugation is mediated by two types of mobile genetic elements that encode a T4SS: conjugative plasmids and integrative and conjugative elements (ICEs), also known as

conjugative transposons (Bhatty et al. 2013). Conjugative elements transfer themselves, as well as other genetic elements that do not encode a T4SS, a phenomenon known as mobilization

(Ramsay & Firth 2017). Conjugative plasmids and ICEs often carry cargo genes that confer a variety of advantageous phenotypes to their host cells (Frost et al. 2005).

Plasmids are extrachromosomal, typically circular DNA that can autonomously replicate in a host cell. They regulate their copy numbers (the number of plasmids per cell), which can range from one to several hundred. Some plasmids, particularly those with low copy numbers,

encode partition mechanisms to ensure equal segregation during cell division and stable vertical transmission (Novick et al. 1976; Ebersbach & Gerdes 2005). Different plasmids that share

similar replication or segregation modules interfere with each other's stable inheritance and therefore cannot coexist or are 'incompatible'. Plasmids are classified and referred to by their

incompatibility (Inc) groups based on this phenomenon (Novick 1987).

ICEs are modular genetic elements similar to plasmids and lysogenic phage. While capable of autonomous replication like plasmids (Lee et al. 2010; Wright & Grossman 2016; Carraro et al. 2015), ICEs generally take a passive approach to persisting in host cells (Bafnuelos-Vazquez et al. 2017) (Fig. 1). ICEs reside integrated in the host chromosome in a mostly

transcriptionally quiescent state, similar to lysogenic phages. Stochastically (Minoia et al. 2008) or under specific conditions (Johnson & Grossman 2015), ICEs excise out of the chromosome to form plasmid-like circular intermediates that express conjugation genes for processing and

transferring DNA into neighboring recipient cells. After successfully entering a recipient cell as ssDNA, ICEs re-circularize, become double-stranded, and integrate into the new host

chromosome. Recipients that stably inherit ICEs (or plasmids) by conjugation are referred to as transconjugants (Wozniak & Waldor 2010; Johnson & Grossman 2015). While not as widely studied as plasmids, ICEs have been found to outnumber conjugative plasmids in an analysis of over 1000 bacterial genomes (Guglielmini et al. 2011), suggesting they play a significant role in bacterial adaptation. Recipient T4SS T4relaxase chromosome

O

0

0

Gene Expression DNA processing

Excision Conjugation machinery 0)

0

0

Fig. 1. ICE lifecycle. See text above for description. Plasmid transfer by conjugation occurs in the same fashion, except they do not integrate into the host chromosome.

Conjugative T4SSs

Conjugative T4SSs comprise the largest of three subfamilies of T4SSs; the other two are dedicated to the secretion of protein effectors by pathogens such as Legionella pneumoniae,

Bordetella pertussis, Helicobacter pylori, and Coxiella burnetti, or the release and uptake of

substrates from the environment without cell contact, such as the GGI system of Neisseria

gonorrhoeae (Bhatty et al. 2013). Recent phylogenetic studies suggest that conjugative T4SSs

evolved first in Gram-negative bacteria before spreading to Gram-positive bacteria and Archaea (Guglielmini et al. 2013; Guglielmini et al. 2014). According to these studies, there are eight evolutionary MPF subclasses of T4SSs. Six are found in Gram-negative bacteria: four in Proteobacteria (MPFF, MPFT, MPFI, and MPFG), one in Bacteroides (MPFB), and one in Cyanobacteria (MPFc). Two are found in Gram-positive bacteria and Archaea: MPFFA and

MPFFATA.

Structurally, all T4SSs share six core components: two cytoplasmic, membrane-associated motor ATPases (including the T4CP that recognizes the relaxosome) that provide energy for secretion, an extracellular cell wall hydrolase that digests peptidoglycan, and three membrane proteins that form the channel that delivers the substrate from the donor into the recipient. These components are described from the prototypical conjugative T4SS, the VirB and VirD proteins of the Ti plasmid from A. tumefaciens (Bhatty et al. 2013; Sharifahmadian &

Baron 2017).

