1887

Microbiology Society logo

Volume 169, Issue 1

Transcriptional organization and regulation of the K1 type VI secretion system gene cluster Open Access

Abstract

The type VI secretion system (T6SS) is an antimicrobial molecular weapon that is widespread in Proteobacteria and offers competitive advantages to T6SS-positive micro-organisms. Three T6SSs have recently been described in KT2440 and it has been shown that one, K1-T6SS, is used to outcompete a wide range of phytopathogens, protecting plants from pathogen infections. Given the relevance of this system as a powerful and innovative mechanism of biological control, it is critical to understand the processes that govern its expression. Here, we experimentally defined two transcriptional units in the K1-T6SS cluster. One encodes the structural components of the system and is transcribed from two adjacent promoters. The other encodes two hypothetical proteins, the tip of the system and the associated adapters, and effectors and cognate immunity proteins, and it is also transcribed from two adjacent promoters. The four identified promoters contain the typical features of σ-dependent promoters. We have studied the expression of the system under different conditions and in a number of mutants lacking global regulators. K1-T6SS expression is induced in the stationary phase, but its transcription does not depend on the stationary σ factor RpoS. In fact, the expression of the system is indirectly repressed by RpoS. Furthermore, it is also repressed by RpoN and the transcriptional regulator FleQ, an enhancer-binding protein typically acting in conjunction with RpoN. Importantly, expression of the K1-T6SS gene cluster is positively regulated by the GacS–GacA two-component regulatory system (TCS) and repressed by the RetS sensor kinase, which inhibits this TCS. Our findings identified a complex regulatory network that governs T6SS expression in general and K1-T6SS in particular, with implications for controlling and manipulating a bacterial agent that is highly relevant in biological control.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): FleQ , GacS–GacA , gene regulation , Pseudomonas , RetS , RpoN , RpoS and type VI secretion system
© 2023 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001295
2023-01-27
2024-05-17
Loading full text...

Full text loading...

/deliver/fulltext/micro/169/1/mic001295.html?itemId=/content/journal/micro/10.1099/mic.0.001295&mimeType=html&fmt=ahah

