Abstract
N-Acylhomoserine lactone (acyl-HSL)-mediated gene expression, also called autoinduction, is conserved among diverse gram-negative bacteria. In the paradigm Vibrio fischeri system, bioluminescence is autoinducible, and the lux operon requires the transcriptional activator LuxR and the acyl-HSL autoinducer for expression. The production of the acyl-HSL signal molecule is conferred by the luxI gene, and luxR encodes the transcriptional regulator. We show here that Erwinia stewartii, the etiological agent of Stewart's wilt of sweet corn, synthesizes an acyl-HSL. Mass spectral analysis identified the signal molecule as N-(-3-oxohexanoyl)-L-homoserine lactone, which is identical to the V. fischeri autoinducer. We have cloned and sequenced the gene that confers acyl-HSL biosynthesis, called esaI, and the linked gene, esaR, that encodes a gene regulator. The two genes are convergently transcribed and show an unusual overlap of 31 bp at their 3' ends. Sequence analysis indicates that EsaI and EsaR are homologs of LuxI and LuxR, respectively. EsaR can repress its own expression but seems not to regulate the expression of esaI. The untranslated 5' region of esaR contains an inverted repeat with similarity to the lux box-like elements located in the promoter regions of other gene systems regulated by autoinduction. However, unlike the other systems, in which the inverted repeats are located upstream of the -35 promoter elements, the esaR-associated repeat overlaps a putative -10 element. We mutagenized the esaI gene in E. stewartii by gene replacement. The mutant no longer produced detectable levels of the acyl-HSL signal, leading to a concomitant loss of extracellular polysaccharide capsule production and pathogenicity. Both phenotypes were restored by complementation with esal or by exogenous addition of the acyl-HSL.
Full Text
The Full Text of this article is available as a PDF (498.8 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adar Y. Y., Ulitzur S. GroESL proteins facilitate binding of externally added inducer by LuxR protein-containing E. coli cells. J Biolumin Chemilumin. 1993 Sep-Oct;8(5):261–266. doi: 10.1002/bio.1170080506. [DOI] [PubMed] [Google Scholar]
- Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
- Bainton N. J., Stead P., Chhabra S. R., Bycroft B. W., Salmond G. P., Stewart G. S., Williams P. N-(3-oxohexanoyl)-L-homoserine lactone regulates carbapenem antibiotic production in Erwinia carotovora. Biochem J. 1992 Dec 15;288(Pt 3):997–1004. doi: 10.1042/bj2880997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baldwin T. O., Devine J. H., Heckel R. C., Lin J. W., Shadel G. S. The complete nucleotide sequence of the lux regulon of Vibrio fischeri and the luxABN region of Photobacterium leiognathi and the mechanism of control of bacterial bioluminescence. J Biolumin Chemilumin. 1989 Jul;4(1):326–341. doi: 10.1002/bio.1170040145. [DOI] [PubMed] [Google Scholar]
- Beck von Bodman S., Hayman G. T., Farrand S. K. Opine catabolism and conjugal transfer of the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):643–647. doi: 10.1073/pnas.89.2.643. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradshaw-Rouse J. J., Whatley M. H., Coplin D. L., Woods A., Sequeira L., Kelman A. Agglutination of Erwinia stewartii Strains with a Corn Agglutinin: Correlation with Extracellular Polysaccharide Production and Pathogenicity. Appl Environ Microbiol. 1981 Aug;42(2):344–350. doi: 10.1128/aem.42.2.344-350.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Choi S. H., Greenberg E. P. Genetic dissection of DNA binding and luminescence gene activation by the Vibrio fischeri LuxR protein. J Bacteriol. 1992 Jun;174(12):4064–4069. doi: 10.1128/jb.174.12.4064-4069.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Close T. J., Rodriguez R. L. Construction and characterization of the chloramphenicol-resistance gene cartridge: a new approach to the transcriptional mapping of extrachromosomal elements. Gene. 1982 Dec;20(2):305–316. doi: 10.1016/0378-1119(82)90048-8. [DOI] [PubMed] [Google Scholar]
- Cubo M. T., Economou A., Murphy G., Johnston A. W., Downie J. A. Molecular characterization and regulation of the rhizosphere-expressed genes rhiABCR that can influence nodulation by Rhizobium leguminosarum biovar viciae. J Bacteriol. 1992 Jun;174(12):4026–4035. doi: 10.1128/jb.174.12.4026-4035.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Devine J. H., Shadel G. S., Baldwin T. O. Identification of the operator of the lux regulon from the Vibrio fischeri strain ATCC7744. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5688–5692. doi: 10.1073/pnas.86.15.5688. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dolph P. J., Majerczak D. R., Coplin D. L. Characterization of a gene cluster for exopolysaccharide biosynthesis and virulence in Erwinia stewartii. J Bacteriol. 1988 Feb;170(2):865–871. doi: 10.1128/jb.170.2.865-871.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dunlap P. V., Greenberg E. P. Control of Vibrio fischeri lux gene transcription by a cyclic AMP receptor protein-luxR protein regulatory circuit. J Bacteriol. 1988 Sep;170(9):4040–4046. doi: 10.1128/jb.170.9.4040-4046.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eberhard A., Burlingame A. L., Eberhard C., Kenyon G. L., Nealson K. H., Oppenheimer N. J. Structural identification of autoinducer of Photobacterium fischeri luciferase. Biochemistry. 1981 Apr 28;20(9):2444–2449. doi: 10.1021/bi00512a013. [DOI] [PubMed] [Google Scholar]
- Eberhard A. Inhibition and activation of bacterial luciferase synthesis. J Bacteriol. 1972 Mar;109(3):1101–1105. doi: 10.1128/jb.109.3.1101-1105.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Engebrecht J., Nealson K., Silverman M. Bacterial bioluminescence: isolation and genetic analysis of functions from Vibrio fischeri. Cell. 1983 Mar;32(3):773–781. doi: 10.1016/0092-8674(83)90063-6. [DOI] [PubMed] [Google Scholar]
- Engebrecht J., Silverman M. Nucleotide sequence of the regulatory locus controlling expression of bacterial genes for bioluminescence. Nucleic Acids Res. 1987 Dec 23;15(24):10455–10467. doi: 10.1093/nar/15.24.10455. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fuqua W. C., Winans S. C. A LuxR-LuxI type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumor metabolite. J Bacteriol. 1994 May;176(10):2796–2806. doi: 10.1128/jb.176.10.2796-2806.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fuqua W. C., Winans S. C., Greenberg E. P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol. 1994 Jan;176(2):269–275. doi: 10.1128/jb.176.2.269-275.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gambello M. J., Iglewski B. H. Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression. J Bacteriol. 1991 May;173(9):3000–3009. doi: 10.1128/jb.173.9.3000-3009.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hanzelka B. L., Greenberg E. P. Evidence that the N-terminal region of the Vibrio fischeri LuxR protein constitutes an autoinducer-binding domain. J Bacteriol. 1995 Feb;177(3):815–817. doi: 10.1128/jb.177.3.815-817.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hwang I., Li P. L., Zhang L., Piper K. R., Cook D. M., Tate M. E., Farrand S. K. TraI, a LuxI homologue, is responsible for production of conjugation factor, the Ti plasmid N-acylhomoserine lactone autoinducer. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4639–4643. doi: 10.1073/pnas.91.11.4639. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jones S., Yu B., Bainton N. J., Birdsall M., Bycroft B. W., Chhabra S. R., Cox A. J., Golby P., Reeves P. J., Stephens S. The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa. EMBO J. 1993 Jun;12(6):2477–2482. doi: 10.1002/j.1460-2075.1993.tb05902.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Keen N. T., Tamaki S., Kobayashi D., Trollinger D. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene. 1988 Oct 15;70(1):191–197. doi: 10.1016/0378-1119(88)90117-5. [DOI] [PubMed] [Google Scholar]
- Kuo A., Blough N. V., Dunlap P. V. Multiple N-acyl-L-homoserine lactone autoinducers of luminescence in the marine symbiotic bacterium Vibrio fischeri. J Bacteriol. 1994 Dec;176(24):7558–7565. doi: 10.1128/jb.176.24.7558-7565.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leigh J. A., Coplin D. L. Exopolysaccharides in plant-bacterial interactions. Annu Rev Microbiol. 1992;46:307–346. doi: 10.1146/annurev.mi.46.100192.001515. [DOI] [PubMed] [Google Scholar]
- Marck C. 'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucleic Acids Res. 1988 Mar 11;16(5):1829–1836. doi: 10.1093/nar/16.5.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meighen E. A. Molecular biology of bacterial bioluminescence. Microbiol Rev. 1991 Mar;55(1):123–142. doi: 10.1128/mr.55.1.123-142.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nag D. K., Huang H. V., Berg D. E. Bidirectional chain-termination nucleotide sequencing: transposon Tn5seq1 as a mobile source of primer sites. Gene. 1988 Apr 15;64(1):135–145. doi: 10.1016/0378-1119(88)90487-8. [DOI] [PubMed] [Google Scholar]
- Nealson K. H. Autoinduction of bacterial luciferase. Occurrence, mechanism and significance. Arch Microbiol. 1977 Feb 4;112(1):73–79. doi: 10.1007/BF00446657. [DOI] [PubMed] [Google Scholar]
- Nealson K. H., Platt T., Hastings J. W. Cellular control of the synthesis and activity of the bacterial luminescent system. J Bacteriol. 1970 Oct;104(1):313–322. doi: 10.1128/jb.104.1.313-322.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Passador L., Cook J. M., Gambello M. J., Rust L., Iglewski B. H. Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science. 1993 May 21;260(5111):1127–1130. doi: 10.1126/science.8493556. [DOI] [PubMed] [Google Scholar]
- Pearson J. P., Gray K. M., Passador L., Tucker K. D., Eberhard A., Iglewski B. H., Greenberg E. P. Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):197–201. doi: 10.1073/pnas.91.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pierson L. S., 3rd, Keppenne V. D., Wood D. W. Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulated by PhzR in response to cell density. J Bacteriol. 1994 Jul;176(13):3966–3974. doi: 10.1128/jb.176.13.3966-3974.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Piper K. R., Beck von Bodman S., Farrand S. K. Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature. 1993 Apr 1;362(6419):448–450. doi: 10.1038/362448a0. [DOI] [PubMed] [Google Scholar]
- Pirhonen M., Flego D., Heikinheimo R., Palva E. T. A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora. EMBO J. 1993 Jun;12(6):2467–2476. doi: 10.1002/j.1460-2075.1993.tb05901.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ruby E. G., McFall-Ngai M. J. A squid that glows in the night: development of an animal-bacterial mutualism. J Bacteriol. 1992 Aug;174(15):4865–4870. doi: 10.1128/jb.174.15.4865-4870.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Seed P. C., Passador L., Iglewski B. H. Activation of the Pseudomonas aeruginosa lasI gene by LasR and the Pseudomonas autoinducer PAI: an autoinduction regulatory hierarchy. J Bacteriol. 1995 Feb;177(3):654–659. doi: 10.1128/jb.177.3.654-659.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stachel S. E., An G., Flores C., Nester E. W. A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J. 1985 Apr;4(4):891–898. doi: 10.1002/j.1460-2075.1985.tb03715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Staskawicz B., Dahlbeck D., Keen N., Napoli C. Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol. 1987 Dec;169(12):5789–5794. doi: 10.1128/jb.169.12.5789-5794.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Swift S., Bainton N. J., Winson M. K. Gram-negative bacterial communication by N-acyl homoserine lactones: a universal language? Trends Microbiol. 1994 Jun;2(6):193–198. doi: 10.1016/0966-842x(94)90110-q. [DOI] [PubMed] [Google Scholar]
- Swift S., Winson M. K., Chan P. F., Bainton N. J., Birdsall M., Reeves P. J., Rees C. E., Chhabra S. R., Hill P. J., Throup J. P. A novel strategy for the isolation of luxI homologues: evidence for the widespread distribution of a LuxR:LuxI superfamily in enteric bacteria. Mol Microbiol. 1993 Nov;10(3):511–520. doi: 10.1111/j.1365-2958.1993.tb00923.x. [DOI] [PubMed] [Google Scholar]
- Tiedeman A. A., Smith J. M. lacZY gene fusion cassettes with KanR resistance. Nucleic Acids Res. 1988 Apr 25;16(8):3587–3587. doi: 10.1093/nar/16.8.3587. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Torres-Cabassa A., Gottesman S., Frederick R. D., Dolph P. J., Coplin D. L. Control of extracellular polysaccharide synthesis in Erwinia stewartii and Escherichia coli K-12: a common regulatory function. J Bacteriol. 1987 Oct;169(10):4525–4531. doi: 10.1128/jb.169.10.4525-4531.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
- Zhang L., Murphy P. J., Kerr A., Tate M. E. Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones. Nature. 1993 Apr 1;362(6419):446–448. doi: 10.1038/362446a0. [DOI] [PubMed] [Google Scholar]