Journal of Medical Microbiology (2013), 62, 663–676 DOI 10.1099/jmm.0.053983-0

Review Type VI system regulation as a consequence of evolutionary pressure

Sarah T. Miyata, Verena Bachmann and Stefan Pukatzki

Correspondence Department of Medical Microbiology and Immunology, 6-22 Heritage Medical Research Centre, Stefan Pukatzki University of Alberta, Edmonton, Alberta T6G 2S2, Canada [email protected]

The type VI secretion system (T6SS) is a mechanism evolved by Gram-negative bacteria to negotiate interactions with eukaryotic and prokaryotic competitors. T6SSs are encoded by a diverse array of bacteria and include plant, animal, human and fish pathogens, as well as environmental isolates. As such, the regulatory mechanisms governing T6SS gene expression vary widely from species to species, and even from strain to strain within a given species. This review concentrates on the four bacterial genera that the majority of recent T6SS regulatory studies have been focused on: Vibrio, Pseudomonas, Burkholderia and Edwardsiella.

Introduction eukaryotes include actin cross-linking (Pukatzki et al., 2007), formation of actin protrusions (Aubert et al., 2008, The type VI secretion system (T6SS) is a multi-functional 2010), limitation of bacterial colonization and intracellular virulence mechanism utilized by Gram-negative bacteria to replication, and adaptation to deoxycholic acid (Parsons & kill prokaryotic and eukaryotic organisms. The T6SS is Heffron, 2005; Robinson et al., 2009; Chow & Mazmanian, hypothesized to form a needle-like appendage that exports 2010; Lertpiriyapong et al., 2012). toxins across the bacterial cell envelope and into an adjacent target cell. Structural T6SS proteins form a spring- More recently, it was discovered that the T6SS enables + loaded inverted -like structure within the T6SS bacteria to kill prokaryotic target cells in addition bacterial cytoplasm that is hypothesized to eject proteins to eukaryotes (Hood et al., 2010; MacIntyre et al., 2010; across the cell envelope (Basler et al., 2012). The T6SS Schwarz et al., 2010; Murdoch et al., 2011; Russell et al., needle complex proteins share similarity with the T4 2011). V. cholerae uses its T6SS to outcompete a variety of bacteriophage tail spike and include the haemolysin co- Gram-negative bacteria (MacIntyre et al., 2010; Ishikawa regulated protein (Hcp) and valine glycine repeat G (VgrG) et al., 2012). encodes three bacterial proteins (Williams et al., 1996; Pukatzki et al., 2006, 2007; toxins, two of which target the peptidoglycan layer and one Leiman et al., 2009). Hcp is predicted to form a nanotube that is probably introduced into the cytoplasm of target that is surrounded by VipA and VipB proteins (Bo¨nemann cells (Hood et al., 2010; Russell et al., 2011). Burkholderia et al., 2009; Basler et al., 2012). Upon contraction of the thailandensis encodes five specialized T6SS clusters, one cytoplasmic VipA/VipB tube, the Hcp tube (postulated to of which is important for inter-bacterial competition be capped with VgrG proteins) is presumably ejected from (Schwarz et al., 2010). Antibacterial T6SS effector proteins the bacterium (Mougous et al., 2006; Pukatzki et al., 2006, are encoded by a wide array of Gram-negative bacteria, 2007; Bo¨nemann et al., 2009; Leiman et al., 2009; Basler implying that the T6SS is commonly used to mediate inter- et al., 2012). ClpV is required to disassemble the contracted bacterial competition (Russell et al., 2012). VipA/VipB sheath (Basler & Mekalanos, 2012). These T6SS gene clusters are present in numerous Gram-negative proteins are highly conserved among T6SSs and are proteobacteria, encompassing an impressive array of imperative for the secretion of toxins from the bacterial pathogens that infect humans (Mougous et al., 2006; cell (Ma et al., 2009). Pukatzki et al., 2006), animals (Schell et al., 2007; Burtnick Initially, the T6SS was described as a virulence mechanism et al., 2011) and plants (Bladergroen et al., 2003; Liu et al., specifically targeting eukaryotic cells. Virulence towards 2008; Records & Gross, 2010). Given the diversity of phagocytic cells like macrophages or the amoeboid host bacteria that utilize the T6SS, it is not surprising that model Dictyostelium discoideum was described in a number numerous regulatory mechanisms govern T6SS gene of bacterial species including (Pukatzki expression. Some common trends in regulatory mechan- et al., 2006, 2007; Miyata et al., 2011), Burkholderia species isms of T6SS include (QS), changes in (Shalom et al., 2007; Aubert et al., 2008; Burtnick et al., temperature and pH, and two-component regulatory 2010; Chen et al., 2011), Yersinia pestis (Robinson et al., systems (TCSs) (Zheng et al., 2005; Aubert et al., 2008; 2009) and Salmonella enterica (Parsons & Heffron, 2005). Liu et al., 2008; Ishikawa et al., 2009; Khajanchi et al., 2009; Phenotypic consequences of T6SS-based interactions with Lesic et al., 2009; Pieper et al., 2009; Wang et al., 2009b;

