Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Apr;179(8):2707–2716. doi: 10.1128/jb.179.8.2707-2716.1997

Identification, cloning, and characterization of the Ima operon, whose gene products are unique to Listeria monocytogenes.

S Schäferkordt 1, T Chakraborty 1
PMCID: PMC179021  PMID: 9098070

Abstract

The lmaA gene of Listeria monocytogenes encodes a protein capable of inducing delayed-type hypersensitivity reactions in L. monocytogenes-immune mice (S. Göhmann, M. Leimeister-Wachter, E. Schiltz, W. Goebel, and T. Chakraborty, M. Microbiol. 4:1091-1099, 1990). Here we show that it is the last gene of the lma operon, which now comprises four genes, lmaDCBA. Maxicell analysis of peptides encoded by the lma operon identified four polypeptides of 16.7, 16.4, 14.9, and 21 kDa which correspond to the gene products encoded by the lmaD, -C, -B, and -A genes, respectively. Northern blot analysis of the lma operon showed that lmaA is expressed by two transcripts: the longer lmaDCBA transcript of 2,100 nucleotides, which was observed at growth temperatures of 37 and 20 degrees C, and a shorter transcript consisting of lmaBA, which is detected only at low temperatures (20 degrees C). Two promoters, one preceding the lmaD gene and another located upstream of the lmaB gene, were detected. An extended stem-loop structure resembling box elements found in other gram-positive pathogens was also present in the lmaC-lmaB intergenic region. By immunoblot analysis, we found that although LmaA was produced at both temperatures (20 and 37 degrees C), it was secreted into culture supernatants only at 20 degrees C. However, LmaA lacks a bona fide signal peptide sequence and could, like flagellin, be secreted by a type III transport system. DNA hybridization studies indicate that the lma operon is species specific and restricted to pathogenic strains of L. monocytogenes.

