臺灣博碩士論文加值系統

在大部分的革蘭氏陰性菌中發現具有第六型分泌系統 (Type VI secretion system, T6SS)，為噬菌體尾部同功的微型分子結構，與Hcp蛋白共同堆疊形成類tail-tube結構；而VgrG蛋白作用則為tail-tube端點形成尖刺的結構。植物病原菌Pseudomonas syrigae pv. tomato DC3000 (Pst DC3000) 推測具有兩群的T6SS基因，HIS-I及HIS-II。Hcp2屬於HIS-II基因群，前人研究顯示Hcp2蛋白在被分泌抵來抗腸桿菌科細菌時扮演必須的角色。然而VgrG在細菌中的功能尚未清楚。在本研究中，預測Pst DC3000具有7個類VgrG基因，其中3個屬於HIS-I及HIS-II基因群，另外4個則分散於細菌的基因組中。本研究利用系統性去除 (systematic deletion) 基因的方式證明各個VgrG蛋白在分泌及細菌間競爭中的貢獻程度。在類VgrG基因中，VgrG-2b (PSPTO_5436)為抵抗E. coli主要的貢獻者；VgrG-2a (PSPTO_5415) 在5種不同的植物病害細菌中則有較小的貢獻。上述兩種VgrG蛋白皆屬於HIS-II基因群。此外，VgrG-2b蛋白在離體試驗 (in vitro) 中可直接與Hcp2蛋白作用。綜合本研究結果顯示HIS-II在Pst DC3000與其他細菌競爭作用中扮演主要的角色，進一步證實Pst DC3000具有較佳競爭能力的原因。此外，在T6SS研究中找到11個與VgrG有關聯的基因，顯示其可能具有成潛力為VgrG-2a下游PSPTO_5413的作用蛋白 (effector)。

The type VI secretion system (T6SS), found in most gram-negative bacteria, is a macromolecular structure analogous to bacteriophage tails, with Hcp stacking to form a tail-tube like structure and VgrG, acting as a puncturing device at the tip of this tube. The plant pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is known to have two putative T6SS gene clusters, HSI-I and HSI-II. Hcp2, encoded in the HSI-II gene cluster, previously shown to be secreted and playing an essential role in antimicrobial activity against enterobacteriaceae. However, the role of VgrG in this bacterium is still unknown. In this study, a total of seven vgrG genes were predicted to be present in Pst DC3000, three of which are located within the HSI-I and HSI-II gene clusters with additional four others scattered in the bacterial genome. Here, we demonstrate the secretion of VgrG proteins, and also, the contribution of each VgrG protein in secretion and interbacterial competition by systematic deletion of these genes. Among these VgrG homologs, VgrG-2b (PSPTO_5436) was the major contributor in secretion and interbacterial competition activity against E. coli and five different phytopathogenic bacteria followed by a small contribution of VgrG-2a (PSPTO_5415). Both VgrG proteins are encoded within the HSI-II gene cluster. Furthermore, VgrG-2b was shown to directly interact with Hcp2 in vitro. All these further support the notion that HSI-II plays a major role in Pst DC3000 to compete for niches, thus conferring this bacterium significant fitness advantage against competitive microbes. Additionally, eleven putative VgrG-associated genes were targeted for deletion to observe its role in the T6SS results showed a potential effector activity for PSPTO_5413, located downstream of VgrG-2a.

Index I
List of Tables and Figures III
Chinese Abstract V
Abstract VI
Introduction 1
Interbacterial Interactions 1
Secretion Systems 2
The Type VI Secretion Systems 4
The components of the Type VI secretion system. 6
Gene cluster architecture and evolution of the Type VI secretion system. 8
Regulation of the Type VI secretion system. 9
Functions of the Type VI secretion system 12
Proteins Secreted by T6SS 14
Hcp 14
VgrG 15
Type VI secreted effector proteins 19
(A) Cell wall-targeting effectors: 19
(B) Membrane-targeting effectors 20
(C) Cytoplasmic- or Nucleic acid-targeting effector 21
P. syringae pv. tomato DC3000 and its Type VI Secretion System 21
Objectives 24
Material and Methods 25
Bacterial strains and growth conditions 25
Bioinformatic Analysis 25
Preparation of electrocompetent cells 26
DNA purification from agarose gel slice 26
DNA manipulation 27
Affinity purification of VgrG antibody 27
GUS reporter Assay 28
Secretion Assay 30
Interbacterial Competitions Assays 31
Protein Purification 32
Gel Filtration Chromatography 33
High Performance Liquid Chromatography 34
Bait-prey Pull-down for Protein-protein Interaction 34
Results 36
In silico analysis predicts the presence of seven copies of vgrG 36
VgrG is expressed and secreted into the culture medium 38
VgrG-2a and VgrG-2b are the only secreted VgrG proteins in Pst DC3000. 39
VgrG-2a and VgrG-2b are the only VgrG proteins contributing to the interbacterial competition activity. 40
PSPTO_5413 is a possible effector protein while other VgrG-associated genes did not affect bacteria killing phenotype in Pst DC3000. 43
VgrG-2b interacts with Hcp2 in vitro 45
Discussion 48
Reference 54
Tables 65
Figures 74

Aschtgen, M.-S., Bernard, C. S., De Bentzmann, S., Lloubes, R. and Cascales, E. (2008). SciN is an outer membrane lipoprotein required for type VI secretion in enteroaggregative Escherichia coli. J. Bacteriol. 190, 7523–7531.
