Abstract
In Salmonella typhimurium, transcription of the glnA gene (encoding glutamine synthetase) is under the control of the nitrogen-regulatory (ntr) system comprising the alternate sigma factor sigma54 (NtrA) and the two-component sensor-transcriptional activator pair NtrB and NtrC. The glnA, ntrB, and ntrC genes form an operon. We measured the virulence of S. typhimurium strains with nitrogen-regulatory mutations after intraperitoneal (i.p.) or oral inoculations of BALB/c mice. Strains with single mutations in glnA, ntrA, ntrB, or ntrC had i.p. 50% lethal doses (LD50s) of <10 bacteria, similar to the wild-type strain. However, a strain with a delta(glnA-ntrC) operon deletion had an i.p. LD50 of >10(5) bacteria, as did delta glnA ntrA and delta glnA ntrC strains, suggesting that glnA strains require an ntr-transcribed gene for full virulence. High-level transcription of the glutamine transport operon (glnHPQ) is dependent upon both ntrA and ntrC, as determined by glnHp-lacZ fusion measurements. Moreover, delta glnA glnH and delta glnA glnQ strains are attenuated, similar to delta glnA ntrA and delta glnA ntrC strains. These results reveal that access of S. typhimurium to host glutamine depends on the ntr system, which apparently is required for the transcription of the glutamine transport genes. The delta(glnA-ntrC) strain exhibited a reduced ability to survive within the macrophage cell line J774, identifying a potential host environment with low levels of glutamine. Finally, the delta(glnA-ntrC) strain, when inoculated at doses as low as 10 organisms, provided mice with protective immunity against challenge by the wild-type strain, demonstrating its potential use as a live vaccine.
Full Text
The Full Text of this article is available as a PDF (320.2 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Alpuche Aranda C. M., Swanson J. A., Loomis W. P., Miller S. I. Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10079–10083. doi: 10.1073/pnas.89.21.10079. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bajaj V., Hwang C., Lee C. A. hilA is a novel ompR/toxR family member that activates the expression of Salmonella typhimurium invasion genes. Mol Microbiol. 1995 Nov;18(4):715–727. doi: 10.1111/j.1365-2958.1995.mmi_18040715.x. [DOI] [PubMed] [Google Scholar]
- Betteridge P. R., Ayling P. D. The regulation of glutamine transport and glutamine synthetase in Salmonella typhimurium. J Gen Microbiol. 1976 Aug;96(2):324–334. doi: 10.1099/00221287-95-2-324. [DOI] [PubMed] [Google Scholar]
- Bäumler A. J., Kusters J. G., Stojiljkovic I., Heffron F. Salmonella typhimurium loci involved in survival within macrophages. Infect Immun. 1994 May;62(5):1623–1630. doi: 10.1128/iai.62.5.1623-1630.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carter P. B., Collins F. M. The route of enteric infection in normal mice. J Exp Med. 1974 May 1;139(5):1189–1203. doi: 10.1084/jem.139.5.1189. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Claverie-Martin F., Magasanik B. Role of integration host factor in the regulation of the glnHp2 promoter of Escherichia coli. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1631–1635. doi: 10.1073/pnas.88.5.1631. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Craig N. L., Nash H. A. E. coli integration host factor binds to specific sites in DNA. Cell. 1984 Dec;39(3 Pt 2):707–716. doi: 10.1016/0092-8674(84)90478-1. [DOI] [PubMed] [Google Scholar]
- Donnenberg M. S., Kaper J. B. Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun. 1991 Dec;59(12):4310–4317. doi: 10.1128/iai.59.12.4310-4317.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Elliott T. A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon. J Bacteriol. 1992 Jan;174(1):245–253. doi: 10.1128/jb.174.1.245-253.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Elliott T. Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium. J Bacteriol. 1989 Jul;171(7):3948–3960. doi: 10.1128/jb.171.7.3948-3960.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Enomoto M., Stocker B. A. Transduction by phage P1kc in Salmonella typhimurium. Virology. 1974 Aug;60(2):503–514. doi: 10.1016/0042-6822(74)90344-4. [DOI] [PubMed] [Google Scholar]
- Fields P. I., Swanson R. V., Haidaris C. G., Heffron F. Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5189–5193. doi: 10.1073/pnas.83.14.5189. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Galán J. E., Ginocchio C., Costeas P. Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family. J Bacteriol. 1992 Jul;174(13):4338–4349. doi: 10.1128/jb.174.13.4338-4349.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hamilton C. M., Aldea M., Washburn B. K., Babitzke P., Kushner S. R. New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol. 1989 Sep;171(9):4617–4622. doi: 10.1128/jb.171.9.4617-4622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