The VirD4 and VirB4 motor ATPases: VirD4 is the T4CP that recognizes and couples the relaxosome to the T4SS. VirD4 is predicted to form a hexamer anchored to the inner membrane by its N-terminal transmembrane domain, and a cytoplasmic C-terminal region

containing ATPase domains and an all alpha domain important for relaxosome specificity (Gomis-Rtith et al. 2001; Whitaker et al. 2015). VirB4 is predicted to form a hexamer with membrane-associating N-terminal domains and C-terminal ATPase domains (Middleton et al. 2005; Draper et al. 2006). Both VirD4 and VirB4 have been shown to interact with membrane channel components and with the transferred DNA (Cascales & Christie 2004; Atmakuri et al. 2004). Both of their ATPase activities are essential for transfer (Atmakuri et al. 2004).

The VirB 1 cell wall hydrolase: VirB 1 is a cell wall hydrolase that localizes to the inner membrane with an N-terminal lytic transglycosylase domain that cleaves peptidoglycan strands. It is thought to aid in localized cell wall degradation for the assembly of the T4SS channel, but is not absolutely required for transfer (Baron et al. 1997). VirB 1 is cleaved at the cell surface to release a soluble C-terminal fragment called VirB 1* that is essential for pilus formation (Baron et al. 1997; Zupan et al. 2007).

The VirB3, VirB6, and VirB8 membrane channel: VirB3 is a small hydrophobic inner membrane protein with two transmembrane domains that is strongly associated with the VirB4 ATPase (Jones et al. 1994; Yuan et al. 2005; Mossey et al. 2010). VirB6 is a polytopic inner membrane protein with five transmembrane domains and a large central periplasmic domain (Jakubowski et al. 2004). VirB8 is a bitopic protein with an N-terminal cytoplasmic region, a transmembrane domain, and large periplasmic C-terminal tail (Thorstenson & Zambryski 1994; Das & Xie 2000). It is thought that VirB6 and VirB8 oligomerize and their periplasmic domains largely form the membrane channel (Cascales & Christie 2004; Jakubowski et al. 2004).

All T4SSs carry related homologs or functionally/structurally similar analogs of these six Vir components from the Ti plasmid. Beyond this common core set, there are many differences

between T4SSs found in Gram-positive versus Gram-negative bacteria. While T4SSs found in Gram-positive bacteria are 'minimal' and mostly composed of the six core components, T4SSs found in Gram-negative bacteria are more complex, and encode up to five additional conserved components (Fig. 2) (Bhatty et al. 2013; Goessweiner-mohr et al. 2014). These differences reflect adaptations to cell envelope architecture. Gram-positive bacteria have a single cell membrane surrounded by a thick peptidoglycan layer, while Gram-negative bacteria have two (an inner and outer) membranes, sandwiching a thin peptidoglycan layer dispersed in periplasmic space (Silhavy et al. 2010). Since T4SSs in Gram-negative bacteria need to secrete across an additional membrane, they encode three outer membrane proteins (VirB7, VirB9, and VirB 10) that form a complex that interacts with the inner membrane channel (Fronzes et al. 2009).

Gram Negptive _r85

viral*

VirfVIr(i

Substrate Seretion Pathway

Gram Positve

Substrate S n Pathm

Fig. 2. Models of T4SSs from Gram-Negative and Gram-Positive bacteria. A model of the VirB/D T4SS is depicted on the left. A generic model of how the six core VirB/D components

might assemble as a T4SS in Gram-positive bacteria is depicted on the left (Figure from (DeWitt 2013), adapted from (Alvarez-Martinez & Christie 2009)).

Some T4SSs in Gram-negative bacteria encode pili components that aid in mediating donor to recipient cell contact (Bhatty et al. 2013; Arutyunov & Frost 2013). T4SSs in Gram-positive bacteria so far have not been found to encode any pili, though sex-pheromone

responsive conjugative plasmids in E.

faecalis

There are also differing requirements for cell wall hydrolytic activity. Most T4SSs in Gram-positive bacteria encode cell wall hydrolases with multiple enzymatic domains that are essential for transfer, as demonstrated for pCF 10, pIP501, pCW3, and ICEBs1 (Laverde Gomez et al. 2014; Arends et al. 2013; Bantwal et al. 2012; Dewitt & Grossman 2014). In contrast, the cell wall hydrolases of T4SSs in Gram-negative bacteria tend to have a single enzymatic domain and are partially dispensable, such as those of the Ti plasmid, R1, and pKM 101 (Baron et al. 1997; Bayer et al. 1995; Winans & Walker 1985).