Aschtgen, M.-S., Gavioli, M., Dessen, A., Lloubes, R. and Cascales, E. (2010a). The SciZ protein anchors the enteroaggregative Escherichia coli Type VI secretion system to the cell wall. Mol. Microbiol. 75, 886–899.
Aschtgen, M.-S., Thomas, M. S. and Cascales, E. (2010b). Anchoring the type VI secretion system to the peptidoglycan: TssL, TagL, TagP... what else? Virulence 1, 535–540.
Ballister, E. R., Lai, A. H., Zuckermann, R. N., Cheng, Y. and Mougous, J. D. (2008). In vitro self-assembly of tailorable nanotubes from a simple protein building block. Proc. Natl. Acad. Sci. U.S.A. 105, 3733–3738.
Barret, M., Egan, F., Fargier, E., Morrissey, J. P. and O''Gara, F. (2011). Genomic analysis of the type VI secretion systems in Pseudomonas spp.: novel clusters and putative effectors uncovered. Microbiology (Reading, Engl.) 157, 1726–1739.
Bartonickova, L., Sterzenbach, T., Nell, S., Kops, F., Schulze, J., Venzke, A., Brenneke, B., Bader, S., Gruber, A. D., Suerbaum, S., et al. (2012). Hcp and VgrG1 are secreted components of the Helicobacter hepaticus type VI secretion system and VgrG1 increases the bacterial colitogenic potential. Cell Microbiol.
Basler, M. and Mekalanos, J. J. (2012). Type 6 secretion dynamics within and between bacterial cells. Science 337, 815.
Basler, M., Ho, B. T. and Mekalanos, J. J. (2013). Tit-for-Tat: Type VI Secretion System Counterattack during Bacterial Cell-Cell Interactions. Cell 152, 884–894.
Basler, M., Pilhofer, M., Henderson, G. P., Jensen, G. J. and Mekalanos, J. J. (2012). Type VI secretion requires a dynamic contractile phage tail-like structure. Nat Protoc 483, 182–186.
Bassler, B. L. and Losick, R. (2006). Bacterially Speaking. Cell 125, 237–246.
Bell, B. L., Mohapatra, N. P. and Gunn, J. S. (2010). Regulation of Virulence Gene Transcripts by the Francisella novicida Orphan Response Regulator PmrA: Role of Phosphorylation and Evidence of MglA/SspA Interaction. Infect. Immun. 78, 2189–2198.
Benz, J., Sendlmeier, C., Barends, T. R. M. and Meinhart, A. (2012). Structural insights into the effector-immunity system Tse1/Tsi1 from Pseudomonas aeruginosa. PLoS ONE 7, e40453.
Bernard, C. S., Brunet, Y. R., Gavioli, M., Lloubes, R. and Cascales, E. (2011). Regulation of type VI secretion gene clusters by sigma54 and cognate enhancer binding proteins. J. Bacteriol. 193, 2158–2167.
Bernard, C. S., Brunet, Y. R., Gueguen, E. and Cascales, E. (2010). Nooks and crannies in type VI secretion regulation. J. Bacteriol. 192, 3850–3860.
Boyer, F., Fichant, G., Berthod, J., Vandenbrouck, Y. and Attree, I. (2009). Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources? BMC Genomics 10, 104.
Bonemann, G., Pietrosiuk, A. and Mogk, A. (2010). Tubules and donuts: a type VI secretion story. Mol. Microbiol. 76, 815–821.
Bonemann, G., Pietrosiuk, A., Diemand, A., Zentgraf, H. and Mogk, A. (2009). Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion. EMBO J 28, 315–325.
Brencic, A. and Lory, S. (2009). Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA. Mol. Microbiol. 72, 612–632.
Brencic, A., McFarland, K. A., McManus, H. R., Castang, S., Mogno, I., Dove, S. L. and Lory, S. (2009). The GacS/GacA signal transduction system of Pseudomonas aeruginosaacts exclusively through its control over the transcription of the RsmY and RsmZ regulatory small RNAs. Mol. Microbiol. 73, 434–445.