Full Text

The Full Text of this article is available as a PDF (2.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. Chakraborty T., Ebel F., Wehland J., Dufrenne J., Notermans S. Naturally occurring virulence-attenuated isolates of Listeria monocytogenes capable of inducing long term protection against infection by virulent strains of homologous and heterologous serotypes. FEMS Immunol Med Microbiol. 1994 Nov;10(1):1–9. doi: 10.1111/j.1574-695X.1994.tb00004.x. [DOI] [PubMed] [Google Scholar]
  3. Chakraborty T., Leimeister-Wächter M., Domann E., Hartl M., Goebel W., Nichterlein T., Notermans S. Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene. J Bacteriol. 1992 Jan;174(2):568–574. doi: 10.1128/jb.174.2.568-574.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Domann E., Leimeister-Wächter M., Goebel W., Chakraborty T. Molecular cloning, sequencing, and identification of a metalloprotease gene from Listeria monocytogenes that is species specific and physically linked to the listeriolysin gene. Infect Immun. 1991 Jan;59(1):65–72. doi: 10.1128/iai.59.1.65-72.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dons L., Rasmussen O. F., Olsen J. E. Cloning and characterization of a gene encoding flagellin of Listeria monocytogenes. Mol Microbiol. 1992 Oct;6(20):2919–2929. doi: 10.1111/j.1365-2958.1992.tb01751.x. [DOI] [PubMed] [Google Scholar]
  6. Dramsi S., Biswas I., Maguin E., Braun L., Mastroeni P., Cossart P. Entry of Listeria monocytogenes into hepatocytes requires expression of inIB, a surface protein of the internalin multigene family. Mol Microbiol. 1995 Apr;16(2):251–261. doi: 10.1111/j.1365-2958.1995.tb02297.x. [DOI] [PubMed] [Google Scholar]
  7. Dramsi S., Kocks C., Forestier C., Cossart P. Internalin-mediated invasion of epithelial cells by Listeria monocytogenes is regulated by the bacterial growth state, temperature and the pleiotropic activator prfA. Mol Microbiol. 1993 Sep;9(5):931–941. doi: 10.1111/j.1365-2958.1993.tb01223.x. [DOI] [PubMed] [Google Scholar]
  8. Farber J. M., Peterkin P. I. Listeria monocytogenes, a food-borne pathogen. Microbiol Rev. 1991 Sep;55(3):476–511. doi: 10.1128/mr.55.3.476-511.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  10. Gellin B. G., Broome C. V. Listeriosis. JAMA. 1989 Mar 3;261(9):1313–1320. [PubMed] [Google Scholar]
  11. Göhmann S., Leimeister-Wächter M., Schiltz E., Goebel W., Chakraborty T. Characterization of a Listeria monocytogenes-specific protein capable of inducing delayed hypersensitivity in Listeria-immune mice. Mol Microbiol. 1990 Jul;4(7):1091–1099. doi: 10.1111/j.1365-2958.1990.tb00683.x. [DOI] [PubMed] [Google Scholar]
  12. Johnson W. M., Tyler S. D., Ewan E. P., Ashton F. E., Wang G., Rozee K. R. Detection of genes coding for listeriolysin and Listeria monocytogenes antigen A (ImaA) in Listeria spp. by the polymerase chain reaction. Microb Pathog. 1992 Jan;12(1):79–86. doi: 10.1016/0882-4010(92)90068-y. [DOI] [PubMed] [Google Scholar]
  13. Leimeister-Wächter M., Chakraborty T. Detection of listeriolysin, the thiol-dependent hemolysin in Listeria monocytogenes, Listeria ivanovii, and Listeria seeligeri. Infect Immun. 1989 Aug;57(8):2350–2357. doi: 10.1128/iai.57.8.2350-2357.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Leimeister-Wächter M., Domann E., Chakraborty T. Detection of a gene encoding a phosphatidylinositol-specific phospholipase C that is co-ordinately expressed with listeriolysin in Listeria monocytogenes. Mol Microbiol. 1991 Feb;5(2):361–366. doi: 10.1111/j.1365-2958.1991.tb02117.x. [DOI] [PubMed] [Google Scholar]
  15. Leimeister-Wächter M., Domann E., Chakraborty T. The expression of virulence genes in Listeria monocytogenes is thermoregulated. J Bacteriol. 1992 Feb;174(3):947–952. doi: 10.1128/jb.174.3.947-952.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Leimeister-Wächter M., Haffner C., Domann E., Goebel W., Chakraborty T. Identification of a gene that positively regulates expression of listeriolysin, the major virulence factor of listeria monocytogenes. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8336–8340. doi: 10.1073/pnas.87.21.8336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lenz L. L., Dere B., Bevan M. J. Identification of an H2-M3-restricted Listeria epitope: implications for antigen presentation by M3. Immunity. 1996 Jul;5(1):63–72. doi: 10.1016/s1074-7613(00)80310-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lingnau A., Chakraborty T., Niebuhr K., Domann E., Wehland J. Identification and purification of novel internalin-related proteins in Listeria monocytogenes and Listeria ivanovii. Infect Immun. 1996 Mar;64(3):1002–1006. doi: 10.1128/iai.64.3.1002-1006.