- Harms E., Gochman N., Schneider J. A. Lysosomal pool of free-amino acids. Biochem Biophys Res Commun. 1981 Apr 15;99(3):830–836. doi: 10.1016/0006-291x(81)91239-0. [DOI] [PubMed] [Google Scholar]
- Higgins C. F., Ames G. F. Two periplasmic transport proteins which interact with a common membrane receptor show extensive homology: complete nucleotide sequences. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6038–6042. doi: 10.1073/pnas.78.10.6038. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hirschman J., Wong P. K., Sei K., Keener J., Kustu S. Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7525–7529. doi: 10.1073/pnas.82.22.7525. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoover T. R., Santero E., Porter S., Kustu S. The integration host factor stimulates interaction of RNA polymerase with NIFA, the transcriptional activator for nitrogen fixation operons. Cell. 1990 Oct 5;63(1):11–22. doi: 10.1016/0092-8674(90)90284-l. [DOI] [PubMed] [Google Scholar]
- Ikeda T. P., Shauger A. E., Kustu S. Salmonella typhimurium apparently perceives external nitrogen limitation as internal glutamine limitation. J Mol Biol. 1996 Jun 21;259(4):589–607. doi: 10.1006/jmbi.1996.0342. [DOI] [PubMed] [Google Scholar]
- Ishimoto K. S., Lory S. Formation of pilin in Pseudomonas aeruginosa requires the alternative sigma factor (RpoN) of RNA polymerase. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1954–1957. doi: 10.1073/pnas.86.6.1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Keener J., Kustu S. Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NTRB and NTRC of enteric bacteria: roles of the conserved amino-terminal domain of NTRC. Proc Natl Acad Sci U S A. 1988 Jul;85(14):4976–4980. doi: 10.1073/pnas.85.14.4976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klimpel K. W., Lesley S. A., Clark V. L. Identification of subunits of gonococcal RNA polymerase by immunoblot analysis: evidence for multiple sigma factors. J Bacteriol. 1989 Jul;171(7):3713–3718. doi: 10.1128/jb.171.7.3713-3718.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klose K. E., Weiss D. S., Kustu S. Glutamate at the site of phosphorylation of nitrogen-regulatory protein NTRC mimics aspartyl-phosphate and activates the protein. J Mol Biol. 1993 Jul 5;232(1):67–78. doi: 10.1006/jmbi.1993.1370. [DOI] [PubMed] [Google Scholar]
- Krajewska-Grynkiewicz K., Kustu S. Evidence that nitrogen regulatory gene ntrC of Salmonella typhimurium is transcribed from the glnA promoter as well as from a separate ntr promoter. Mol Gen Genet. 1984;193(1):135–142. doi: 10.1007/BF00327426. [DOI] [PubMed] [Google Scholar]
- Krajewska-Grynkiewicz K., Kustu S. Regulation of transcription of glnA, the structural gene encoding glutamine synthetase, in glnA::Mu d1 (ApR, lac) fusion strains of Salmonella typhimurium. Mol Gen Genet. 1983;192(1-2):187–197. doi: 10.1007/BF00327665. [DOI] [PubMed] [Google Scholar]
- Kustu S. G., Ames G. F. The hisP protein, a known histidine transport component in Salmonella typhimurium, is also an arginine transport component. J Bacteriol. 1973 Oct;116(1):107–113. doi: 10.1128/jb.116.1.107-113.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kustu S. G., McFarland N. C., Hui S. P., Esmon B., Ames G. F. Nitrogen control of Salmonella typhimurium: co-regulation of synthesis of glutamine synthetase and amino acid transport systems. J Bacteriol. 1979 Apr;138(1):218–234. doi: 10.1128/jb.138.1.218-234.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee C. A., Jones B. D., Falkow S. Identification of a Salmonella typhimurium invasion locus by selection for hyperinvasive mutants. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1847–1851. doi: 10.1073/pnas.89.5.1847. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leong J. M., Nunes-Düby S., Lesser C. F., Youderian P., Susskind M. M., Landy A. The phi 80 and P22 attachment sites. Primary structure and interaction with Escherichia coli integration host factor. J Biol Chem. 1985 Apr 10;260(7):4468–4477. [PubMed] [Google Scholar]
- Lissner C. R., Swanson R. N., O'Brien A. D. Genetic control of the innate resistance of mice to Salmonella typhimurium: expression of the Ity gene in peritoneal and splenic macrophages isolated in vitro. J Immunol. 1983 Dec;131(6):3006–3013. [PubMed] [Google Scholar]