053983 G 2013 SGM Printed in Great Britain 663 S. T. Miyata, V. Bachmann and S. Pukatzki

Chakraborty et al., 2010, 2011; Records & Gross, 2010; concentration in the environment. At low-cell density, the Zheng et al., 2010; Gode-Potratz & McCarter, 2011; QS response regulator LuxO is phosphorylated and induces Moscoso et al., 2011; Rogge & Thune, 2011; Zhang et al., the transcription of small quorum-regulatory RNAs (Qrrs). 2011; Ishikawa et al., 2012; Sheng et al., 2012). Other Qrrs bind and destabilize mRNA of the central output environmental cues such as the concentration of iron, regulator, HapR (LuxR in Vibrio alginolyticus, or OpaR in phosphate and magnesium have also been implicated in Vibrio parahaemolyticus). Conversely, at high-cell density, T6SS regulation (Mueller et al., 2009; Wang et al., 2009b; LuxO is inactivated, allowing expression of the master Chakraborty et al., 2010, 2011; Rogge & Thune, 2011; Lv regulator. LuxO, in combination with the regulator TsrA et al., 2012). A summary of T6SS regulatory mechanisms in (VC0070), represses the T6SS in C6706 and mutations in select Gram-negative bacteria is provided in Table 1. both genes are required for Hcp secretion in this strain Although these mechanisms exhibit tight control over the (Zheng et al., 2010). In addition to T6SS regulation, TsrA T6SS of some bacterial species, other species or strains represses TCP and CT, but activates expression of the possess constitutively active T6SSs. It is curious why haemagglutinin protease HapA (Zheng et al., 2010). Thus, bacteria with constitutive T6SSs dedicate a vast energy TsrA acts as a global regulator of virulence genes in the El expenditure to the production of T6SS proteins. Recently, Tor strain C6706, exerting both positive and negative much knowledge of T6SS regulatory mechanisms has been influences on gene expression. gained regarding environmental signals and the ensuing In some V. cholerae strains, T6SS genes are upregulated at a regulatory cascades that trigger T6SS gene activation. Here, high-cell density. Expression of Hcp in O1 V. cholerae we review recent advances in the field of T6SS regulation strains A1552, AJ3 and AJ5 (but not C6706) is dependent focusing on the genera Vibrio, Pseudomonas, Burkholderia on QS networks, as the deletion of hapR or the AI synthase and Edwardsiella. Furthermore, we provide a rationale for genes (cqsA and luxS) resulted in a loss of Hcp production the disparity that some bacterial species possess constitu- (Ishikawa et al., 2009). Hcp is also important for pellicle tively active T6SSs, while others maintain strict regulation formation in V. parahaemolyticus, implying that hcp gene over T6SS genes. expression occurs under high-cell density (Enos-Berlage et al., 2005). Interestingly, OpaR oppositely regulates two Vibrio species distinct T6SS gene clusters in V. parahaemolyticus (Gode- Potratz & McCarter, 2011), implying that one cluster is The majority of Vibrio T6SS research has focused on V. active at high-cell density and the other is active at low-cell cholerae, the aetiological agent of the diarrhoeal disease density. More specifically, OpaR represses transcription . This bacterial genus is extremely diverse and is and expression of Hcp1 indirectly as OpaR does not bind classified into more than 200 serogroups. Of the seven the hcp1 promoter region (Ma et al., 2012). OpaR was recorded cholera pandemics, the strains responsible for the also found to positively regulate cyclic-di-GMP levels; a last two (and possibly three) pandemics are believed to phenotype associated with increased T6SS gene expres- belong to the O1 serogroup, including the El Tor strains sion in Pseudomonas aeruginosa (Moscoso et al., 2011). V. C6706 and N16961 (responsible for the current seventh alginolyticus also encodes two T6SS gene clusters; however, pandemic) (Cvjetanovic & Barua, 1972; Kamal, 1974). only one of these is regulated via QS (Sheng et al., 2012). These strains utilize the toxin co-regulated pilus (TCP) and Hcp1 expression in V. alginolyticus peaks at mid-exponen- cholera toxin (CT) as their prominent virulence factors. tial phase and mutation of luxO resulted in lower Hcp Conversely, non-O1/non-O139 serogroup strains cause transcript levels, while the opposite was true for a luxR small-scale outbreaks of gastroenteritis, often indepen- mutant. These results are opposite to those observed dently of the CT and TCP. The overwhelming majority of for V. cholerae A1552, AJ3 and AJ5, and imply that V. sequenced V. cholerae strains possess the full complement alginolyticus upregulates Hcp expression under low-cell of T6SS genes, suggesting that the T6SS is part of the V. density. Similarly, Vibrio anguillarum upregulates T6SS cholerae 1500-gene core genome and plays a crucial role in genes under low AI concentrations (Weber et al., 2009, V. cholerae’s lifestyle. However, T6SS gene regulation 2011). It is plausible that differences in T6SS QS regulation differs between strains, as some strains constitutively between strains arise from variations in the signal express T6SS genes and others restrict T6SS gene transduction pathways of QS. expression. Numerous publications have reported T6SS regulation in V. cholerae (Pukatzki et al., 2006; Ishikawa V. cholerae spends the majority of its life cycle in the ocean or et al., 2009, 2012; Mueller et al., 2009; Zheng et al., 2010, in brackish waters, where it constantly experiences fluxes in environmental conditions. Fittingly, V. cholerae alters its gene 2011; Kitaoka et al., 2011) and the T6SS regulon in this expression in response to changes in temperature and salinity. pathogen is complex. Both the environmental cues and the Ishikawa et al. (2012) demonstrated that the osmoregulator ensuing regulatory cascades leading to T6SS gene expres- OscR (VCA0029) represses the expression of Hcp in A1552. sion will be reviewed here. V. cholerae El Tor strains A1552, E7946 and 93Ag49 produce QS regulates V. cholerae virulence factors such as CT, TCP and secrete Hcp when grown in medium with high salt and chemotaxis genes in C6706 (Zhu et al., 2002). QS concentration, as opposed to standard laboratory conditions communication is mediated via sensing autoinducer (AI) where Hcp is produced (in a QS-dependent manner) but not