Brian M Peters, M. A. J.-R. G. A. O. J. W. C. M. E. S. (2012). Polymicrobial Interactions: Impact on Pathogenesis and Human Disease. Clinical Microbiology Reviews 25, 193.
Campanaro, S., Vezzi, A., Vitulo, N., Lauro, F. M., D''Angelo, M., Simonato, F., Cestaro, A., Malacrida, G., Bertoloni, G., Valle, G., et al. (2005). Laterally transferred elements and high pressure adaptation in Photobacterium profundum strains. BMC Genomics 6, 122.
Carruthers, M. D., Nicholson, P. A., Tracy, E. N. and Munson, R. S. (2013). Acinetobacter baumannii Utilizes a Type VI Secretion System for Bacterial Competition. PLoS ONE 8, e59388.
Casabona, M. G., Silverman, J. M., Sall, K. M., Boyer, F., Coute, Y., Poirel, J., Grunwald, D., Mougous, J. D., Elsen, S. and Attree, I. (2012). An ABC transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas aeruginosa. Environ Microbiol.
Cascales, E. (2008). The type VI secretion toolkit. Audio, Transactions of the IRE Professional Group on 9, 735–741.

Cascales, E. and Cambillau, C. (2012). Structural biology of type VI secretion systems. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 367, 1102–1111.
Castang, S., McManus, H. R., Turner, K. H. and Dove, S. L. (2008). H-NS family members function coordinately in an opportunistic pathogen. Proc. Natl. Acad. Sci. U.S.A. 105, 18947–18952.
Cathelyn, J. S., Crosby, S. D., Lathem, W. W., Goldman, W. E. and Miller, V. L. (2006). RovA, a global regulator of Yersinia pestis, specifically required for bubonic plague. Proc. Natl. Acad. Sci. U.S.A. 103, 13514–13519.
Chou, S., Bui, N. K., Russell, A. B., Lexa, K. W., Gardiner, T. E., LeRoux, M., Vollmer, W. and Mougous, J. D. (2012). Structure of a Peptidoglycan Amidase Effector Targeted to Gram-Negative Bacteria by the Type VI Secretion System. Cell Rep 1, 656–664.
Das, S. and Chaudhuri, K. (2003). Identification of a Unique IAHP (IcmF Associated Homologous Proteins) Cluster in Vibrio cholerae and other Proteobacteria through In Silico Analysis. In Silico Biology Volume 3, 287–300.
Deziel, E., Gopalan, S., Tampakaki, A. P., Lepine, F., Padfield, K. E., Saucier, M., Xiao, G. and Rahme, L. G. (2004). The contribution of MvfR to Pseudomonas aeruginosa pathogenesis and quorum sensing circuitry regulation: multiple quorum sensing-regulated genes are modulated without affecting lasRI, rhlRI or the production of N-acyl- l-homoserine lactones. Mol. Microbiol. 55, 998–1014.
Dong, T. G., Ho, B. T., Yoder-Himes, D. R. and Mekalanos, J. J. (2013). Identification of T6SS-dependent effector and immunity proteins by Tn-seq in Vibrio cholerae. Proc. Natl. Acad. Sci. U.S.A. 110, 2623–2628.
Dudley, E. G., Thomson, N. R., Parkhill, J., Morin, N. P. and Nataro, J. P. (2006). Proteomic and microarray characterization of the AggR regulon identifies a pheU pathogenicity island in enteroaggregative Escherichia coli. Mol. Microbiol. 61, 1267–1282.
English, G., Byron, O., Cianfanelli, F. R., Prescott, A. R. and Coulthurst, S. J. (2014). Biochemical analysis of TssK, a core component of the bacterial Type VI secretion system, reveals distinct oligomeric states of TssK and identifies a TssK-TssFG sub-complex. Biochem. J.
Evans, M. L. and Chapman, M. R. (2014). Curli biogenesis: Order out of disorder. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1843, 1551–1558.
Felisberto-Rodrigues, C. C., Durand, E. E., Aschtgen, M.-S. M., Blangy, S. S., Ortiz-Lombardia, M. M., Douzi, B. B., Cambillau, C. C. and Cascales, E. (2011). Towards a structural comprehension of bacterial type VI secretion systems: characterization of the TssJ-TssM complex of an Escherichia coli pathovar. PLoS Pathog 7, e1002386–e1002386.
Filiatrault, M. J., Stodghill, P. V., Bronstein, P. A., Moll, S., Lindeberg, M., Grills, G., Schweitzer, P., Wang, W., Schroth, G. P., Luo, S., et al. (2010). Transcriptome Analysis of Pseudomonas syringae Identifies New Genes, Noncoding RNAs, and Antisense Activity. J. Bacteriol. 192, 2359–2372.