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lingnau A., Domann E., Hudel M., Bock M., Nichterlein T., Wehland J., Chakraborty T. Expression of the Listeria monocytogenes EGD inlA and inlB genes, whose products mediate bacterial entry into tissue culture cell lines, by PrfA-dependent and -independent mechanisms. Infect Immun. 1995 Oct;63(10):3896–3903. doi: 10.1128/iai.63.10.3896-3903.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Martin B., Humbert O., Camara M., Guenzi E., Walker J., Mitchell T., Andrew P., Prudhomme M., Alloing G., Hakenbeck R. A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae. Nucleic Acids Res. 1992 Jul 11;20(13):3479–3483. doi: 10.1093/nar/20.13.3479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mengaud J., Dramsi S., Gouin E., Vazquez-Boland J. A., Milon G., Cossart P. Pleiotropic control of Listeria monocytogenes virulence factors by a gene that is autoregulated. Mol Microbiol. 1991 Sep;5(9):2273–2283. doi: 10.1111/j.1365-2958.1991.tb02158.x. [DOI] [PubMed] [Google Scholar]
  22. Mengaud J., Vicente M. F., Cossart P. Transcriptional mapping and nucleotide sequence of the Listeria monocytogenes hlyA region reveal structural features that may be involved in regulation. Infect Immun. 1989 Dec;57(12):3695–3701. doi: 10.1128/iai.57.12.3695-3701.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Michel E., Cossart P. Physical map of the Listeria monocytogenes chromosome. J Bacteriol. 1992 Nov;174(22):7098–7103. doi: 10.1128/jb.174.22.7098-7103.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ménard R., Dehio C., Sansonetti P. J. Bacterial entry into epithelial cells: the paradigm of Shigella. Trends Microbiol. 1996 Jun;4(6):220–226. doi: 10.1016/0966-842X(96)10039-1. [DOI] [PubMed] [Google Scholar]
  25. Park S. F., Stewart G. S. High-efficiency transformation of Listeria monocytogenes by electroporation of penicillin-treated cells. Gene. 1990 Sep 28;94(1):129–132. doi: 10.1016/0378-1119(90)90479-b. [DOI] [PubMed] [Google Scholar]
  26. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Peel M., Donachie W., Shaw A. Temperature-dependent expression of flagella of Listeria monocytogenes studied by electron microscopy, SDS-PAGE and western blotting. J Gen Microbiol. 1988 Aug;134(8):2171–2178. doi: 10.1099/00221287-134-8-2171. [DOI] [PubMed] [Google Scholar]
  28. Saluja S. K., Weiser J. N. The genetic basis of colony opacity in Streptococcus pneumoniae: evidence for the effect of box elements on the frequency of phenotypic variation. Mol Microbiol. 1995 Apr;16(2):215–227. doi: 10.1111/j.1365-2958.1995.tb02294.x. [DOI] [PubMed] [Google Scholar]
  29. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schuchat A., Swaminathan B., Broome C. V. Epidemiology of human listeriosis. Clin Microbiol Rev. 1991 Apr;4(2):169–183. doi: 10.1128/cmr.4.2.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schäferkordt S., Chakraborty T. Vector plasmid for insertional mutagenesis and directional cloning in Listeria spp. Biotechniques. 1995 Nov;19(5):720-2, 724-5. [PubMed] [Google Scholar]
  32. Sokolovic Z., Schüller S., Bohne J., Baur A., Rdest U., Dickneite C., Nichterlein T., Goebel W. Differences in virulence and in expression of PrfA and PrfA-regulated virulence genes of Listeria monocytogenes strains belonging to serogroup 4. Infect Immun. 1996 Oct;64(10):4008–4019. doi: 10.1128/iai.64.10.4008-4019.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  34. Van Gijsegem F., Genin S., Boucher C. Conservation of secretion pathways for pathogenicity determinants of plant and animal bacteria. Trends Microbiol. 1993 Aug;1(5):175–180. doi: 10.1016/0966-842x(93)90087-8. [DOI] [PubMed] [Google Scholar]
  35. Walker J. K., Morgan J. H., McLauchlin J., Grant K. A., Shallcross J. A. Listeria innocua isolated from a case of ovine meningoencephalitis. Vet Microbiol. 1994 Nov;42(2-3):245–253. doi: 10.1016/0378-1135(94)90023-x. [DOI] [PubMed] [Google Scholar]
  36. Walker S. J., Archer P., Banks J. G. Growth of Listeria monocytogenes at refrigeration temperatures. J Appl Bacteriol. 1990 Feb;68(2):157–162. doi: 10.1111/j.1365-2672.1990.tb02561.x. [DOI] [PubMed] [Google Scholar]
  37. Watarai M., Tobe T., Yoshikawa M., Sasakawa C. Contact of Shigella with host cells triggers release of Ipa invasins and is an essential function of invasiveness. EMBO J. 1995 Jun 1;14(11):2461–2470. doi: 10.1002/j.1460-2075.1995.tb07243.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wuenscher M. D., Köhler S., Goebel W., Chakraborty T. Gene disruption by plasmid integration in Listeria monocytogenes: insertional inactivation of the listeriolysin determinant lisA. Mol Gen Genet. 1991 Aug;228(1-2):177–182. doi: 10.1007/BF00282463. [DOI] [PubMed] [Google Scholar]
  39. 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]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES