1887

Abstract

While flagellum-driven motility is hypothesized to play a role in the virulence of species, there is no direct evidence that genes involved in flagellum assembly regulate the synthesis of virulence factors. The purpose of this study was to identify genes that affect the production or secretion of necrosis-inducing protein (Nip) in the strain SCC3193. Transposon mutagenesis of an RpoS strain overexpressing Nip was performed, and a mutant associated with decreased necrosis of tobacco leaves was detected. The mutant contained a transposon in the regulatory region upstream of the flagellar genes and . Additional mutants were generated related to the flagellar genes and . The mutation in , but not those in and , inhibited transcription. Moreover, the regulatory effect of the mutation on transcription was partially dependent on the Rcs phosphorelay. Secretion of Nip was also dependent on a type II secretion mechanism. Overall, the results of this study indicate that the mutation is responsible for reduced motility and lower levels of expression.

Funding
This study was supported by the:
  • Estonian Science Foundation (Award SF0180088s08)
  • Academy of Finland (Award 128566)
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.071092-0
2014-01-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/160/1/179.html?itemId=/content/journal/micro/10.1099/mic.0.071092-0&mimeType=html&fmt=ahah

References

  1. Andersson R. A., Kõiv V., Norman-Setterblad C., Pirhonen M. ( 1999). Role of RpoS in virulence and stress tolerance of the plant pathogen Erwinia carotovora subsp. carotovora.. Microbiology 145:3547–3556[PubMed]
    [Google Scholar]
  2. Andresen L., Kõiv V., Alamäe T., Mäe A. ( 2007). The Rcs phosphorelay modulates the expression of plant cell wall degrading enzymes and virulence in Pectobacterium carotovorum ssp. carotovorum. FEMS Microbiol Lett 273:229–238 [View Article][PubMed]
    [Google Scholar]
  3. Andresen L., Sala E., Kõiv V., Mäe A. ( 2010). A role for the Rcs phosphorelay in regulating expression of plant cell wall degrading enzymes in Pectobacterium carotovorum subsp. carotovorum.. Microbiology 156:1323–1334 [View Article][PubMed]
    [Google Scholar]
  4. Bouillaut L., Ramarao N., Buisson C., Gilois N., Gohar M., Lereclus D., Nielsen-Leroux C. ( 2005). FlhA influences Bacillus thuringiensis PlcR-regulated gene transcription, protein production, and virulence. Appl Environ Microbiol 71:8903–8910 [View Article][PubMed]
    [Google Scholar]
  5. Caetano-Anollés G. ( 1993). Amplifying DNA with arbitrary oligonucleotide primers. PCR Methods Appl 3:85–94 [View Article][PubMed]
    [Google Scholar]
  6. Chatterjee A., Cui Y., Liu Y., Dumenyo C. K., Chatterjee A. K. ( 1995). Inactivation of rsmA leads to overproduction of extracellular pectinases, cellulases, and proteases in Erwinia carotovora subsp. carotovora in the absence of the starvation/cell density-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone. Appl Environ Microbiol 61:1959–1967[PubMed]
    [Google Scholar]
  7. Chatterjee A., Cui Y., Chatterjee A. K. ( 2009). RsmC of Erwinia carotovora subsp. carotovora negatively controls motility, extracellular protein production, and virulence by binding FlhD and modulating transcriptional activity of the master regulator, FlhDC. J Bacteriol 191:4582–4593 [View Article][PubMed]
    [Google Scholar]
  8. Chen M. H., Takeda S., Yamada H., Ishii Y., Yamashino T., Mizuno T. ( 2001). Characterization of the RcsC–>YojN–>RcsB phosphorelay signaling pathway involved in capsular synthesis in Escherichia coli.. Biosci Biotechnol Biochem 65:2364–2367 [View Article][PubMed]
    [Google Scholar]
  9. Clarke D. J. ( 2010). The Rcs phosphorelay: more than just a two-component pathway. Future Microbiol 5:1173–1184 [View Article][PubMed]
    [Google Scholar]
  10. Corbett M., Virtue S., Bell K., Birch P., Burr T., Hyman L., Lilley K., Poock S., Toth I., Salmond G. ( 2005). Identification of a new quorum-sensing-controlled virulence factor in Erwinia carotovora subsp. atroseptica secreted via the type II targeting pathway. Mol Plant Microbe Interact 18:334–342 [View Article][PubMed]
    [Google Scholar]
  11. Coulthurst S. J., Lilley K. S., Hedley P. E., Liu H., Toth I. K., Salmond G. P. ( 2008). DsbA plays a critical and multifaceted role in the production of secreted virulence factors by the phytopathogen Erwinia carotovora subsp. atroseptica.. J Biol Chem 283:23739–23753 [View Article][PubMed]
    [Google Scholar]
  12. Cui Y., Chatterjee A., Chatterjee A. K. ( 2001). Effects of the two-component system comprising GacA and GacS of Erwinia carotovora subsp. carotovora on the production of global regulatory rsmB RNA, extracellular enzymes, and harpinEcc. Mol Plant Microbe Interact 14:516–526 [View Article][PubMed]
    [Google Scholar]
  13. Cui Y., Chatterjee A., Yang H., Chatterjee A. K. ( 2008). Regulatory network controlling extracellular proteins in Erwinia carotovora subsp. carotovora: FlhDC, the master regulator of flagellar genes, activates rsmB regulatory RNA production by affecting gacA and hexA (lrhA) expression. J Bacteriol 190:4610–4623 [View Article][PubMed]
    [Google Scholar]
  14. Datsenko K. A., Wanner B. L. ( 2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645 [View Article][PubMed]
    [Google Scholar]
  15. Espinosa A., Alfano J. R. ( 2004). Disabling surveillance: bacterial type III secretion system effectors that suppress innate immunity. Cell Microbiol 6:1027–1040 [View Article][PubMed]
    [Google Scholar]
  16. Friesen T. L., Faris J. D., Solomon P. S., Oliver R. P. ( 2008). Host-specific toxins: effectors of necrotrophic pathogenicity. Cell Microbiol 10:1421–1428 [View Article][PubMed]
    [Google Scholar]
  17. Gauger E. J., Leatham M. P., Mercado-Lubo R., Laux D. C., Conway T., Cohen P. S. ( 2007). Role of motility and the flhDC Operon in Escherichia coli MG1655 colonization of the mouse intestine. Infect Immun 75:3315–3324 [View Article][PubMed]
    [Google Scholar]
  18. Ghelardi E., Celandroni F., Salvetti S., Beecher D. J., Gominet M., Lereclus D., Wong A. C., Senesi S. ( 2002). Requirement of flhA for swarming differentiation, flagellin export, and secretion of virulence-associated proteins in Bacillus thuringiensis.. J Bacteriol 184:6424–6433 [View Article][PubMed]
    [Google Scholar]
  19. Hossain M. M., Shibata S., Aizawa S.-I., Tsuyumu S. ( 2005). Motility is an important determinant for pathogenesis of Erwinia carotovora sunsp. carotovora. Physiol Mo.. Plant Pathol 66:134–143
    [Google Scholar]
  20. Huynh T. V., Dahlbeck D., Staskawicz B. J. ( 1989). Bacterial blight of soybean: regulation of a pathogen gene determining host cultivar specificity. Science 245:1374–1377 [View Article][PubMed]
    [Google Scholar]
  21. Hyytiäinen H., Montesano M., Palva E. T. ( 2001). Global regulators ExpA (GacA) and KdgR modulate extracellular enzyme gene expression through the RsmA-rsmB system in Erwinia carotovora subsp. carotovora.. Mol Plant Microbe Interact 14:931–938 [View Article][PubMed]
    [Google Scholar]
  22. Hyytiäinen H., Sjöblom S., Palomäki T., Tuikkala A., Palva E. T. ( 2003). The PmrA-PmrB two-component system responding to acidic pH and iron controls virulence in the plant pathogen Erwinia carotovora ssp. carotovora. Mol Microbiol 50:795–807 [View Article][PubMed]
    [Google Scholar]
  23. Karlinsey J. E., Tanaka S., Bettenworth V., Yamaguchi S., Boos W., Aizawa S. I., Hughes K. T. ( 2000). Completion of the hook-basal body complex of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription. Mol Microbiol 37:1220–1231 [View Article][PubMed]
    [Google Scholar]
  24. Kõiv V., Andresen L., Broberg M., Frolova J., Somervuo P., Auvinen P., Pirhonen M., Tenson T., Mäe A. ( 2013). Lack of RsmA-mediated control results in constant hypervirulence, cell elongation, and hyperflagellation in Pectobacterium wasabiae.. PLoS ONE 8:e54248 [View Article][PubMed]
    [Google Scholar]
  25. Konkel M. E., Klena J. D., Rivera-Amill V., Monteville M. R., Biswas D., Raphael B., Mickelson J. ( 2004). Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagellar export apparatus. J Bacteriol 186:3296–3303 [View Article][PubMed]
    [Google Scholar]
  26. Liu X., Matsumura P. ( 1994). The FlhD/FlhC complex, a transcriptional activator of the Escherichia coli flagellar class II operons. J Bacteriol 176:7345–7351[PubMed]
    [Google Scholar]
  27. Marits R., Kõiv V., Laasik E., Mäe A. ( 1999). Isolation of an extracellular protease gene of Erwinia carotovora subsp. carotovora strain SCC3193 by transposon mutagenesis and the role of protease in phytopathogenicity. Microbiology 145:1959–1966 [View Article][PubMed]
    [Google Scholar]
  28. Marits R., Tshuikina M., Pirhonen M., Laasik E., Mäe A. ( 2002). Regulation of the expression of prtW:gusA fusions in Erwinia carotovora subsp. carotovora.. Microbiology 148:835–842[PubMed]
    [Google Scholar]
  29. Matsumoto H., Muroi H., Umehara M., Yoshitake Y., Tsuyumu S. ( 2003). Peh production, flagellum synthesis, and virulence reduced in Erwinia carotovora subsp. carotovora by mutation in a homologue of cytR.. Mol Plant Microbe Interact 16:389–397 [View Article][PubMed]
    [Google Scholar]
  30. Mattinen L., Tshuikina M., Mäe A., Pirhonen M. ( 2004). Identification and characterization of Nip, necrosis-inducing virulence protein of Erwinia carotovora subsp. carotovora.. Mol Plant Microbe Interact 17:1366–1375 [View Article][PubMed]
    [Google Scholar]
  31. Miller J. H. ( 1972). Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, NY: Cold Spring Harbor;
    [Google Scholar]
  32. Mukherjee A., Cui Y., Ma W., Liu Y., Chatterjee A. K. ( 2000). hexA of Erwinia carotovora ssp. carotovora strain Ecc71 negatively regulates production of RpoS and rsmB RNA, a global regulator of extracellular proteins, plant virulence and the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone. Environ Microbiol 2:203–215 [View Article][PubMed]
    [Google Scholar]
  33. Novel G., Didier-Fichet M. L., Stoeber F. ( 1974). Inducibility of beta-glucuronidase in wild-type and hexuronate-negative mutants of Escherichia coli K-12. J Bacteriol 120:89–95[PubMed]
    [Google Scholar]
  34. Ottmann C., Luberacki B., Küfner I., Koch W., Brunner F., Weyand M., Mattinen L., Pirhonen M., Anderluh G. & other authors ( 2009). A common toxin fold mediates microbial attack and plant defense. Proc Natl Acad Sci U S A 106:10359–10364 [View Article][PubMed]
    [Google Scholar]
  35. Pavel H., Forsman M., Shingler V. ( 1994). An aromatic effector specificity mutant of the transcriptional regulator DmpR overcomes the growth constraints of Pseudomonas sp. strain CF600 on para-substituted methylphenols. J Bacteriol 176:7550–7557[PubMed]
    [Google Scholar]
  36. Pemberton C. L., Whitehead N. A., Sebaihia M., Bell K. S., Hyman L. J., Harris S. J., Matlin A. J., Robson N. D., Birch P. R. & other authors ( 2005). Novel quorum-sensing-controlled genes in Erwinia carotovora subsp. carotovora: identification of a fungal elicitor homologue in a soft-rotting bacterium. Mol Plant Microbe Interact 18:343–353 [View Article][PubMed]
    [Google Scholar]
  37. Pérombelon M. C. M. ( 2002). Potato diseases caused by soft rot erwinias: an overview of pathogenesis. Plant Pathol 51:1–12 [View Article]
    [Google Scholar]
  38. Pirhonen M., Heino P., Helander I., Harju P., Palva E. T. ( 1988). Bacteriophage T4 resistant mutants of the plant pathogen Erwinia carotovora.. Microb Pathog 4:359–367 [View Article][PubMed]
    [Google Scholar]
  39. Pirhonen M., Saarilahti H., Karlsson M.-B., Palva E. T. ( 1991). Identification of pathogenicity determinants of Erwinia carotovora subspecies carotovora by transposon mutagenesis. Mol Plant Microbe Interact 4:276–283 [View Article]
    [Google Scholar]
  40. Põllumaa L., Alamäe T., Mäe A. ( 2012). Quorum sensing and expression of virulence in pectobacteria. Sensors (Basel) 12:3327–3349 [View Article][PubMed]
    [Google Scholar]
  41. Prüß B. M. ( 2000). FlhD, a transcriptional regulator in bacteria. Recent Res Dev Microbiol 4:31–42
    [Google Scholar]
  42. Reeves P. J., Douglas P., Salmond G. P. ( 1994). beta-Lactamase topology probe analysis of the OutO NMePhe peptidase, and six other Out protein components of the Erwinia carotovora general secretion pathway apparatus. Mol Microbiol 12:445–457 [View Article][PubMed]
    [Google Scholar]
  43. Sambrook J., Russell D. W. ( 2001). Molecular Cloning: A Laboratory Manual New York: Cold Spring Harbor Laboratory Press;
    [Google Scholar]
  44. Shiba Y., Matsumoto K., Hara H. ( 2006). DjlA negatively regulates the Rcs signal transduction system in Escherichia coli.. Genes Genet Syst 81:51–56 [View Article][PubMed]
    [Google Scholar]
  45. Studier F. W., Moffatt B. A. ( 1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130 [View Article][PubMed]
    [Google Scholar]
  46. Toth I. K., Birch P. R. ( 2005). Rotting softly and stealthily. Curr Opin Plant Biol 8:424–429 [View Article][PubMed]
    [Google Scholar]
  47. Wang S., Fleming R. T., Westbrook E. M., Matsumura P., McKay D. B. ( 2006). Structure of the Escherichia coli FlhDC complex, a prokaryotic heteromeric regulator of transcription. J Mol Biol 355:798–808 [View Article][PubMed]
    [Google Scholar]
  48. Young G. M., Schmiel D. H., Miller V. L. ( 1999). A new pathway for the secretion of virulence factors by bacteria: the flagellar export apparatus functions as a protein-secretion system. Proc Natl Acad Sci U S A 96:6456–6461 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.071092-0
Loading
/content/journal/micro/10.1099/mic.0.071092-0
Loading

Data & Media loading...

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