Folkesson, A., Lofdahl, S. and Normark, S. (2002). The Salmonella enterica subspecies I specific centisome 7 genomic island encodes novel protein families present in bacteria living in close contact with eukaryotic cells. Res. Microbiol. 153, 537–545.
Geibel, S. and Waksman, G. (2014). The molecular dissection of the chaperone–usher pathway. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1843, 1559–1567.
Goodman, A. L., Kulasekara, B., Rietsch, A., Boyd, D., Smith, R. S. and Lory, S. (2004). A Signaling Network Reciprocally Regulates Genes Associated with Acute Infection and Chronic Persistence in Pseudomonas aeruginosa. Dev Cell 7, 745–754.
Goosens, V. J., Monteferrante, C. G. and van Dijl, J. M. (2014). The Tat system of Gram-positive bacteria. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1843, 1698–1706.
Haapalainen, M., Mosorin, H., Dorati, F., Wu, R.-F., Roine, E., Taira, S., Nissinen, R., Mattinen, L., Jackson, R., Pirhonen, M., et al. (2012). Hcp2, a Secreted Protein of the Phytopathogen Pseudomonas syringae pv. Tomato DC3000, Is Required for Fitness for Competition against Bacteria and Yeasts. J. Bacteriol. 194, 4810–4822.
Hachani, A., Allsopp, L. P., Oduko, Y. and Filloux, A. (2014). The VgrG proteins are “A la carte” delivery systems for bacterial type VI effectors. J. Biol. Chem.
Hachani, A., Lossi, N. S., Hamilton, A., Jones, C., Bleves, S., Albesa-Jove, D. and Filloux, A. (2011). Type VI secretion system in Pseudomonas aeruginosa: secretion and multimerization of VgrG proteins. J. Biol. Chem. 286, 12317–12327.
Hall-Stoodley, L., Costerton, J. W. and Stoodley, P. (2004). Bacterial biofilms: from the Natural environment to infectious diseases. Nat. Rev. Microbiol. 2, 95–108.
Hamburger, Z. A. (1999). Crystal Structure of Invasin: A Bacterial Integrin-Binding Protein. Science 286, 291–295.
Hayes, C. S., Aoki, S. K. and Low, D. A. (2010). Bacterial Contact-Dependent Delivery Systems. Annu. Rev. Genet. 44, 71–90.
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. and Pease, L. R. (1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59.



Hood, R. D., Singh, P., Hsu, F., Guvener, T., Carl, M. A., Trinidad, R. R. S., Silverman, J. M., Ohlson, B. B., Hicks, K. G., Plemel, R. L., et al. (2010). A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria. Cell Host Microbe 7, 25–37.
Hunt, T. A., Kooi, C., Sokol, P. A. and Valvano, M. A. (2004). Identification of Burkholderia cenocepacia Genes Required for Bacterial Survival In Vivo. Infect. Immun. 72, 4010–4022.
Ishikawa, T., Rompikuntal, P. K., Lindmark, B., Milton, D. L. and Wai, S. N. (2009). Quorum sensing regulation of the two hcp alleles in Vibrio cholerae O1 strains. PLoS ONE 4, e6734.
Jiang, F., Waterfield, N. R., Yang, J., Yang, G. and Jin, Q. (2014). A Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells. Cell Host Microbe 15, 600–610.
Jingyi Wang, C. L. H. Y. A. M. S. J. (1998). A Novel Serine/Threonine Protein Kinase Homologue of Pseudomonas aeruginosa Is Specifically Inducible within the Host Infection Site and Is Required for Full Virulence in Neutropenic Mice. J. Bacteriol. 180, 6764.
Joshi, H., Dave, R. and Venugopalan, V. P. (2009). Competition Triggers Plasmid-Mediated Enhancement of Substrate Utilisation in Pseudomonas putida. PLoS ONE 4, e6065.
Kang, Y., Nguyen, D. T., Son, M. S. and Hoang, T. T. (2008). The Pseudomonas aeruginosa PsrA responds to long-chain fatty acid signals to regulate the fadBA5 -oxidation operon. Microbiology (Reading, Engl.) 154, 1584–1598.
Kitaoka, M., Miyata, S. T., Brooks, T. M., Unterweger, D. and Pukatzki, S. (2011). VasH is a transcriptional regulator of the type VI secretion system functional in endemic and pandemic Vibrio cholerae. J. Bacteriol. 193, 6471–6482.
Konovalova, A. and Sogaard-Andersen, L. (2011). Close encounters: contact-dependent interactions in bacteria. Mol. Microbiol. 81, 297–301.
Koskiniemi, S., Lamoureux, J. G., Nikolakakis, K. C., T''kint de Roodenbeke, C., Kaplan, M. D., Low, D. A. and Hayes, C. S. (2013). Rhs proteins from diverse bacteria mediate intercellular competition. Proc. Natl. Acad. Sci. U.S.A. 110, 7032–7037.
Kostakioti, M., Newman, C. L., Thanassi, D. G. and Stathopoulos, C. (2005). Mechanisms of Protein Export across the Bacterial Outer Membrane. J. Bacteriol. 187, 4306–4314.
Lapouge, K., Schubert, M., Allain, F. H. T. and Haas, D. (2007). Gac/Rsm signal transduction pathway of γ-proteobacteria: from RNA recognition to regulation of social behaviour. Mol. Microbiol. 67, 241–253.

Leiman, P. G. and Shneider, M. M. (2011). Contractile Tail Machines of Bacteriophages. In link.springer.com, pp. 93–114. Boston, MA: Springer US.
Leiman, P. G., Basler, M., Ramagopal, U. A., Bonanno, J. B., Sauder, J. M., Pukatzki, S., Burley, S. K., Almo, S. C. and Mekalanos, J. J. (2009). Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin. Proc. Natl. Acad. Sci. U.S.A. 106, 4154–4159.
Lertpiriyapong, K., Gamazon, E. R., Feng, Y., Park, D. S., Pang, J., Botka, G., Graffam, M. E., Ge, Z. and Fox, J. G. (2012). Campylobacter jejuni type VI secretion system: roles in adaptation to deoxycholic acid, host cell adherence, invasion, and in vivo colonization. PLoS ONE 7, e42842.
Li, J.-F., Li, L. and Sheen, J. (2010). Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology. Plant Methods 6, 1.
Li, L., Zhang, W., Liu, Q., Gao, Y., Gao, Y., Wang, Y., Wang, D. Z., Li, Z. and Wang, T. (2013). Structural Insights on the Bacteriolytic and Self-protection Mechanism of Muramidase Effector Tse3 in Pseudomonas aeruginosa. J. Biol. Chem. 288, 30607–30613.
Lin, J.-S., Ma, L.-S. and Lai, E.-M. (2013). Systematic Dissection of the Agrobacterium Type VI Secretion System Reveals Machinery and Secreted Components for Subcomplex Formation. PLoS ONE 8, e67647.
Lloyd, A. L., Rasko, D. A. and Mobley, H. L. T. (2007). Defining Genomic Islands and Uropathogen-Specific Genes in Uropathogenic Escherichia coli. J. Bacteriol. 189, 3532–3546.
Lossi, N. S., Dajani, R., Freemont, P. and Filloux, A. (2011). Structure-function analysis of HsiF, a gp25-like component of the type VI secretion system, in Pseudomonas aeruginosa. Microbiology (Reading, Engl.) 157, 3292–3305.
Lucchini, S., Rowley, G., Goldberg, M. D., Hurd, D., Harrison, M. and Hinton, J. C. D. (2006). H-NS Mediates the Silencing of Laterally Acquired Genes in Bacteria. PLoS Pathog 2, e81.
Ma, A. T. and Mekalanos, J. J. (2010). In vivo actin cross-linking induced by Vibrio cholerae type VI secretion system is associated with intestinal inflammation. Proc. Natl. Acad. Sci. U.S.A. 107, 4365–4370.
Ma, L.-S., Hachani, A., Lin, J.-S., Filloux, A. and Lai, E.-M. (2014). Agrobacterium tumefaciens Deploys a Superfamily of Type VI Secretion DNase Effectors as Weapons for Interbacterial Competition In Planta. Cell Host Microbe 16, 94–104.
MacIntyre, D. L., Miyata, S. T., Kitaoka, M. and Pukatzki, S. (2010). The Vibrio cholerae type VI secretion system displays antimicrobial properties. Proc. Natl. Acad. Sci. U.S.A. 107, 19520–19524.

Mary Purcell, H. A. S. (1998). The Legionella pneumophila icmGCDJBF Genes Are Required for Killing of Human Macrophages. Infect. Immun. 66, 2245.
Miyata, S. T., Bachmann, V. and Pukatzki, S. (2013). Type VI secretion system regulation as a consequence of evolutionary pressure. J. Med. Microbiol. 62, 663–676.
Miyata, S. T., Kitaoka, M., Brooks, T. M., McAuley, S. B. and Pukatzki, S. (2011). Vibrio cholerae requires the type VI secretion system virulence factor VasX to kill Dictyostelium discoideum. Infect. Immun. 79, 2941–2949.
Miyata, S. T., Kitaoka, M., Wieteska, L., Frech, C., Chen, N. and Pukatzki, S. (2010). The Vibrio Cholerae Type VI Secretion System: Evaluating its Role in the Human Disease Cholera. Front Microbiol 1, 117.
Mougous, J. D., Cuff, M. E., Raunser, S., Shen, A., Zhou, M., Gifford, C. A., Goodman, A. L., Joachimiak, G., Ordonez, C. L., Lory, S., et al. (2006). A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 312, 1526–1530.
Mougous, J. D., Gifford, C. A., Ramsdell, T. L. and Mekalanos, J. J. (2007). Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa. Nat. Cell Biol. 9, 797–803.
Murdoch, S. L., Trunk, K., English, G., Fritsch, M. J., Pourkarimi, E. and Coulthurst, S. J. (2011). The opportunistic pathogen Serratia marcescens utilizes type VI secretion to target bacterial competitors. J. Bacteriol. 193, 6057–6069.
Murzin, A. G. (1993). OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences. EMBO J 12, 861.
Natale, P., Bruser, T. and Driessen, A. J. M. (2008). Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane—Distinct translocases and mechanisms. Biochimica et Biophysica Acta (BBA) - Biomembranes 1778, 1735–1756.
Oganesyan, N., Ankoudinova, I., Kim, S.-H. and Kim, R. (2007). Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization. Protein Expr. Purif. 52, 280–285.
Osipiuk, J., Xu, X., Cui, H., Savchenko, A., Edwards, A. and Joachimiak, A. (2011). Crystal structure of secretory protein Hcp3 from Pseudomonas aeruginosa. J. Struct. Funct. Genomics 12, 21–26.
Pallen, M., Chaudhuri, R. and Khan, A. (2002). Bacterial FHA domains: neglected players in the phospho-threonine signalling game? Trends Microbiol. 10, 556–563.
Parsons, D. A. and Heffron, F. (2005). sciS, an icmF Homolog in Salmonella enterica Serovar Typhimurium, Limits Intracellular Replication and Decreases Virulence. Infect. Immun. 73, 4338–4345.
Pukatzki, S. S., Ma, A. T. A., Revel, A. T. A., Sturtevant, D. D. and Mekalanos, J. J. (2007). Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin. Proc. Natl. Acad. Sci. U.S.A. 104, 15508–15513.
Pukatzki, S., Ma, A. T., Sturtevant, D., Krastins, B., Sarracino, D., Nelson, W. C., Heidelberg, J. F. and Mekalanos, J. J. (2006). Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc. Natl. Acad. Sci. U.S.A. 103, 1528–1533.
Renzi, F., Rescalli, E., Galli, E. and Bertoni, G. (2010). Identification of genes regulated by the MvaT-like paralogues TurA and TurB of Pseudomonas putidaKT2440. Environ Microbiol 12, 254–263.
Robb, C. S., Assmus, M., Nano, F. E. and Boraston, A. B. (2013). Structure of the T6SS lipoprotein TssJ1 from Pseudomonas aeruginosa. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 69, 607–610.
Roest, H. P., Mulders, I. H. M., Spaink, H. P., Wijffelman, C. A. and Ben J J Lugtenberg (1997). A Rhizobium leguminosarum Biovar trifolii Locus Not Localized on the Sym Plasmid Hinders Effective Nodulation on Plants of the Pea Cross-Inoculation Group. http://dx.doi.org/10.1094/MPMI.1997.10.7.938.
Russell, A. B., Hood, R. D., Bui, N. K., LeRoux, M., Vollmer, W. and Mougous, J. D. (2011). Type VI secretion delivers bacteriolytic effectors to target cells. Nature 475, 343–347.
Russell, A. B., LeRoux, M., Hathazi, K., Agnello, D. M., Ishikawa, T., Wiggins, P. A., Wai, S. N. and Mougous, J. D. (2013). Diverse type VI secretion phospholipases are functionally plastic antibacterial effectors. Nat Protoc.
Russell, A. B., Peterson, S. B. and Mougous, J. D. (2014). Type VI secretion system effectors: poisons with a purpose. Nat. Rev. Microbiol.
Russell, A. B., Singh, P., Brittnacher, M., Bui, N. K., Hood, R. D., Carl, M. A., Agnello, D. M., Schwarz, S., Goodlett, D. R., Vollmer, W., et al. (2012). A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach. Cell Host Microbe 11, 538–549.
Sabala, I., Jagielska, E., Bardelang, P. T., Czapinska, H., Dahms, S. O., Sharpe, J. A., James, R., Than, M. E., Thomas, N. R. and Bochtler, M. (2014). Crystal structure of the antimicrobial peptidase lysostaphin from Staphylococcus simulans. FEBS J. 281, 4112–4122.
Salomon, D., Gonzalez, H., Updegraff, B. L. and Orth, K. (2013). Vibrio parahaemolyticus Type VI Secretion System 1 Is Activated in Marine Conditions to Target Bacteria, and Is Differentially Regulated from System 2. PLoS ONE 8, e61086.
Salomon, D., Klimko, J. A. and Orth, K. (2014). H-NS regulates the Vibrio parahaemolyticus type VI secretion system 1. Microbiology (Reading, Engl.).
Sana, T. G., Soscia, C., Tonglet, C. M., Garvis, S. and Bleves, S. (2013). Divergent Control of Two Type VI Secretion Systems by RpoN in Pseudomonas aeruginosa. PLoS ONE 8, e76030.
Santic, M., Molmeret, M., Barker, J. R., Klose, K. E., Dekanic, A., Doric, M. and Abu Kwaik, Y. (2007). A Francisella tularensis pathogenicity island protein essential for bacterial proliferation within the host cell cytosol. Cell Microbiol 9, 2391–2403.
Sarris, P. F., Skandalis, N., Kokkinidis, M. and Panopoulos, N. J. (2010). In silico analysis reveals multiple putative type VI secretion systems and effector proteins in Pseudomonas syringae pathovars. Mol. Plant Pathol. 11, 795–804.
Schell, M. A., Ulrich, R. L., Ribot, W. J., Brueggemann, E. E., Hines, H. B., Chen, D., Lipscomb, L., Kim, H. S., Mrazek, J., Nierman, W. C., et al. (2007). Type VI secretion is a major virulence determinant in Burkholderia mallei. Mol. Microbiol. 64, 1466–1485.
Schlieker, C., Zentgraf, H., Dersch, P. and Mogk, A. (2005). ClpV, a unique Hsp100/Clp member of pathogenic proteobacteria. Biol. Chem. 386.
Schwarz, S., Hood, R. D. and Mougous, J. D. (2010a). What is type VI secretion doing in all those bugs? Trends Microbiol. 18, 531–537.
Schwarz, S., West, T. E., Boyer, F., Chiang, W.-C., Carl, M. A., Hood, R. D., Rohmer, L., Tolker-Nielsen, T., Skerrett, S. J. and Mougous, J. D. (2010b). Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions. PLoS Pathog 6, e1001068.
Shneider, M. M., Buth, S. A., Ho, B. T., Basler, M., Mekalanos, J. J. and Leiman, P. G. (2013). PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nat Protoc.
Shrivastava, S. and Mande, S. S. (2008). Identification and functional characterization of gene components of Type VI Secretion system in bacterial genomes. PLoS ONE 3, e2955.
Silverman, J. M., Agnello, D. M., Zheng, H., Andrews, B. T., Li, M., Catalano, C. E., Gonen, T. and Mougous, J. D. (2013). Haemolysin Coregulated Protein Is an Exported Receptor and Chaperone of Type VI Secretion Substrates. Mol. Cell.
Silverman, J. M., Austin, L. S., Hsu, F., Hicks, K. G., Hood, R. D. and Mougous, J. D. (2011). Separate inputs modulate phosphorylation-dependent and -independent type VI secretion activation. Mol. Microbiol. 82, 1277–1290.
Srinivasa Rao, P. S., Yamada, Y., Tan, Y. P. and Leung, K. Y. (2004). Use of proteomics to identify novel virulence determinants that are required for Edwardsiella tarda pathogenesis. Mol. Microbiol. 53, 573–586.

Stathopoulos, C., Hendrixson, D. R., Thanassi, D. G., Hultgren, S. J., St Geme, J. W., III and Curtiss, R., III (2000). Secretion of virulence determinants by the general secretory pathway in Gram-negative pathogens: an evolving story. Microbes and Infection 2, 1061–1072.
Suarez, G., Sierra, J. C., Erova, T. E., Sha, J., Horneman, A. J. and Chopra, A. K. (2010). A type VI secretion system effector protein, VgrG1, from Aeromonas hydrophila that induces host cell toxicity by ADP ribosylation of actin. J. Bacteriol. 192, 155–168.
Suarez, G., Sierra, J. C., Sha, J., Wang, S., Erova, T. E., Fadl, A. A., Foltz, S. M., Horneman, A. J. and Chopra, A. K. (2008). Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophila. Microbial Pathogenesis 44, 344–361.
Suerbaum, S., Josenhans, C., Sterzenbach, T., Drescher, B., Brandt, P., Bell, M., Droge, M., Fartmann, B., Fischer, H. P., Ge, Z., et al. (2003). The complete genome sequence of the carcinogenic bacterium Helicobacter hepaticus. Proc. Natl. Acad. Sci. U.S.A. 100, 7901–7906.