ICEBsJ - an integrative and conjugative element in B. subtifis

ICEBs1 is a ~20 kb ICE found integrated at the 3' end of the tRNA gene trnS-leu2 in the

Gram-positive bacteria Bacillus subtilis and related species (Burrus et al. 2002; Auchtung et al. 2005; Earl et al. 2007). ICEBs1 is a robust model to study ICE biology. It resides in a genetically tractable host (making it amenable to experimental manipulation) and can be induced to transfer

at remarkably high frequencies. Over-expression of the ICEBs1 activator RapI results in excision in >90% of donor cells (Auchtung et al. 2005; Lee & Grossman 2007), and recipients that

to -20% transconjugants per donor cell (Chapter 2). ICEBs] encodes 25 genes (Table 1) organized in a modular fashion, with regulatory and accessory genes on the left and right sides flanking conjugation genes in the middle (Fig. 3).

immR Pxi Py'kJ PyWdK Prpi PphrI

VirD4 VirB8 VirB3 VirB4 VirB6 VirB1

mooL

attR

int A R xis helP conQ nicK B C D E yddF conG cwlT I J K rapi phril yddM

imm con ydd

DNA Regulation I Conjugation Unknown

processing

Fig. 3. Genetic map of ICEBsJ. Open reading frames are indicated by horizontal arrows pointing in the direction of transcription, with the gene name indicated below. The color and patterns indicate the gene's function as DNA processing (diagonal stripes), regulation (black), conjugation (gray), or unknown (white). Conjugation genes encoding proteins

homologous/analogous to the VirB/D T4SS are indicated by the corresponding Vir protein names in bold above the arrows. The positions of the promoters for immR, xis, yddl, yddK, rapI,

phr, and an uncharacterized small antisense RNA are indicated by vertical arrows with the

arrow head pointing in the direction of transcription. Black boxes indicate the 60 bp att repeats marking the ends of the element (Auchtung et al. 2016). See Table 1 for detailed functions and references.

Table 1. ICEBsJ genes and functions.

ORF Function References

int Site-specific recombinase (integration and excision) (Lee et al. 2007)

(Bose et al. 2008; Bose & immA Anti-repressor protease (ImmR degradation) Grossman 2011)

irnmR Transcriptional repressor; Immunity repressor (Int inhibition) (Auchtung et al. 2007) Xis Recombination-directionality-factor (promotes Int towards excision) (Lee et al. 2007) ydzL Unknown

ydcO Unknown

Helicase processivity factor (unwinding of DNA for

helP replication/transfer) (Thomas et al. 2013)

conQ Coupling protein ATPase (VirD4-like) (Lee et al. 2012)

nicK Relaxase (nicks DNA at oriT for replication/transfer) (Lee & Grossman 2007) ydcS Unknown

ydcT Unknown

yddA Unknown

conB Membrane channel (VirB8-like) (Leonetti et al. 2015)

conC Membrane channel (Leonetti et al. 2015)

(Berkmen et al. 2010;

conD Membrane channel (VirB3-like) Leonetti et al. 2015)

(Berkmen et al. 2010;

conE ATPase (VirB4-like) Leonetti et al. 2015)

yddF Unknown; Mild effect on mobilization (Leonetti et al. 2015) (Leonetti et al. 2015), conG Membrane channel (VirB6-like); Exclusion target This thesis

(Fukushima et al. 2008; cwiT Cell wall hydrolase (VirBI-like) Dewitt & Grossman 2014)

yddI Unknown (Leonetti et al. 2015)

ICEBsJ encodes a MPFFA class T4SS (Guglielmini et al. 2013) consisting of ConQ/the

VirD4 T4CP, ConE/the VirB4 ATPase, Cw1T/the VirB 1 cell wall hydrolase, and the membrane channel comprised of ConC, ConD/VirB3, ConG/VirB6, and ConB/VirB8 (Fig. 4). All of these are essential for ICEBsJ transfer. ConQ has a predicted structure matching that of VirD4 and other T4CPs, N-terminal transmembrane domains and cytoplasmic, C-terminal ATPase domains. ConE is likely a peripheral membrane ATPase that depends on ConB and ConD to localize properly (Berkmen et al. 2010; Leonetti et al. 2015). CwlT is a bifunctional cell wall hydrolase containing muramidase and peptidase domains that cleave peptidoglycan strands and peptide crosslinks respectively (Dewitt & Grossman 2014). ConC is a predicted small membrane protein that does not resemble any Vir component and may represent a Gram-positive-specific

adaptation, as proteins similar to it in topology exist in other T4SSs in Gram-positive bacteria (Alvarez-Martinez & Christie 2009). ConD is a small membrane channel protein analogous to VirB3 that interacts with ConE. The membrane channel components ConG and ConB (analogous to VirB6 and VirB8) are respectively predicted to be polytopic and bitopic membrane proteins with extracellular C-terminal tails that likely form the bulk of the channel (Leonetti et al. 2015; Auchtung et al. 2016).

yddK. Unknown

(Auchtung et al. 2005; Bose

rapI Cell-cell signaling (Activates ImmA) et al. 2008)

hrI Cell-cell signaling (Inhibits Rapl) (Auchtung et al. 2005)

Cell wall Cell membrane Cytoplasm ---- - -- - -- ---- -- - - - -- C- --- C--- n--- - - -- - - - ---C-- nC-- --- -- - -- - -- C-- p- ng- -- - -- --- - -- - --- ---------- -------p r- --------------- --- ---- --- - - ---------------------C- ----E -- --A TP--- N- ------ --- ----K- ---~~~~A - - - -- - - - -P-- - - - -- - - - -D +-- Conjugative ssDNA ATP

Fig. 4. The T4SS of ICEBsJ. The predicted model for the T4SS of ICEBs] (Adapted from (Auchtung et al. 2016)). Oligomerization and topologies of the components are not confirmed and inferred from what is known of analogous proteins from T4SSs in Gram-negative bacteria (Alvarez-Martinez & Christie 2009). Refer to Table 1 and text above for description and references.

Regulation of ICEBsJ

The ICEBsJ transcriptional repressor ImmR autoregulates its expression and represses expression from the Pxis promoter, maintaining the integrated, quiescent state (Auchtung et al. 2007). The activation of ICEBsJ requires the degradation of ImmR by the ICE-encoded protease ImmA (Bose et al. 2008). ImmA can be activated through two pathways - the DNA damage response or the ICE-encoded RapI-PhrI cell signaling system (Fig. 5). In the former, the

presence of ssDNA (which can result from double-stranded breaks or replicative fork collapses) activates RecA, a host protein crucial for both homologous recombination and DNA repair (Bell & Kowalczykowski 2016). Activated RecA in turn activates ImmA, resulting in the degradation of ImmR and ICEBsJ activation (Auchtung et al. 2005; Bose et al. 2008; Bose & Grossman 2011).

In the absence of damage, ImmA is regulated by the ICE-encoded RapI-PhrL cell signaling system. RapI is a cytoplasmic activator of ImmA. Expression of RapI from its promoter is repressed by the host transcriptional factor AbrB (Auchtung et al. 2005). AbrB is active during low cell density (typically during exponential phase) and inactivated by high cell density and low nutrient availability (typical during stationary phase) (Phillips & Strauch 2002). The regulation of RapI by AbrB ensures that no RapI is produced (thus no ICE transfer) during conditions that favor rapid host cell growth. Instead, RapI is produced when cells are slow growing, crowded, and nutrient-limited.

PhrI is a small peptide inhibitor of RapI that must be exported out of the cell to be processed into its active pentapeptide form. If present in sufficient extracellular concentration, the processed PhrI is reimported into the cell, where it prevents RapI activity (Auchtung et al. 2005). This blocks ICEBsJ from initiating excision and transfer when concentrations of donor cells (containing ICEBsJ) are high. PhrI is expressed from its own promoter which is regulated by the stationary phase regulator sigma factor H (McQuade et al. 2001) and co-expressed from

the rapI promoter (Auchtung et al. 2005). The regulation of RapI expression in combination with

PhrI signaling ensures that ICEBsJ is activated specifically under conditions where host survival is not guaranteed and/or unoccupied recipient cells are available.