- Mahan M. J., Slauch J. M., Mekalanos J. J. Bacteriophage P22 transduction of integrated plasmids: single-step cloning of Salmonella typhimurium gene fusions. J Bacteriol. 1993 Nov;175(21):7086–7091. doi: 10.1128/jb.175.21.7086-7091.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mahan M. J., Slauch J. M., Mekalanos J. J. Selection of bacterial virulence genes that are specifically induced in host tissues. Science. 1993 Jan 29;259(5095):686–688. doi: 10.1126/science.8430319. [DOI] [PubMed] [Google Scholar]
- McCarter L., Krajewska-Grynkiewicz K., Trinh D., Wei G., Kustu S. Characterization of mutations that lie in the promoter-regulatory region for glnA, the structural gene encoding glutamine synthetase. Mol Gen Genet. 1984;197(1):150–160. doi: 10.1007/BF00327936. [DOI] [PubMed] [Google Scholar]
- McFarland N., McCarter L., Artz S., Kustu S. Nitrogen regulatory locus "glnR" of enteric bacteria is composed of cistrons ntrB and ntrC: identification of their protein products. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2135–2139. doi: 10.1073/pnas.78.4.2135. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miller S. I., Kukral A. M., Mekalanos J. J. A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5054–5058. doi: 10.1073/pnas.86.13.5054. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ninfa A. J., Magasanik B. Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5909–5913. doi: 10.1073/pnas.83.16.5909. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nohno T., Saito T., Hong J. S. Cloning and complete nucleotide sequence of the Escherichia coli glutamine permease operon (glnHPQ). Mol Gen Genet. 1986 Nov;205(2):260–269. doi: 10.1007/BF00430437. [DOI] [PubMed] [Google Scholar]
- Nohno T., Saito T. Two transcriptional start sites found in the promoter region of Escherichia coli glutamine permease operon, glnHPQ. Nucleic Acids Res. 1987 Mar 25;15(6):2777–2777. doi: 10.1093/nar/15.6.2777. [DOI] [PMC free article] [PubMed] [Google Scholar]
- North A. K., Weiss D. S., Suzuki H., Flashner Y., Kustu S. Repressor forms of the enhancer-binding protein NrtC: some fail in coupling ATP hydrolysis to open complex formation by sigma 54-holoenzyme. J Mol Biol. 1996 Jul 19;260(3):317–331. doi: 10.1006/jmbi.1996.0403. [DOI] [PubMed] [Google Scholar]
- O'Toole R., Milton D. L., Wolf-Watz H. Chemotactic motility is required for invasion of the host by the fish pathogen Vibrio anguillarum. Mol Microbiol. 1996 Feb;19(3):625–637. doi: 10.1046/j.1365-2958.1996.412927.x. [DOI] [PubMed] [Google Scholar]
- Popham D. L., Szeto D., Keener J., Kustu S. Function of a bacterial activator protein that binds to transcriptional enhancers. Science. 1989 Feb 3;243(4891):629–635. doi: 10.1126/science.2563595. [DOI] [PubMed] [Google Scholar]
- Porter S. C., North A. K., Wedel A. B., Kustu S. Oligomerization of NTRC at the glnA enhancer is required for transcriptional activation. Genes Dev. 1993 Nov;7(11):2258–2273. doi: 10.1101/gad.7.11.2258. [DOI] [PubMed] [Google Scholar]
- Reitzer L. J., Magasanik B. Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter. Cell. 1986 Jun 20;45(6):785–792. doi: 10.1016/0092-8674(86)90553-2. [DOI] [PubMed] [Google Scholar]
- Rose R. E. The nucleotide sequence of pACYC184. Nucleic Acids Res. 1988 Jan 11;16(1):355–355. doi: 10.1093/nar/16.1.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sasse-Dwight S., Gralla J. D. Probing the Escherichia coli glnALG upstream activation mechanism in vivo. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8934–8938. doi: 10.1073/pnas.85.23.8934. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schmieger H. Phage P22-mutants with increased or decreased transduction abilities. Mol Gen Genet. 1972;119(1):75–88. doi: 10.1007/BF00270447. [DOI] [PubMed] [Google Scholar]
- Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
- Wang R. F., Kushner S. R. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene. 1991 Apr;100:195–199. [PubMed] [Google Scholar]
- Watson N. A new revision of the sequence of plasmid pBR322. Gene. 1988 Oct 30;70(2):399–403. doi: 10.1016/0378-1119(88)90212-0. [DOI] [PubMed] [Google Scholar]
- Wedel A., Weiss D. S., Popham D., Dröge P., Kustu S. A bacterial enhancer functions to tether a transcriptional activator near a promoter. Science. 1990 Apr 27;248(4954):486–490. doi: 10.1126/science.1970441. [DOI] [PubMed] [Google Scholar]
- Willis R. C., Iwata K. K., Furlong C. E. Regulation of Glutamine Transport in Escherichia coli. J Bacteriol. 1975 Jun;122(3):1032–1037. doi: 10.1128/jb.122.3.1032-1037.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Xu K., Elliott T. Cloning, DNA sequence, and complementation analysis of the Salmonella typhimurium hemN gene encoding a putative oxygen-independent coproporphyrinogen III oxidase. J Bacteriol. 1994 Jun;176(11):3196–3203. doi: 10.1128/jb.176.11.3196-3203.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]