Toesca, I. J., French, C. T. and Miller, J. F. (2014). The T6SS-5 VgrG spike protein mediates membrane fusion during intercellular spread by pseudomallei-group Burkholderia species. Infect. Immun.
Tomaz, A. (1965). Control of the Competent State in Pneumococcus by a Hormone-Like Cell Product: An Example for a New Type of Regulatory Mechanism in Bacteria. Nature 208, 155–159.
Tseng, T.-T., Tyler, B. M. and Setubal, J. C. (2009). Protein secretion systems in bacterial-host associations, and their description in the Gene Ontology. BMC Microbiol. 9, S2.
Uchida, K., Leiman, P. G., Arisaka, F. and Kanamaru, S. (2013). Structure and properties of the C-terminal β-helical domain of VgrG protein from Escherichia coli O157. J. Biochem.
Vanderlene L Kung, S. K. C. S. E. M. B. A. J. H. A. R. H. (2012). An rhs gene of Pseudomonas aeruginosa encodes a virulence protein that activates the inflammasome. Proc. Natl. Acad. Sci. U.S.A. 109, 1275–1280.
Veesler, D. and Cambillau, C. (2011). A common evolutionary origin for tailed-bacteriophage functional modules and bacterial machineries. Microbiol Mol Biol Rev 75, 423–33– first page of table of contents.
Wang, X., Wang, Q., Xiao, J., Liu, Q., Wu, H., Xu, L. and Zhang, Y. (2009). Edwardsiella tarda T6SS component evpP is regulated by esrB and iron, and plays essential roles in the invasion of fish. Fish Shellfish Immunol. 27, 469–477.
Wang, Y. D., Zhao, S. and Hill, C. W. (1998). Rhs elements comprise three subfamilies which diverged prior to acquisition by Escherichia coli. J. Bacteriol. 180, 4102–4110.
Whalen, M. C., Innes, R. W., Bent, A. F. and Staskawicz, B. J. (1991). Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean.
Wilderman, P. J., Vasil, A. I., Johnson, Z. and Vasil, M. L. (2001). Genetic and biochemical analyses of a eukaryotic-like phospholipase D of Pseudomonas aeruginosa suggest horizontal acquisition and a role for persistence in a chronic pulmonary infection model. Mol. Microbiol. 39, 291–304.
Williams, S. G., Varcoe, L. T., Attridge, S. R. and Manning, P. A. (1996). Vibrio cholerae Hcp, a secreted protein coregulated with HlyA. Infect. Immun. 64, 283–289.
Winsor, G. L., Lam, D. K. W., Fleming, L., Lo, R., Whiteside, M. D., Yu, N. Y., Hancock, R. E. W. and Brinkman, F. S. L. (2010). Pseudomonas Genome Database: improved comparative analysis and population genomics capability for Pseudomonas genomes. Nucleic Acids Res 39, D596–D600.
Wu, H.-Y., Chung, P.-C., Shih, H.-W., Wen, S.-R. and Lai, E.-M. (2008). Secretome analysis uncovers an Hcp-family protein secreted via a type VI secretion system in Agrobacterium tumefaciens. J. Bacteriol. 190, 2841–2850.
Young, J. M., Takikawa, Y., Gardan, L. and Stead, D. E. (1992). Changing Concepts in the Taxonomy of Plant Pathogenic Bacteria. Annu. Rev. Phytopathol. 30, 67–105.
Zhang, W., Wang, Y., Song, Y., Wang, T., Xu, S., Peng, Z., Lin, X., Zhang, L. and Shen, X. (2013). A type VI secretion system regulated by OmpR in Yersinia pseudotuberculosis functions to maintain intracellular pH homeostasis. Environ Microbiol 15, 557–569.
Zheng (2010). Quorum sensing and a global regulator TsrA control expression of type VI secretion and virulence in Vibrio cholerae. Proc. Natl. Acad. Sci. U.S.A. 107, 21128–21133.
Zheng, J. and Leung, K. Y. (2007). Dissection of a type VI secretion system in Edwardsiella tarda. Mol. Microbiol. 66, 1192–1206.
Zobell, C. E. (1943). The Effect of Solid Surfaces upon Bacterial Activity. J. Bacteriol. 46, 39.
Zoued, A., Durand, E., Bebeacua, C., Brunet, Y. R., Douzi, B., Cambillau, C., Cascales, E. and Journet, L. (2013). TssK is a trimeric cytoplasmic protein interacting with components of both phage-like and membrane anchoring complexes of the Type VI secretion system. J. Biol. Chem.