PhrI

AM Phrl

RecA -> RecA* Rapi

DNA \ damage ImmA I lmmR P. R is

/z7 7z MA6K

Ye;

e

o

4W I-4

K2o

I

Y~

H

Fig. 5. Regulation of ICEBsJ. The big rectangle represents a donor cell with an abbreviated genetic map of ICEBsJ inside. The gray circle and cylinder represent protein import and export channels in the membrane. Open reading frames are indicated by horizontal arrows pointing in the direction of transcription, with gene names below. The color and patterns indicate the gene's function as DNA processing (diagonal stripes), regulation (black), or unknown (white).

Promoters are indicated by vertical arrows pointing in the direction of transcription. Black arrows indicate activation while red bars indicate inhibition. See text above for description of regulation. Figure adapted from (Auchtung et al. 2016).

Phrl

In addition to tightly regulating the activation of transfer, ICEBsJ regulates the entry and establishment of ICEBs] into new cells. Specifically, ICEBs] prevents transfer and integration of additional copies into a host cell that already contains ICEBsJ. One mechanism (exclusion) prevents transfer and will be discussed in greater detail below and in Chapter 2. The other mechanism is called immunity, which prevents the integration of additional copies that enter an occupied host (Auchtung et al. 2007). ImmR, the transcriptional repressor in the 'recipient' cell, blocks the expression and/or activity of the integrase of the incoming ICEBs], which is required for successful integration into the chromosome. Without active integrase, the additional copy of

ICEBsJ is forced to remain extrachromosomal, and is presumably lost through subsequent cell

divisions.

Exclusion

Exclusion describes mechanisms employed by conjugative elements to prevent redundant transfer of identical or closely related elements (Garcillan-Barcia & de la Cruz 2008). Surface exclusion (limited to the F plasmid and related elements) prevents the formation of a stable mating pair between two donor cells, and depends on a small outer membrane lipoprotein TraT. The detailed mechanism of surface exclusion is unknown. Entry exclusion (widespread among many conjugative elements) prevents the transfer of DNA through the T4SS. The general mechanism of entry exclusion involves a small inner membrane protein in the 'recipient' donor cell that recognizes and blocks its cognate T4SS from the donating cell. The remainder of this thesis will focus entirely on entry exclusion, here-on referred to as exclusion.

Exclusion has mostly been characterized in conjugative plasmids found in Gram-negative bacteria, with the majority of studies only identifying the exclusion gene acting in the recipient

cell. Only three exclusion systems have been characterized in detail, identifying both the

exclusion protein acting in the recipient and its target component of the T4SS in the donor (Fig. 6). These are 1) the F/Ri 00 plasmids (Anthony et al. 1999; Audette et al. 2007) and 2) the SXT/R391 ICEs (Marrero & Waldor 2005; Marrero & Waldor 2007), two pairs of homologous elements that encode MPFF T4SSs (Guglielmini et al. 2014), and 3) the R64/R621a plasmids (Sakuma et al. 2013), homologous elements that encode MPFI T4SSs (Guglielmini et al. 2014).

Periplasm

TraGF

TraGR391

N NC Cytoplasm TraYR₆₄ C N Periplasm NopN N N Cytoplasm

TraSF TraSR100

EexS

Fig. 6. Topologies of F/R100, SXT/R391, and R64/R621a exclusion proteins and targets. Exclusion proteins (green) are on the bottom and their corresponding donor targets (purple) on

top. White triangles indicate functional PhoA insertions, green triangles indicate functional GFP insertions. Red boxes represent mapped regions of exclusion specificity. The dotted red box indicates the region of exclusion specificity for TraY from R62 1a. TraS from the F and R100 plasmids are shown separately because they have significantly different predicted topologies. All other proteins are predicted to have similar topologies to their corresponding homologs. All topologies were generated using CCTOP (Dobson et al. 2015), and match the predictions made in their respective studies (see text below for details and references).