664 Journal of Medical Microbiology 62 T6SS regulation and evolutionary pressure secreted (Ishikawa et al., 2012). Furthermore, A1552 kills C-terminal helix–turn–helix DNA-binding domain similar Escherichia coli MC4100, and this T6SS-mediated killing is to other bEBPs (Bernard et al., 2011; Kitaoka et al.,2011). enhanced with high salt conditions (Ishikawa et al.,2012). Importantly, polymorphisms in VasH are not responsible for This implies that the V. cholerae T6SS is important for inter- the differential regulation of T6SS genes in strains like V52 bacterial competition in the species’ native environment. (constitutively active T6SS) and N16961 (repressed T6SS Interestingly, E7946 was also used in a study employing in under laboratory conditions), as vasH from N16961 is vivo expression technology, which determined that T6SS functional when expressed in trans in both V52DvasH and genes were upregulated as the bacterium traversed the wild-type N16961 (Kitaoka et al., 2011). Sequence align- human and murine gastrointestinal tract (Lombardo et al., ments of vasH from V52, N16961 and four V. cholerae 2007). Furthermore, T6SS gene expression in strain C6706 isolates from the Rio Grande indicate that vasH is conserved was increased during an infection of mouse intestine among not only pandemic and endemic clinical strains, but (Mandlik et al., 2011). This adds additional complexity to also those isolates from environmental sources (Unterweger the T6SS regulatory cascade in O1 V. cholerae strains, as T6SS et al., 2012). Taken together, a universal role for VasH in + genes appear to be upregulated not only in their native several T6SS bacterial species has been established. environment, but also during host infection. T6SS regulation in V. cholerae is complex and can differ Many environmental cues feed into the T6SS regulatory depending on the strain studied. It is currently unclear how networks of V. cholerae. Once an external signal is received, all the internal regulators (VasH, HlyU, VCA0122, TsrA, the bacterium translates this signal to alter gene expression OscR) function in concert within the cell to influence gene accordingly. A number of transcriptional regulators have expression. At present, it appears that different extra- been identified in V. cholerae that positively or negatively cellular signals (osmolarity, cell density, salinity, temper- influence T6SS gene expression. Microarray and qPCR data ature) are responsible for regulating T6SS gene expression by Syed et al. (2009) indicated that V. cholerae O395 flagellar within the genus Vibrio. mutants had increased T6SS gene expression. This supports the hypothesis that virulence and flagellar genes are inversely regulated. Zheng et al. (2011) identified the transcriptional Pseudomonas species regulator VCA0122 (encoded within the large V. cholerae Bacteria of the genus Pseudomonas belong to the family T6SS gene cluster on the small chromosome) which, when Pseudomonadaceae which has nearly 200 described species deleted, results in reduced hcp expression and attenuated D. (Euze´by, 1997). They inhabit a wide variety of environments discoideum killing, but is dispensable for killing E. coli. such as soil, water and host organisms, and therefore have Another positive regulator of the T6SS in V. cholerae is the very versatile biology (Palleroni, 1992). Genomic informa- alternate s54 factor RpoN (Pukatzki et al., 2006; Ishikawa tion has been compiled for 90 strains (Silby et al., 2011), and et al., 2009; Kitaoka et al., 2011; Dong & Mekalanos, 2012). recently in silico genomic analysis of 34 Pseudomonas RpoN and one of its activators, VasH, are crucial for V. genomes identified 70 T6SSs with at least one locus per cholerae virulence towards D. discoideum and for killing E. strain (except Pseudomonas stutzeri A1501) (Barret et al., coli (Pukatzki et al.,2006;Kitaokaet al., 2011; Zheng et al., 2011). Six pathovars of the plant pathogen Pseudomonas 2011; Dong & Mekalanos, 2012). VasH acts as a bacterial- syringae carry one or two T6SS gene clusters in addition to a enhancer-binding protein (bEBP) and is required for T3SS (Records & Gross, 2010; Sarris et al., 2010; O’Brien recruiting s54 to 224/212 promoter sequences. s54- et al., 2011). In contrast, the human opportunistic pathogen dependent T6SS promoters were found upstream of putative Pseudomonas aeruginosa encodes nearly all secretion systems T6SS operons in V. cholerae, Pseudomonas aeruginosa and described thus far, including three evolutionarily distinct Aeromonas hydrophila,implyingthatregulationbybEBPsis T6SS clusters (designated Hcp secretion island (HSI) I–III) common in T6SS gene clusters (Bernard et al.,2011). and an orphaned hcp2/vgrG2 locus (Mougous et al., 2006; However, there exists a discrepancy in the literature Lesic et al., 2009; Termine & Michel, 2009; Bleves et al., regarding the specific operons regulated by RpoN in V. 2010). Toxins critical for inter-bacterial competition are co- cholerae (Bernard et al., 2011; Dong & Mekalanos, 2012). regulated with HSI-I genes (Hood et al., 2010; Russell et al., Using electromobility shift assays and b-glucuronidase 2011); however, HSI-I also plays a crucial role in chronic fusions as a reporter of transcriptional activity, Bernard infections as shown in the rat model of chronic respiratory et al. (2011) determined that VasH/RpoN regulates the large infections, and in chronically infected cystic fibrosis patients T6SS gene cluster, as well as the small auxiliary clusters which (Potvin et al., 2003; Mougous et al., 2006). For HSI-I, PppA encode T6SS secreted proteins in V. cholerae O395. and PpkA (serine–threonine phosphatase and kinase, Contradictory to this, RNA-seq, ChIP-seq and qPCR data respectively) exert their effects on the T6SS scaffold protein indicate that in strain V52, RpoN strictly regulates the Fha1, which mediates the export of T6SS proteins (Mougous smaller hcp-encoding clusters and not the large T6SS cluster et al., 2007; Casabona et al., 2012). HSI-II and HSI-III are (Bernard et al., 2011; Dong & Mekalanos, 2012). Differences important for pathogenesis in the Arabidopsis thaliana in the promoter sequences of El Tor and classical strains may model (Lesic et al., 2009) and furthermore, HSI-II deletion explain these different findings. VasH has an N-terminal mutants are attenuated in mouse infection and the HSI-III regulatory domain, a central s54-activating domain and a gene cluster is upregulated in the presence of epithelial cell http://jmm.sgmjournals.org 665 S. T. Miyata, V. Bachmann and S. Pukatzki

Table 1. Summary of regulatory mechanisms in select Gram-negative bacteria

Organism Gene cluster Regulator Regulatory mechanism Effect Reference

V. cholerae VCA0106-VCA0124, High cell QS (Regulation of HapR) Induction Ishikawa et al. (2009) VC1415-VC1419, density (strain VCA0017-VCA0021 dependent) High osmolarity Unknown Induction Ishikawa et al. (2012) (decreased OscR) High salinity Unknown Induction Ishikawa et al. (2012) Low temperature Unknown Induction Ishikawa et al. (2012) (decreased TsrA) HlyU Unknown Induction Williams et al. (1996) VasH s54 (RpoN) activator Induction Pukatzki et al. (2006); Kitaoka et al. (2011) Indole Unknown Induction Mueller et al. (2009) TsrA QS LuxO Repression Zheng et al. (2010) In vivo (human Unknown Induction Lombardo et al. (2007); and mouse) Mandlik et al. (2011) Flagella mutant Unknown Induction Syed et al. (2009) VCA0122 mutant Unknown Repression Zheng et al. (2011) V. parahaemolyticus T6SS2 High cell density QS (Regulation of OpaR) Induction Enos-Berlage et al. (2005); Gode-Potratz et al. (2011); McCarter et al. (2011) T6SS1 Low cell density QS (Regulation of OpaR) Induction Gode-Potratz et al. (2011) V. alginolyticus T6SSVA1 RpoN Unknown Induction Sheng et al. (2012) Growth phase QS LuxO–LuxR Repression or Sheng et al. (2012) induction V. anguillarum VtsE-H High cell density QS (Regulation of VanT) Repression Weber et al. (2009, 2011) Pseudomonas LadS TCS GacS-GacA & c-di- Induction Ventre et al. (2006) aeruginosa GMP WspR c-di-GMP Induction Moscoso et al. (2011) HSI-I Surface growth Post-translational Induction Silverman et al. (2011) (threonine phosphorylation) HSI-I RetS TCS GacS-GacA & c-di- Repression Bordi et al. (2010) GMP HSI-I PpkA Post-translational Induction Mougous et al. (2007; (phosphorylation) Hsu et al. (2009) HSI-I PppA Post-translational Repression Mougous et al. (2007) (phosphorylation) HSI-I TagF Unknown Repression Silverman et al. (2011) HSI-I MvfR QS PQS Repression Lesic et al. (2009) HSI-II/III MvfR QS PQS Induction Lesic et al. (2009) HSI-I LasR QS LasR-LasI Repression Lesic et al. (2009) HSI-II/III LasR QS LasR-LasI Induction Lesic et al. (2009) HSI-II PsrA DNA binding Repression Kang et al. (2008) (transcription factor) HSI-II/III Unknown s54 (RpoN) Induction Hendrickson et al. (2001) HSI-II/III MvaT Transcriptional silencing Repression Castang et al. (2008) (H-NS) Pseudomonas RetS Post-transcriptional Repression Records & Gross (2010) syringae (RsmA regulon) LadS Post-transcriptional Induction Records & Gross (2010) (RsmA regulon) s54 Induction Bernard et al. (2011) Pseudomonas HSI-I/HSI-III gacS-mutant TCS GacS-GacA Repression Lalaouna et al. (2012) brassicacearum

666 Journal of Medical Microbiology 62 T6SS regulation and evolutionary pressure

Table 1. cont.

Organism Gene cluster Regulator Regulatory mechanism Effect Reference

Pseudomonas gacA-mutant TCS GacS-GacA Repression Hassan et al. (2010) fluorescens Pf-5 B. mallei Phagocytosis Unknown Induction Shanks et al. (2009); Burtnick et al. (2010) B. pseudomallei AtsR TCS Repression Aubert et al. (2008) T6SS-1 Phagocytosis TCS VirA-VirG Induction Shalom et al. (2007); Chen et al. (2011) T6SS-1 BspR Unknown; via regulator Induction Sun et al. (2010) BprC T6SS1-5 Rich medium Unknown Induction Burtnick et al. (2011) B. cenocepacia AtsR Unknown (might be Repression Aubert et al. (2008) connected to BCAM0381) E. tarda Acidic pH TCS PhoP-PhoQ Induction Chakraborty et al. (2010) Temperature TCS PhoP-PhoQ Induction Chakraborty et al. (2010) (23-35uC) Iron (Fur) Crosstalk with Pho Reduction or Wang et al. (2009b); regulon and evpP induction Chakraborty et al. (2011) High Mg2+ (10mM) TCS PhoP-PhoQ Reduction Chakraborty et al. (2010) EsrC TCS EsrA-EsrB Induction Lv et al. (2012) Phosphate TCS PhoB-PhoR Reduction Chakraborty et al. (2011) Antimicrobial peptides TCS PhoP-PhoQ Induction Chakraborty et al. (2010) PST operon mutants Unknown; via phosphate Repression Rao et al. (2004) acquisition proteins E. ictaluri EsrC TCS EsrA-EsrB Induction Rogge & Thune (2011) Low pH EsrB Induction Rogge & Thune (2011) Low phosphate Unknown Induction Rogge & Thune (2011) Pectobacterium Plant host extract Unknown Induction Mattinen et al. (2008) atrosepticum Oxygen Unknown Repression Babujee et al. (2012) S. marcescens Rich medium, 37uC, Unknown Reduction Murdoch et al. (2011) stationary phase Enteroaggregative sci1 Fur DNA binding Repression Brunet et al. (2011) E. coli (transcription factor) sci1 Dam DNA methylation Induction Brunet et al. (2011) Avian pathogenic Host contact Unknown Induction de Pace et al. (2011) E. coli Y. pseudotuberculosis T6SS4 Growth phase QS (AHL-dependent) Induction Zhang et al. (2011) T6SS4 Temperature (26uC) QS (AHL-dependent) Induction Zhang et al. (2011) S. enterica serovar sci PmrA TCS PmrA-PmrB Induction Wang et al. (2011) Typhi sci RcsB TCS PmrA-PmrB Induction Wang et al. (2011) sciK Hfq Unknown Induction Wang et al. (2011)

extracts (Chugani & Greenberg, 2007; Lesic et al., 2009; (Veesenmeyer et al., 2009). The two major QS systems las Starkey & Rahme, 2009). HSI-I and HSI-III clusters are also and rhl are each composed of an N-acyl-L-homoserine induced during different stages of biofilm development synthase (LasI or RhlI) and its cognate transcriptional (Southey-Pillig et al., 2005), which implies differential regulator (LasR or RhlR) (De´ziel et al., 2005). Pathogenic regulation via QS systems. Therefore, strict regulation of the Pseudomonas aeruginosa also synthesizes a quinolone- different T6SS loci in Pseudomonas species seems to be crucial signalling molecule 2-heptyl-3-hydroxy-4-quinolone (PQS), for a functional virulence mechanism (Mougous et al., 2006; which is a regulatory link between the Las and Rhl systems. Lesic et al., 2009; Bernard et al., 2010; Leung et al., 2011). Regulation of PQS biosynthesis genes is mediated by the QS systems regulate ~350 genes in Pseudomonas aeruginosa transcriptional regulator MvfR (De´ziel et al., 2004; De´ziel and are involved in a wide variety of cellular processes et al., 2005; Venturi, 2006). Importantly, QS systems play a http://jmm.sgmjournals.org 667 S. T. Miyata, V. Bachmann and S. Pukatzki role in differentially regulating gene expression in all three that of the wild-type (Hassan et al., 2010; Lalaouna et al., T6SS loci in Pseudomonas aeruginosa (Lesic et al.,2009; 2012). Therefore, it is conceivable that there is a connection Bernard et al., 2010). HSI-I gene expression is suppressed by between GacS/GacA TCS and T6SS in Pseudomonas species. LasR and MvfR, whereas HSI-II and HSI-III are positively Altogether, the complex regulation of the T6SS in regulated by both. Pseudomonas is controlled at many levels and by different RpoN (discussed above for V. cholerae) positively regulates regulatory systems, highlighting the importance of this synthesis of RhlI and is crucial in the regulation of virulence factor in the life cycle of different Pseudomonas virulence genes in Pseudomonas aeruginosa (Buck et al., species. Currently, Pseudomonas entomophila and Pseudo- 2000; Hendrickson et al., 2001; Leung et al., 2011). monas mendocina are species used as simplified bacterial Bioinformatic analysis suggests that the Pseudomonas models for understanding/identifying regulatory mechan- syringae and Pseudomonas aeruginosa HSI-II and HSI-III isms in silico (Sarris & Scoulica, 2011). clusters contain s54-binding sites; however, it remains to be shown conclusively whether s54-dependent gene regulation in Pseudomonas is required for T6SS-mediated virulence Burkholderia species (Hendrickson et al., 2001; Alarco´n-Chaidez et al., 2003; The Burkholderia genus encompasses several pathogenic Bernard et al., 2011). Using microarray analysis, the TetR- organisms including Burkholderia mallei (Glanders disease), like transcription factor PsrA was shown to upregulate the Burkholderia pseudomallei (melioidosis) and Burkholderia HSI-II locus in stationary phase (Kang et al., 2008) and in a cepacia (human lung infections). Burkholderia thailandensis separate report, microarray analysis identified MvaT (a is a close relative of B. pseudomallei but rarely infects histone-like nucleoid structuring protein) as a negative humans or animals (Glass et al., 2006). Burkholderia species regulator of clpV3, hcp2 and vgrG2 in the HSI-II and HSI- possess multiple T6SS gene clusters: B. mallei and B. III clusters (Castang et al., 2008). thailandensis have five T6SS clusters, while B. pseudomallei The RetS/LadS/GacS pathway coordinately regulates gene has six (Schell et al., 2007). The T6SS-5 of B. thailandensis expression of HSI-I and the T3SS during acute and chronic targets eukaryotes and is important for murine pneumonic Pseudomonas aeruginosa infections (Goodman et al., 2004; melioidosis, whereas T6SS-1 mediates inter-species bacterial Coggan & Wolfgang, 2012). RetS and LadS are orphan competition (Schwarz et al., 2010). It is currently unclear hybrid sensor kinases that counteract each other’s effects. what the other T6SS gene clusters in Burkholderia are RetS downregulates biofilm formation and T6SS gene important for and whether each one targets different species. expression, but induces low levels of the second messenger Encoding multiple T6SS gene clusters that potentially target c-di-GMP and upregulates T3SS genes, whereas LadS has the different species would require strict regulation over the reciprocal effect (Goodman et al., 2004; Ventre et al., 2006; various gene clusters. Recent advances in the regulation of Bordi et al., 2010). LadS also has a role in Pseudomonas Burkholderia T6SS clusters are discussed here. aeruginosa PA14 cytotoxicity towards HeLa cells (Mikkelsen The response regulator AtsR negatively regulates T6SS et al., 2011). Regulation of both secretion systems is genes in B. cenocepacia (Aubert et al., 2008). AtsR shares mediated by the GacS/GacA TCS. Phosphorylation of similarity and functions similarly to the Pseudomonas GacA leads to transcription of the small RNAs rsmZ and aeruginosa regulator RetS, which is important for switching rsmY, which sequester the post-transcriptional regulator between acute and chronic stages of lung infection in cystic RsmA, resulting in opposite regulation of the T3SS and fibrosis patients (Mougous et al., 2006). Inactivation of T6SS. The same regulatory effect can be achieved by artificial AtsR results in increased biofilm production and expres- modulation of c-di-GMP levels by overexpressing phospho- sion, and hypersecretion of Hcp (Aubert et al., 2008). diesterases or the diguanylate cyclase WspR, suggesting that the pathways are linked (Goodman et al., 2004; Ventre et al., The T6SS-1 gene cluster in both B. mallei and B. 2006; Bordi et al., 2010; Moscoso et al., 2011). The HptB pseudomallei are strictly regulated, becoming active when signalling pathway also controls biofilm formation and the the VirAG TCS is overexpressed or upon internalization by T3SS via a response regulator and an anti-s factor and has phagocytic cells (Schell et al., 2007; Shalom et al., 2007; an impact on the expression of RsmY. However, regulation Burtnick et al., 2010, 2011; Chen et al., 2011). The VirAG of T6SS genes seems to be specifically controlled via the RetS TCS is crucial for B. mallei virulence and regulates genes pathway, as overproduction of RsmY alone does not have an involved in actin tail formation, capsule production and impact on T6SS gene expression (Bordi et al., 2010). RetS the T6SS (Schell et al., 2007). Recently, a T3SS cluster 3 and LadS sensor kinases also antagonistically regulate the (T3SS-3) regulator, BspR, was identified in B. pseudomallei T6SS and virulence factors in Pseudomonas syringae, but that also regulates the T6SS-1 cluster (Sun et al., 2010). the RetS/LadS regulatory pathway seems to be parallel to, Mutation of bspR reduced T6SS-1 gene expression rather than upstream of, the GacS/GacA TCS (Records & compared with that of the parent strain when grown in Gross, 2010). Also, in Pseudomonas fluorescens Pf-5 and liquid culture (Sun et al., 2010). BspR is a TetR family Pseudomonas brassicacearum the GacS/GacA TCS plays a regulator involved in a complex regulatory cascade that critical role for the expression of T6SS proteins, as gacA and includes downstream regulators BprP, BsaN, BicA and gacS mutants reduce T6SS gene expression compared with BprC. BprP is a membrane-localized DNA-binding protein