References

  1. Mougous JD, Cuff ME, Raunser S, Shen A, Zhou M et al. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 2006; 312:1526–1530 [View Article]
     [Google Scholar]
  2. Pukatzki S, Ma AT, Sturtevant D, Krastins B, Sarracino D et al. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci 2006; 103:1528–1533 [View Article]
     [Google Scholar]
  3. Journet L, Cascales E. The type VI secretion system in Escherichia coli and related species. EcoSal Plus 2016; 7:ESP0009-20 [View Article]
     [Google Scholar]
  4. Hernandez RE, Gallegos-Monterrosa R, Coulthurst SJ. Type VI secretion system effector proteins: effective weapons for bacterial competitiveness. Cell Microbiol 2020; 22:e13241 [View Article] [PubMed]
     [Google Scholar]
  5. Bernal P, Allsopp LP, Filloux A, Llamas MA. The Pseudomonas putida T6SS is a plant warden against phytopathogens. ISME Journal 2017; 11:972–987 [View Article]
     [Google Scholar]
  6. Durán D, Bernal P, Vazquez-Arias D, Blanco-Romero E, Garrido-Sanz D et al. Pseudomonas fluorescens F113 type VI secretion systems mediate bacterial killing and adaption to the rhizosphere microbiome. Sci Rep 2021; 11:5772
     [Google Scholar]
  7. Marchi M, Boutin M, Gazengel K, Rispe C, Gauthier J-P et al. Genomic analysis of the biocontrol strain Pseudomonas fluorescens Pf29Arp with evidence of T3SS and T6SS gene expression on plant roots. Environ Microbiol Rep 2013; 5:393–403 [View Article]
     [Google Scholar]
  8. Cherrak Y, Flaugnatti N, Durand E, Journet L, Cascales E. Structure and activity of the type VI secretion system. Microbiol Spectr 2019; 7: [View Article]
     [Google Scholar]
  9. Boyer F, Fichant G, Berthod J, Vandenbrouck Y, Attree I. 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 2009; 10:104–114 [View Article] [PubMed]
     [Google Scholar]
  10. Bernal P, Llamas MA, Filloux A. Type VI secretion systems in plant-associated bacteria. Environ Microbiol 2018; 20:1–15 [View Article]
     [Google Scholar]
  11. Bernal P, Furniss RCD, Fecht S, Leung RCY, Spiga L et al. A novel stabilization mechanism for the type VI secretion system sheath. Proc Natl Acad Sci U S A 2021; 118:e2008500118 [View Article]
     [Google Scholar]
  12. Santin YG, Doan T, Lebrun R, Espinosa L, Journet L et al. In vivo TssA proximity labelling during type VI secretion biogenesis reveals TagA as a protein that stops and holds the sheath. Nat Microbiol 2018; 3:1304–1313 [View Article] [PubMed]
     [Google Scholar]
  13. Aschtgen M-S, Thomas MS, Cascales E. Anchoring the type VI secretion system to the peptidoglycan: TssL, TagL, TagP… what else?. Virulence 2010; 1:535–540 [View Article]
     [Google Scholar]
  14. Mougous JD, Gifford CA, Ramsdell TL, Mekalanos JJ. Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa. Nat Cell Biol 2007; 9:797–803 [View Article] [PubMed]
     [Google Scholar]
  15. Allsopp LP, Collins ACZ, Hawkins E, Wood TE, Filloux A. RpoN/Sfa2-dependent activation of the Pseudomonas aeruginosa H2-T6SS and its cognate arsenal of antibacterial toxins. Nucleic Acids Res 2022; 50:227–243 [View Article] [PubMed]
     [Google Scholar]
  16. Lin J-S, Pissaridou P, Wu H-H, Tsai M-D, Filloux A et al. TagF-mediated repression of bacterial type VI secretion systems involves a direct interaction with the cytoplasmic protein Fha. J Biol Chem 2018; 293:8829–8842 [View Article]
     [Google Scholar]
  17. Bernal P, Murillo-Torres M, Allsopp LP. Integrating signals to drive type VI secretion system killing. Environ Microbiol 2020; 22:4520–4523 [View Article] [PubMed]
     [Google Scholar]
  18. Seibt H, Aung KM, Ishikawa T, Sjöström A, Gullberg M et al. Elevated levels of VCA0117 (VasH) in response to external signals activate the type VI secretion system of Vibrio cholerae O1 El Tor A1552. Environ Microbiol 2020; 22:4409–4423 [View Article] [PubMed]
     [Google Scholar]
  19. Hsu F, Schwarz S, Mougous JD. TagR promotes PpkA-catalysed type VI secretion activation in Pseudomonas aeruginosa. Mol Microbiol 2009; 72:1111–1125 [View Article] [PubMed]
     [Google Scholar]
  20. Casabona MG, Silverman JM, Sall KM, Boyer F, Couté Y et al. An ABC transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas aeruginosa. Environ Microbiol 2013; 15:471–486 [View Article]
     [Google Scholar]
  21. Whitney JC, Beck CM, Goo Y, Russell AB, Harding BN et al. Genetically distinct pathways guide effector export through the type VI secretion system. Mol Microbiol 2014; 92:529–542 [View Article]
     [Google Scholar]
  22. Fritsch MJ, Trunk K, Diniz JA, Guo M, Trost M et al. Proteomic identification of novel secreted antibacterial toxins of the Serratia marcescens type VI secretion system. Mol Cell Proteomics 2013; 12:2735–2749 [View Article]
     [Google Scholar]
  23. Lin J-S, Wu H-H, Hsu P-H, Ma L-S, Pang Y-Y et al. Fha interaction with phosphothreonine of TssL activates type VI secretion in Agrobacterium tumefaciens. PLoS Pathog 2014; 10:e1003991 [View Article]
     [Google Scholar]
  24. Silverman JM, Austin LS, Hsu F, Hicks KG, Hood RD et al. Separate inputs modulate phosphorylation-dependent and -independent type VI secretion activation. Mol Microbiol 2011; 82:1277–1290 [View Article] [PubMed]
     [Google Scholar]
  25. Ostrowski A, Cianfanelli FR, Porter M, Mariano G, Peltier J et al. Killing with proficiency: Integrated post-translational regulation of an offensive type VI secretion system. PLoS Pathog 2018; 14:e1007230 [View Article] [PubMed]
     [Google Scholar]
  26. Bernard CS, Brunet YR, Gueguen E, Cascales E. Nooks and crannies in type VI secretion regulation. J Bacteriol 2010; 192:3850–3860 [View Article] [PubMed]
     [Google Scholar]
  27. Lesic B, Starkey M, He J, Hazan R, Rahme LG. Quorum sensing differentially regulates Pseudomonas aeruginosa type VI secretion locus I and homologous loci II and III, which are required for pathogenesis. Microbiology 2009; 155:2845–2855 [View Article]
     [Google Scholar]
  28. Alves JA, Leal FC, Previato-Mello M, da Silva Neto JF. A quorum sensing-regulated type VI secretion system containing multiple nonredundant VgrG proteins is required for interbacterial competition in Chromobacterium violaceum. Microbiol Spectr 2022; 10:e0157622 [View Article] [PubMed]
     [Google Scholar]
  29. Gooderham WJ, Hancock REW. Regulation of virulence and antibiotic resistance by two-component regulatory systems in Pseudomonas aeruginosa. FEMS Microbiology Reviews 2009; 33:279–294 [View Article]
     [Google Scholar]
  30. Allsopp LP, Wood TE, Howard SA, Maggiorelli F, Nolan LM et al. RsmA and AmrZ orchestrate the assembly of all three type VI secretion systems in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 2017; 114:7707–7712 [View Article]
     [Google Scholar]
  31. Castang S, McManus HR, Turner KH, Dove SL. H-NS family members function coordinately in an opportunistic pathogen. Proc Natl Acad Sci 2008; 105:18947–18952 [View Article]
     [Google Scholar]
  32. Renzi F, Rescalli E, Galli E, Bertoni G. Identification of genes regulated by the MvaT-like paralogues TurA and TurB of Pseudomonas putida KT2440. Environ Microbiol 2010; 12:254–263 [View Article] [PubMed]
     [Google Scholar]
  33. Brunet YR, Bernard CS, Cascales E. Fur-dam regulatory interplay at an internal promoter of the enteroaggregative Escherichia coli type VI secretion sci1 gene cluster. J Bacteriol 2020; 202:e00075-20 [View Article] [PubMed]
     [Google Scholar]
  34. Battesti A, Majdalani N, Gottesman S. The RpoS-mediated general stress response in Escherichia coli. Annu Rev Microbiol 2011; 65:189–213 [View Article] [PubMed]
     [Google Scholar]
  35. Storey D, McNally A, Åstrand M, Santos JS-PG, Rodriguez-Escudero I et al. Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent. PLoS Pathog 2020; 16:e1007969 [View Article]
     [Google Scholar]
  36. Guan J, Xiao X, Xu S, Gao F, Wang J et al. Roles of RpoS in Yersinia pseudotuberculosis stress survival, motility, biofilm formation and type VI secretion system expression. Journal of Microbiology 2015; 53:633–642 [View Article]
     [Google Scholar]
  37. Otero-Asman JR, Wettstadt S, Bernal P, Llamas MA. Diversity of extracytoplasmic function sigma (σECF) factor-dependent signaling in Pseudomonas. Mol Microbiol 2019; 112:356–373 [View Article] [PubMed]
     [Google Scholar]
  38. Bayer-Santos E, Lima L dos, Ceseti L de, Ratagami CY, Santana E de et al. Xanthomonas citri T6SS mediates resistance to Dictyostelium predation and is regulated by an ECF σ factor and cognate Ser/Thr kinase. Environ Microbiol 2018; 20:1562–1575 [View Article]
     [Google Scholar]
  39. Barrios H, Valderrama B, Morett E. Compilation and analysis of sigma(54)-dependent promoter sequences. Nucleic Acids Res 1999; 27:4305–4313 [View Article] [PubMed]
     [Google Scholar]
  40. Rappas M, Bose D. Bacterial enhancer-binding proteins: unlocking sigma54-dependent gene transcription. Curr Opin Struct Biol 2007; 17:110–116 [View Article]
     [Google Scholar]
  41. Kitaoka M, Miyata ST, Brooks TM, Unterweger D, Pukatzki S. VasH is a transcriptional regulator of the type VI secretion system functional in endemic and pandemic Vibrio cholerae. J Bacteriol 2011; 193:6471–6482 [View Article] [PubMed]
     [Google Scholar]
  42. Wang Y, Li Y, Wang J, Wang X. FleQ regulates both the type VI secretion system and flagella in Pseudomonas putida. Biotechnol Appl Biochem 2018; 65:419–427 [View Article] [PubMed]
     [Google Scholar]
  43. Zhou T, Huang J, Liu Z, Lin Q, Xu Z et al. The two-component system FleS/FleR represses H1-T6SS via cyclic di-GMP signaling in Pseudomonas aeruginosa. Appl Environ Microbiol 2022; 88:e0165521 [View Article] [PubMed]
     [Google Scholar]
  44. Bouteiller M, Gallique M, Bourigault Y, Kosta A, Hardouin J et al. Crosstalk between the type VI secretion system and the expression of class IV flagellar genes in the Pseudomonas fluorescens MFE01 strain. Microorganisms 2020; 8:80506–80522 [View Article] [PubMed]
     [Google Scholar]
  45. Espinosa-Urgel M, Salido A, Ramos J-L. Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds. J Bacteriol 2000; 182:2363–2369 [View Article] [PubMed]
     [Google Scholar]
  46. Weller DM. Pseudomonas biocontrol agents of soilborne pathogens: looking back over 30 years. Phytopathology 2007; 97:250–256 [View Article]
     [Google Scholar]
  47. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd edition. NY: Molecular Cloning: A Laboratory Manual; 1989
     [Google Scholar]
  48. Choi K-H, Kumar A, Schweizer HP. A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 2006; 64:391–397 [View Article] [PubMed]
     [Google Scholar]
  49. Miller JH. n.d. Experiments in molecular genetics bacterial genetics - E. coli. Cold Spring Harbor Laboratory 50:
     [Google Scholar]
  50. Duque E, García V, de la Torre J, Godoy P, Bernal P et al. Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida. Environ Microbiol 2004; 6:1021–1031 [View Article] [PubMed]
     [Google Scholar]
  51. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29:e45 [View Article] [PubMed]
     [Google Scholar]
  52. Pacheco-Sánchez D, Molina-Fuentes Á, Marín P, Díaz-Romero A, Marqués S. DbdR, a new member of the LysR family of transcriptional regulators, coordinately controls four promoters in the Thauera aromatica AR-1 3,5-dihydroxybenzoate anaerobic degradation pathway. Appl Environ Microbiol 2019; 85:e02295-18 [View Article] [PubMed]
     [Google Scholar]
  53. Tillett D, Burns BP, Neilan BA. Optimized rapid amplification of cDNA ends (RACE) for mapping bacterial mRNA transcripts. Biotechniques 2000; 28:448 [View Article] [PubMed]
     [Google Scholar]
  54. Normark S, Bergström S, Edlund T, Grundström T, Jaurin B et al. Overlapping genes. Annu Rev Genet 1983; 17:499–525 [View Article] [PubMed]
     [Google Scholar]
  55. Sana TG, Hachani A, Bucior I, Soscia C, Garvis S et al. The second type VI secretion system of Pseudomonas aeruginosa strain PAO1 is regulated by quorum sensing and fur and modulates internalization in epithelial cells. J Biol Chem 2012; 287:27095–27105 [View Article]
     [Google Scholar]
  56. Huang Y, Du P, Zhao M, Liu W, Du Y et al. Functional characterization and conditional regulation of the type VI secretion system in Vibrio fluvialis. Front Microbiol 2017; 8:528 [View Article] [PubMed]
     [Google Scholar]
  57. Hachani A, Lossi NS, Filloux A. A visual assay to monitor T6SS-mediated bacterial competition. J Vis Exp 2013e50103 [View Article]
     [Google Scholar]
  58. Mauri M, Klumpp S, Rao CV. A model for sigma factor competition in bacterial cells. PLoS Comput Biol 2014; 10:e1003845 [View Article] [PubMed]
     [Google Scholar]
  59. Llamas MA, Imperi F, Visca P, Lamont IL. Cell-surface signaling in Pseudomonas: stress responses, iron transport, and pathogenicity. FEMS Microbiology Reviews 2014; 38:569–597 [View Article]
     [Google Scholar]
  60. Llamas MA, Mooij MJ, Sparrius M, Vandenbroucke-Grauls CMJE, Ratledge C et al. Characterization of five novel Pseudomonas aeruginosa cell-surface signalling systems. Mol Microbiol 2008; 67:458–472 [View Article] [PubMed]
     [Google Scholar]
  61. Unni R, Pintor KL, Diepold A, Unterweger D. Presence and absence of type VI secretion systems in bacteria. Microbiology 2022; 168:001151–13 [View Article]
     [Google Scholar]
  62. Schneider JP, Nazarov S, Adaixo R, Liuzzo M, Ringel PD et al. Diverse roles of TssA-like proteins in the assembly of bacterial type VI secretion systems. EMBO J 2019; 38:e100825 [View Article] [PubMed]
     [Google Scholar]
  63. Gonzalez D, Mavridou DAI. Making the best of aggression: the many dimensions of bacterial toxin regulation. Trends in Microbiology 2019; 27:897–905 [View Article]
     [Google Scholar]
  64. Molina L, Bernal P, Udaondo Z, Segura A, Ramos J-L. Complete Genome Sequence of a Pseudomonas putida Clinical Isolate, Strain H8234. Genome Announc 2013; 1:e00496-13 [View Article]
     [Google Scholar]
  65. Molina L, Udaondo Z, Duque E, Fernández M, Bernal P et al. Specific gene loci of clinical Pseudomonas putida isolates. PLoS One 2016; 11:e0147478 [View Article] [PubMed]
     [Google Scholar]
  66. Borrero de Acuña JM, Bernal P. Plant holobiont interactions mediated by the type VI secretion system and the membrane vesicles: promising tools for a greener agriculture. Environ Microbiol 2021; 23:1830–1836 [View Article] [PubMed]
     [Google Scholar]
  67. Weber BS, Ly PM, Irwin JN, Pukatzki S, Feldman MF. A multidrug resistance plasmid contains the molecular switch for type VI secretion in Acinetobacter baumannii. Proc Natl Acad Sci U S A 2015; 112:9442–9447 [View Article]
     [Google Scholar]
  68. Ramos C, Mølbak L, Molin S. Bacterial activity in the rhizosphere analyzed at the single-cell level by monitoring ribosome contents and synthesis rates. Appl Environ Microbiol 2000; 66:801–809 [View Article] [PubMed]
     [Google Scholar]
  69. Hug S, Liu Y, Heiniger B, Bailly A, Ahrens CH et al. Differential expression of Paraburkholderia phymatum type VI secretion systems (T6SS) suggests a role of T6SS-b in early symbiotic interaction. Front Plant Sci 2021; 12:69590 [View Article]
     [Google Scholar]
  70. Jiang F, Waterfield NR, Yang J, Yang G, Jin Q. A Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells. Cell Host Microbe 2014; 15:600–610 [View Article] [PubMed]
     [Google Scholar]
  71. Pini C, Godoy P, Bernal P, Ramos J, Segura A. Regulation of the cyclopropane synthase cfaB gene in Pseudomonas putida KT2440. FEMS Microbiol Lett 2011; 321:107–114 [View Article]
     [Google Scholar]
  72. Ramos-González MI, Molin S. Cloning, sequencing, and phenotypic characterization of the rpoS gene from Pseudomonas putida KT2440. J Bacteriol 1998; 180:3421–3431 [View Article] [PubMed]
     [Google Scholar]
  73. Schuster M, Hawkins AC, Harwood CS, Greenberg EP. The Pseudomonas aeruginosa RpoS regulon and its relationship to quorum sensing. Mol Microbiol 2004; 51:973–985 [View Article] [PubMed]
     [Google Scholar]
  74. Heurlier K, Dénervaud V, Pessi G, Reimmann C, Haas D. Negative control of quorum sensing by RpoN (sigma54) in Pseudomonas aeruginosa PAO1. J Bacteriol 2003; 185:2227–2235 [View Article] [PubMed]
     [Google Scholar]
  75. Baraquet C, Harwood CS. Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the walker a motif of the enhancer-binding protein FleQ. Proc Natl Acad Sci 2013; 110:18478–18483 [View Article]
     [Google Scholar]
  76. Leal-Morales A, Pulido-Sánchez M, López-Sánchez A, Govantes F. Transcriptional organization and regulation of the Pseudomonas putida flagellar system. Environ Microbiol 2022; 24:137–157 [View Article] [PubMed]
     [Google Scholar]
  77. Nie H, Xiao Y, Song M, Wu N, Peng Q et al. Wsp system oppositely modulates antibacterial activity and biofilm formation via FleQ-FleN complex in Pseudomonas putida. Environ Microbiol 2022; 24:1543–1559 [View Article] [PubMed]
     [Google Scholar]
  78. Ferreiro MD, Gallegos MT. Distinctive features of the Gac-Rsm pathway in plant-associated Pseudomonas. Environ Microbiol 2021; 23:5670–5689 [View Article]
     [Google Scholar]
  79. Wang BX, Wheeler KM, Cady KC, Lehoux S, Cummings RD et al. Mucin glycans signal through the sensor kinase rets to inhibit virulence-associated traits in Pseudomonas aeruginosa. Current Biology 2021; 31:90–102 [View Article]
     [Google Scholar]
  80. Records AR, Gross DC. Sensor kinases RetS and LadS regulate Pseudomonas syringae type VI secretion and virulence factors. J Bacteriol 2010; 192:3584–3596 [View Article] [PubMed]
     [Google Scholar]
  81. Heeb S, Haas D. Regulatory roles of the GacS/GacA two-component system in plant-associated and other gram-negative bacteria. Mol Plant Microbe Interact 2001; 14:1351–1363 [View Article]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.001295
Loading
Transcriptional organization and regulation of the Pseudomonas putida K1 type VI secretion system gene cluster
Microbiology 169, 001295 (2023); https://doi.org/10.1099/mic.0.001295
/content/journal/micro/10.1099/mic.0.001295
/content/journal/micro/10.1099/mic.0.001295
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error