1) F and R100 are ~100 kb plasmids isolated from E. coli and Shigellaflexneri

respectively (Frost et al. 1994; Womble & Rownd 1988). The exclusion protein in the recipient was determined to be TraS (Achtman et al. 1977). TraSF and TraSR are ~150 aa and only 17% similar, and predicted to have 3-4 transmembrane domains (Audette et al. 2007). The donor target was determined to be TraG, the VirB6 homolog (Anthony et al. 1999). TraGF and TraGs are ~940 aa, and 93% similar. Audette et al mapped the region of specificity by generating chimeric TraG proteins and testing whether they could by excluded by F or R100 TraS proteins. They found that a ~60 aa region in the C-terminal tail only 56% similar between the TraG homologs accounted for specificity. TraG is predicted to have three or five transmembrane domains, with the C-terminal tail in the periplasmic space. They confirmed the periplasmic localization of the C-terminal tail by creating and demonstrating functional insertions of PhoA (an enzyme active when extracellular) at aa 716 and 739 (Audette et al. 2007).

2) SXT and R391 are ~100 and -89 kb ICEs isolated from Vibrio cholerae and

Providencia rettgeri respectively (Waldor et al. 1996; Coetzee et al. 1972). The exclusion protein

in the recipient was determined to be Eex (Marrero & Waldor 2005). EexS and EexR are -140 aa and 77% similar. The similarity is disproportionately concentrated at the N-terminal regions

-the first 86 aa are 87% identical, while -the last ~56 aa are only 41% identical. Marrero et al determined that overlapping regions in the dissimilar C-terminus (aa 121-137 for EexS and aa 114-130 for EexR) accounted for specificity by generating chimeric Eex proteins and testing whether they could exclude SXT or R391 (Marrero & Waldor 2007). EexS has four

transmembrane domains, confirmed by insertions of PhoA or GFP (fluorescent only when cytoplasmic) and cysteine accessibility assays. This confirmed that the region of specificity for Eex resides in the cytoplasm. Similar to the F and R100 plasmids, the donor target was

determined to be TraG. TraGs and TraGR are ~1189 aa and incredibly similar, 98% identical. In fact, only three amino acids in the C-terminal tail (aa 606-608) determined exclusion specificity, and the authors determined by functional C-terminal GFP fusion and cysteine accessibility assays that the C-terminal tail of TraGR also resides in the cytoplasm.

3) R64 and R62Ia are ~122 and ~93 kb plasmids first isolated from Salmonella

typhimurium (Coetzee et al. 1972; Hedges & Datta 1973). The exclusion protein in the recipient

was determined to be ExcA (Sakuma et al. 2013). ExcAR64 and ExcAR621a are ~210 aa and 53% similar, disproportionately in the C-terminal regions - there is no identity in the N-terminus, the middle ~120 aa are 40% identical, and the last C-terminal 40 aa are 95% identical. Both ExcA proteins are predicted to have two transmembrane domains, with the N-terminus localized in the cytoplasm. If this dissimilar region is responsible for exclusion specificity, then it would

resemble the SXT/R391 exclusion system. The donor target was determined to be TraY (Sakuma et al. 2013), a polytopic inner membrane protein required for transfer (Komano et al. 2000). MPFi T4SSs lack obvious VirB6 homologs, and it was proposed that TraY is a VirB6 analog based on its topology and involvement in entry exclusion (Guglielmini et al. 2014). TraY

2000), an eight transmembrane protein involved in the dot/icm (a protein effector -secreting) T4SS important for the pathogenesis of Legionellapneumoniae (Roy & Isberg 1997). TraYR64 and TraYR62Ia are ~745 aa and over 86% identical in all but two regions: an internal variable

region that is 49% identical and a C-terminal variable region that is 41% identical. Sakuma et al swapped these dissimilar regions between the TraY homologs and tested for exclusion by ExcA proteins. They found that ExcAR64 specificity resides in the internal variable region of TraYR64 (aa 430-523), while ExcAR621a specificity resides in the C-terminal variable region of TraYR621a

(aa 715-745) (Sakuma et al. 2013).

The selective advantages of exclusion

A handful of studies have addressed the selective advantages of exclusion for the

elements that encode them and/or their host cells. It has been proposed that exclusion 1) provides a competitive advantage for the propagation of elements, 2) prevents unwanted recombination between redundant elements 3) promotes diverse elements to coexist (potentially creating new elements and conferring novel benefits to the host), and 4) protects host cells from death caused by excessive transfer.

1) Exclusion provides a competitive advantage for the propagation of elements.

A mathematical model looking at competing incompatible conjugative plasmids with and without exclusion predicted that an exclusion-competent plasmid could spread through a cell population containing an exclusion-deficient plasmid, sometimes eliminating the latter from the population (van der Hoeven 1985). The advantage of exclusion was found to increase with higher transfer rate, lower copy number, and lower fitness cost to the host cell for executing

exclusion. These results suggest that exclusion is prevalent in many conjugative plasmids because it contributes to effective and competitive dissemination across a cell population.

2) Exclusion prevents unwanted recombination between redundant elements.

Several studies of F and related (F-like) plasmids in E.coli suggest that exclusion might benefit an element by keeping out redundant elements from its host cell, thus preventing unwanted recombination that could render it defective. Recombination between F-like plasmid sequences is frequent compared to that between chromosomal sequences (Boyd et al. 1996). Furthermore, transfer stimulates recombination of mobile elements. Transfer of F and F' plasmids (F plasmids carrying chromosomal DNA) results in the excision of transposons and recombination of plasmids in recipient cells; these effects are heightened if the F plasmids lack exclusion (Hopkins et al. 1980; Syvanen et al. 1986; Peters & Benson 1995). These results suggest that, at least in the case of F-like plasmids, recombination between elements is frequent and often stimulated by transfer and exclusion functions to keep redundant elements from recombining with each other.

3) Exclusion promotes element diversity within populations or individual cells

Mathematical modeling predicts that incompatible conjugative plasmids of the same exclusion specificity would have to adopt different strategies (high transfer rate with low fitness cost or vice versa) in order to coexist (within separate cells) in a population (van der Hoeven

1984; van der Hoeven 1986). These results suggest exclusion could exert adaptive pressure on elements and promote their evolution. These results also suggest that exclusion favors

combinations of diverse elements within a population, also benefiting the host cells as they carry a variety of elements that may confer different benefits.

There is some experimental evidence for exclusion leading to diverse combinations of elements within a single cell. SXT and R391 are both ICEs that exclude themselves but not each other (Marrero & Waldor 2005). Transfer from donor strains carrying both SXT and R391 results in the formation of functional SXT/R391 hybrid ICEs in transconjugants (Burrus & Waldor 2004). Hybrid ICEs carried different combinations of cargo genes (encoding antibiotic resistance) from both original ICEs, and were capable of transfer into new recipients.

Exclusion protects recipient cells from death caused by excessive transfer.

The most extensive experimental evidence demonstrating the benefits of exclusion have focused on the phenomenon of lethal zygosis observed with the F plasmid from E. coli. Lethal

zygosis describes recipient cell death in the presence of excess donors that are incapable of exclusion, such as Hfr donors (F plasmid that has integrated into and can transfer the host chromosome) or exclusion-null F plasmid donors (Skurray & Reeves 1973; Skurray & Reeves 1974; Skurray et al. 1976; Ou 1980). Unidirectional transfer from donors to recipients was sufficient to cause recipient killing. The mechanism of killing was hypothesized to be recipient cell wall damage, based on the observation that radioactively labeled recipient cell wall

components could be detected in the media during lethal zygosis matings (Ou 1980). These studies indicate that exclusion benefits both the element and host cell by preventing suicidal levels of element transfer that result in its potential host cell's death.

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Chapter 2

Identification, characterization, and benefits of

an exclusion system in an integrative and

conjugative element of Bacillus subtilis

Monika Avello*, Kathleen P. Davis**, and Alan D. Grossman

This chapter is being prepared for publication.

CONTRIBUTIONS

*MA characterized ICEBs] exclusion, identified yddJ as the exclusion protein, and demonstrated selective advantages of exclusion.

**KPD performed the mutagenesis screens, identified conG as the donor target, and demonstrated ConG and YddJ specificity.