668 Journal of Medical Microbiology 62 T6SS regulation and evolutionary pressure that binds the promoter region of the AraC-type regulator The AraC-type transcriptional activator EsrC is encoded bsaN. Consequently, BsaN (encoded within the T3SS-3 gene within the E. tarda and E. ictaluri T3SS and is directly cluster) and its co-activator BicA positively influence the regulated by EsrB. EsrC in turn regulates the T3SS and expression of BprC, which strictly regulates T6SS-1 and not T6SS (Chakraborty et al., 2011), which implies a co- T3SS-3 in B. pseudomallei (Sun et al., 2010). Interestingly, dependent regulatory mechanism for the T3SS and T6SS, BprC is the critical regulator of T6SS-1 when B. pseudomallei given that EsrC is encoded within the T3SS gene cluster. is grown in liquid culture; however, following phagocytosis Mutants in esrB are markedly attenuated in zebrafish (Lv by RAW 264.7 macrophages, the crucial regulator of T6SS-1 et al., 2012) and mutation of esrC significantly increases genes becomes the VirA–VirG TCS (Chen et al., 2011). VirG LD50 levels in blue gourami fish (Zheng et al., 2005), and BprC have unique transcriptional start sites, with VirG implicating the T3SS and the T6SS as crucial virulence acting directly at the hcp1 promoter and the VirG regulatory factors. In support of this, an evpP (which encodes a crucial cascade acting upstream of tssA (Chen et al., 2011). T6SS effector protein) mutant is attenuated in zebrafish and Delineation of this regulatory cascade implies there is Japanese flounder infection models (Wang et al., 2009b) and cross-talk between the T3SS-3 and T6SS-1 (via BsaN) and is defective for internalization by epithelial papilloma of carp that both systems contribute to virulence in the B. cells (Wang et al., 2009b). Severe replication deficiencies in pseudomallei mouse infection model. head kidney-derived macrophages (HKDM), and an avirulent phenotype in the channel catfish model in esrA In B. pseudomallei, deletion of hcp1 resulted in an increased and esrB mutants of E. ictaluri was also reported (Rogge & LD in the Syrian hamster infection model, and the hcp1 50 Thune, 2011). Interestingly, an esrC mutant maintained the mutant was impaired for growth and in the formation of ability to replicate within HKDM, but lost its virulence in giant multi-nucleated cells when used to infect RAW 264.7 catfish (Rogge & Thune, 2011). Therefore, the T3SS appears macrophages (Burtnick et al., 2011). Importantly, Hcp1 important for establishing infection and replication within expression was not detected when cells were grown in macrophages, while the T6SS is required for virulence. liquid culture, supporting the idea that the T6SS-1 gene Alternatively, the T6SS may limit intracellular replication cluster is important for intracellular growth/survival and is within macrophages similar to Salmonella, since EsrB shares not active when grown in the absence of eukaryotic host similarity with the Salmonella TCS response regulator SsrB cells. Conversely, Hcp6 was constitutively expressed (but (Parsons & Heffron, 2005). not secreted) when B. pseudomallei was grown in liquid culture, and microarray analysis indicated that T6SS-6 Several triggers have been identified that activate the genes were upregulated 100-fold compared with those of expression of T3SS and T6SS genes in Edwardsiella species T6SS-1–T6SS-5 when grown in rich medium (Burtnick including temperature, pH and the extracellular concentra- et al., 2011). Thus, T6SS gene clusters in B. pseudomallei are tions of magnesium, iron and phosphate (Zheng et al., 2005; under control of different regulators and are expressed at Wang et al., 2009b; Chakraborty et al., 2010, 2011; Rogge & different times during the bacterium’s life cycle. It will be Thune, 2011; Lv et al., 2012). In E. ictaluri, T6SS genes are interesting to determine what environmental cues activate activated under conditions that mimic those of a phagosome; T6SS-2–T6SS-5 and whether T6SS-6 is important for that is, low pH and limited phosphate (Rogge & Thune, microbial competition. 2011). In contrast, the E. tarda T3SS (and therefore the T6SS) is active at neutral pH and is repressed under acidic conditions (Srinivasa Rao et al., 2003; Rao et al., 2004; Edwardsiella species Zheng et al., 2005; Okuda et al., 2009). The PhoP-PhoQ TCS Edwardsiella tarda and Edwardsiella ictaluri are both fish responds to changes in temperature and magnesium. pathogens in which the T6SS is crucial for virulence (Rao Secretion of EvpC (Hcp homologue) and EvpP was observed et al., 2004; Zheng et al., 2005; Zheng & Leung, 2007; Wang at 23–35 uC, with maximal secretion at 30 uCandno et al., 2009a, 2009b; Chakraborty et al., 2010, 2011; Rogge & secretion at 20 or 37 uC (Chakraborty et al., 2010). Between Thune, 2011; Lv et al., 2012). E. tarda is an emerging 23 and 35 uC, PhoQ autophosphorylates and the phosphate is pathogen that causes septicaemia in fish and gastrointestinal transferred to the response regulator PhoP, which binds the illness in humans. This versatile organism encodes 33 PhoP box within the esrB promoter (Chakraborty et al., response regulators (Wang et al., 2009a), allowing it to 2010). In turn, phosphorylated EsrB upregulates EsrC, leading respond to a variety of environmental stimuli. The TCSs to T6SS gene expression. Interestingly, PhoP has varying EsrA-EsrB, PhoP-PhoQ and PhoB-PhoR have been asso- degrees of regulation over esrB depending on the strain. In ciated with T6SS regulation, along with EsrC and the ferric- strain EIB202, mutation of phoP reduced the expression of uptake regulator protein Fur (Zheng et al., 2005; Wang et al., EsrB and EsrC by ~one third (Lv et al.,2012).Thisisin 2009a, 2009b, 2010; Chakraborty et al., 2010, 2011; Lv et al., contrast with PPD130/91, where PhoP strictly regulates EsrB 2012). The response regulators EsrB and PhoP are crucial for (Chakraborty et al., 2010). Elevated magnesium concentra- E. tarda pathogenesis in zebrafish and the Japanese flounder tion (10 as opposed to 1 mM) reduces the expression of T6SS (Wang et al., 2009b; Lv et al., 2012). E. ictaluri causes enteric genes (Chakraborty et al., 2010), whereas elevated copper septicaemia in catfish and also regulates its T6SS via EsrA- levels have no significant effect on T6SS gene expression (Hu EsrB (Rogge & Thune, 2011). et al., 2010). Importantly, temperature and magnesium http://jmm.sgmjournals.org 669 S. T. Miyata, V. Bachmann and S. Pukatzki concentrations conducive to T6SS gene expression are discoideum began to prey upon bacteria, and with the physiologically relevant and this implies that T6SS genes are emergence of higher eukaryotes, macrophages started to upregulated during infection (Chakraborty et al., 2010). engulf and destroy bacteria. We postulate that constitu- tively active T6SSs initially provided a fitness advantage for Phosphate and iron are sequestered within the host and bacteria during environmental inter-bacterial interactions low concentrations can prompt invading pathogens to and that, over time, the T6SS evolved to additionally target activate virulence genes. In PPD130/91, high-iron concen- microbial flora inside a host environment and eukaryotic tration lowered the expression levels of EsrC, EvpP and target cells, which coincided with the evolution of stricter EvpC (Chakraborty et al., 2011). Assuming that iron T6SS regulatory mechanisms (summarized in Table 2). depletion results in the opposite phenotype, this coincides with the hypothesis that virulence genes become activated Several studies have reported T6SS antibacterial effects in a once inside the host. Conversely, EIB202 increases evpP variety of species (Hood et al., 2010; MacIntyre et al., 2010; expression when grown in iron-rich media (Wang et al., Schwarz et al., 2010; Murdoch et al., 2011; Haapalainen 2009b). Obviously there are marked differences in T6SS et al., 2012), suggesting that the T6SS may have evolved as regulatory cascades between these two strains and it is a prokaryotic-killing mechanism. Furthermore, strong currently unclear why EIB202 upregulates EvpP under similarity between T6SS structural proteins and the T4 iron-rich conditions. bacteriophage-puncturing device (Pukatzki et al., 2006, 2007; Leiman et al., 2009; Bernard et al., 2010; Basler et al., The E. tarda PhoB-PhoR TCS responds to phosphate in the 2012) further suggests that T6SSs evolved to target other extracellular environment. The response regulator PhoB bacteria. Notable bacteria that possess constitutively active activates transcription of the pstSCAB-phoU operon which T6SSs (under standard laboratory conditions) include V. is responsible for phosphate acquisition (Chakraborty et al., cholerae V52, Serratia marcescens and B. thailandensis (T6SS- 2011). In PPD130/91, PhoB and EsrC simultaneously bind 1) (Pukatzki et al., 2006; Schwarz et al., 2010; Murdoch et al., the promoter region of evpA (Chakraborty et al., 2011). 2011). Importantly, each of these organisms has been shown T6SS genes are repressed at high phosphate concentration to target other prokaryotes using their T6SSs (MacIntyre and this effect was exacerbated when PPD130/91 was et al., 2010; Schwarz et al., 2010; Murdoch et al., 2011). subjected to both high iron and phosphate concentrations, indicating a synergistic effect (Chakraborty et al., 2011). S. marcescens strain Db10 uses its constitutively active T6SS Interestingly, transposon insertions in pstB, pstC and pstS to target a closely related S. marcescens strain ATCC274; abolished the production of both T3SS and T6SS proteins however, Db10 does not target the eukaryotic organisms D. (Rao et al., 2004; Zheng et al., 2005). Thus, not only the discoideum, Caenorhabditis elegans or Galleria mellonella PhoB-PhoR TCS, but also the phosphate acquisition (Murdoch et al., 2011). Thus, Db10 encodes an unevolved proteins are involved in the regulation of virulence factors. or archaic T6SS according to our proposed model. V. cholerae V52 encodes a multi-functional T6SS that can Why do some bacteria possess constitutively active target both prokaryotic and eukaryotic cells using the same T6SSs, whereas others maintain strict regulation of set of T6SS proteins. Other V. cholerae strains encode a similar T6SS gene cluster but lack constant production of T6SS genes? T6SS proteins. Interestingly, V. cholerae strains responsible T6SS gene clusters have been identified in nearly 100 for cholera pandemics are those that lack constitutive T6SS different bacteria, implying that this virulence factor expression. This may be indicative of a closer evolutionary provides a competitive advantage and has been selected relationship between pandemic strains and eukaryotic for throughout evolution (Boyer et al., 2009). As discussed hosts compared with strain V52. above, the regulatory mechanisms governing T6SS gene expression vary widely from species to species, and even T6SSs are encoded by all sequenced V. cholerae genomes, from strain to strain. Interestingly, some bacteria consti- unlike CT that is encoded only by toxigenic strains. Host tutively produce T6SS proteins when grown under cues are required for V. cholerae to produce toxin- standard laboratory conditions, whereas others maintain coregulated pili that are recognized as receptors for the stricter regulation of T6SS genes. We propose that this CTX phage, allowing the delivery of the CT genes to the disparity arose as an adaptation, as bacteria co-evolve with bacterial genome (Waldor & Mekalanos, 1996). Thus, eukaryotic organisms. toxigenic conversion of V. cholerae in the host suggests that the acquisition of CT genes co-evolved with eukaryotes. In Prokaryotic organisms have been in existence for approxi- contrast, the T6SS appears to have been acquired earlier, mately 3.8 billion years, whereas the first single-celled probably before the advent of eukaryotic cells, as they are eukaryote appeared ~2 billion years ago (Sogin, 1991; found in the ancestral V. cholerae genome of all Mojzsis et al., 1996). Thus, inter-bacterial interactions were environmental (non-toxigenic) and clinical (toxigenic) V. a significant driving force behind prokaryotic evolution for cholerae strains sequenced to date. Ma & Mekalanos (2010) the first ~1.8 billion years. With the advent of eukaryotic previously suggested that the T6SS is a core virulence organisms ~2 billion years ago, bacteria were faced with an determinant and could have facilitated the acquisition of additional survival challenge: single-celled amoebae like D. additional virulence factors like CT and TCP in V. cholerae,

670 Journal of Medical Microbiology 62 http://jmm.sgmjournals.org

Table 2. Characteristics of T6SS regulatory mechanisms

Organism Strain No. distinct Constitutively active Prokaryotic interaction/ Eukaryotic Reference T6SS clusters under laboratory competition? interactions? conditions?

V. cholerae V52 1 Yes Yes Yes Pukatzki et al. (2006) C6706 1 No Unknown Yes Zheng et al. (2010) N16961/A1552 1/1 No Unknown/yes Unknown Ishikawa et al. (2009); Ishikawa et al. (2012) B. mallei ATCC 23344 5 No Unknown T6SS-1; yes Schell et al. (2007); Burtnick et al. (2010) B. thailandensis E264 5 T6SS-1; yes, T6SS-2 T6SS-1; yes T6SS-5; yes Schwarz et al. (2010) –T6SS-5; no B. cenocepacia K56-2 1 No Unknown Yes Aubert et al. (2008) B. pseudomallei K96243 6 T6SS21–T6SS5; no, Unknown T6SS-1; yes Shalom et al. (2007); Hcp6; yes Burtnick et al. (2011); Chen et al. (2011) E. tarda EIB202 1 EvpP; yes Unknown Yes Wang et al. (2009b) PPD130/91 1 Yes Unknown Yes Zheng & Leung (2007) E. ictaluri 93-146 1 EvpC; yes Unknown Yes Rogge & Thune (2011) Pseudomonas aeruginosa PAK, PAO1 3 No HSI-I; yes HSI-I, HSI-II, Mougous et al. (2006, 2007); HSI-III; yes Chugani & Greenberg (2007); Lesic et al. (2009); Starkey & Rahme (2009); Hood et al. (2010); Russell et al. (2011, 2012) E. coli APEC SEPT362 1 Yes Unknown Yes de Pace et al. (2011) EAEC 042 2 Yes Unknown Unknown Dudley et al. (2006) RS218 1 Hcp-1; yes Unknown Yes Zhou et al. (2012)

Pectobacterium atrosepticum Pba1043 1 No Unknown Yes Liu et al. (2008) pressure evolutionary and regulation T6SS Y. pseudotuberculosis YPIII 4 No Unknown Unknown Zhang et al. (2011) Y. pestis KIM6+ 5 No Unknown Unknown Pieper et al. (2009) CO92 5 No Unknown YPO0499–YPO0516; Robinson et al. (2009) yes EV76 6 No ypo0499-0516; Yes No Podladchikova et al. (2011) A. hydrophila SSU 1 Yes Unknown Yes Suarez et al. (2008) S. enterica Typhimurium ATCC 1 No Unknown SPI-6; yes Folkesson et al. (2002); 14028 Parsons & Heffron (2005) Typhi GIFU10007 1 Implied Unknown SPI-6; yes Wang et al. (2011) Francisella novicida U112 1 No Unknown Yes de Bruin et al. (2007) S. marcescens Db10 1 Yes Yes No Murdoch et al. (2011) 671 S. T. Miyata, V. Bachmann and S. Pukatzki leading to the development of highly pathogenic V. & McCarter, 2011; Sheng et al., 2012). It will be interesting cholerae strains. In this case, the acquisition and use of to determine whether the different T6SSs in these strains other key virulence factors such as CT lessen the need for a target different subsets of organisms. At the current time, constitutively active T6SS, leading to the utilization of we observe the trend that organisms with constitutively negative regulators such as TsrA in V. cholerae (Zheng et al., active T6SSs kill prokaryotes but not all organisms that kill 2010). prokaryotes have a constitutively active T6SS. Conversely, organisms with more strictly regulated T6SSs target B. thailandensis is similar to V52 in that it has the ability to eukaryotic organisms, but not all organisms with highly kill both prokaryotic and eukaryotic targets. However, regulated T6SSs target eukaryotes. In the case of strictly unlike V52, B. thailandensis encodes multiple evolutiona- regulated T6SSs, the incorporation of additional regulators rily distinct T6SS gene clusters with different functions may help the pathogen to coordinate the T6SS to prevent (Boyer et al., 2009). T6SS-1 is constitutively active and identification by the host immune system. This hypothesis is targets prokaryotes, whereas T6SS-5 strictly targets eukar- supported by the fact that infection of experimental animals yotes (Schwarz et al., 2010; Russell et al., 2012). The with V. cholerae strains harbouring a constitutive T6SS remaining T6SSs are uncharacterized at this time, but causes major inflammation (Ma & Mekalanos, 2010; Zheng bioinformatic analyses suggest that these systems are more et al., 2010), further highlighting the need for tight T6SS closely related to the T6SS-1 (Schwarz et al., 2010). It control in a host environment. Based on these trends, we would seem that B. thailandensis uses its T6SS for inter- hypothesize that the T6SS evolved as a mechanism for bacterial interactions and over time has acquired a new bacteria to outcompete their prokaryotic neighbours and T6SS gene cluster which allows the organism to compete that constitutive expression of this molecular weapon with eukaryotes as well. conferred a survival advantage. Over time, the emergence In contrast to bacteria that constantly produce T6SS of multicellular/eukaryotic hosts introduced bacteria to the + proteins and target prokaryotes are those which have immune system and made it crucial for T6SS pathogens to developed stricter regulatory mechanisms (e.g. via the use their T6SS more wisely to compete with eukaryotic addition of a negative regulator) and use the T6SS only predators (phagocytic immune cells) and bacterial compe- under specific conditions (such as following phagocytosis). titors (commensal flora) in a host environment, as was Bacteria that fall into this category, all of which are previously postulated (Miyata et al., 2010). pathogens, include Pectobacterium atrosepticum (Liu et al., 2008), Agrobacterium tumefaciens (Yuan et al., 2008), Concluding remarks V. cholerae (e.g. strains C6706 and A1552) (Ishikawa et al., 2009, 2012; Zheng et al., 2010), B. cenocepacia Recent work towards understanding T6SS regulatory (Aubert et al., 2008), B. pseudomallei (Shalom et al., 2007; cascades has demonstrated that the regulation of T6SS gene Burtnick et al., 2011; Chen et al., 2011) and B. mallei clusters is quite complex. As discussed here, the majority of (Schell et al., 2007; Shanks et al., 2009; Burtnick et al., recent work in T6SS regulation has focused on the bacterial 2010). We propose that these bacterial T6SSs evolved to genera Edwardsiella, Vibrio, Burkholderia and Pseudomonas. target eukaryotes through close interaction with their These organisms are found in a wide variety of envir- host(s). This idea is supported by the case of B. mallei onmental niches, and cause disease in a diverse array of which, similar to B. thailandensis, encodes multiple T6SSs; eukaryotes. Thus, it is not surprising that these bacteria have however, the T6SS-1 (responsible for prokaryotic competi- evolved mechanisms, such as the T6SS, to promote their tion in B. thailandensis) cluster in B. mallei is significantly own survival. Depending on the bacterial species, the T6SS degraded and is unlikely to function (Schell et al., 2007). can serve as a eukaryotic virulence factor, a prokaryotic Furthermore, the T6SS-5 of B. mallei is crucial for killing mechanism, or both. Regardless of the cellular target, competition against eukaryotic competitors and it was the T6SS clearly provides a competitive advantage at some + suggested that T6SS-5 could be the reason for B. mallei stage in the life cycle of T6SS bacteria. Further under- transitioning into an obligate pathogen, as opposed to B. standing of the T6SS regulatory networks will aid in thailandensis which has an active T6SS-1 (Schell et al., discerning the forces driving T6SS evolution as a fitness 2007; Schwarz et al., 2010). tool for competing against both prokaryotic and eukaryotic + Not all T6SS bacteria can be strictly segregated into either organisms. one of the two classifications we have just outlined. For example, Pseudomonas aeruginosa confers toxicity in Acknowledgements eukaryotes and prokaryotes with its multiple T6SS gene clusters; however, none of these appear to be constitutively The authors thank Teresa Brooks, Daniel Unterweger and Marcia expressed under laboratory conditions (Mougous et al., Craig for helpful discussions and the critical review of the manuscript. Work in S. P.’s laboratory was supported by the Canadian Institute 2006; Hood et al., 2010). Furthermore, organisms harbour- for Health Research Operating grant MOP-84473, Alberta Innovates – ing multiple T6SS gene clusters have just begun to be Health Solutions, and the Canadian Foundation for Innovation. characterized, including those in V. parahaemolyticus, V. S. T. M. was supported by a PhD studentship from Alberta Innovates alginolyticus and Y. pestis (Pieper et al., 2009; Gode-Potratz – Health Solutions.

672 Journal of Medical Microbiology 62 T6SS regulation and evolutionary pressure

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