Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster (original) (raw)

Abstract

A fragment of the Salmonella typhimurium ethanolamine utilization operon was cloned and characterized. The 6.3-kb nucleotide sequence encoded six complete open reading frames, termed cchA, cchB, eutE, eutJ, eutG, and eutH. In addition, the nucleotide sequences of two incomplete open reading frames, termed eutX and eutI, were also determined. Comparison of the deduced amino acid sequences and entries in the GenBank database indicated that eutI encodes a phosphate acetyltransferase-like enzyme. The deduced amino acid sequences of the EutE and EutG proteins revealed a significant degree of homology with the Escherichia coli alcohol dehydrogenase AdhE sequence. Mutations in eutE or eutG completely abolished the ability of mutants to utilize ethanolamine as a carbon source and reduced the ability to utilize ethanolamine as a nitrogen source. The product of eutE is most probably an acetaldehyde dehydrogenase catalyzing the conversion of acetaldehyde into acetyl coenzyme A. The product of the eutG gene, an uncommon iron-containing alcohol dehydrogenase, may protect the cell from unconverted acetaldehyde by converting it into an alcohol. The deduced amino acid sequence of cchA resembles that of carboxysome shell proteins from Thiobacillus neapolitanus and Synechococcus sp. as well as that of the PduA product from S. typhimurium. CchA and CchB proteins may be involved in the formation of an intracellular microcompartment responsible for the metabolism of ethanolamine. The hydrophobic protein encoded by the eutH gene possesses some characteristics of bacterial permeases and might therefore be involved in the transport of ethanolamine. Ethanolamine-utilization mutants were slightly attenuated in a mouse model of S. typhimurium infection, indicating that ethanolamine may be an important source of nitrogen and carbon for S. typhimurium in vivo.

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  1. Boylen C. W., Ensign J. C. Intracellular substrates for endogenous metabolism during long-term starvation of rod and spherical cells of Arthrobacter crystallopoietes. J Bacteriol. 1970 Sep;103(3):578–587. doi: 10.1128/jb.103.3.578-587.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Chang G. W., Chang J. T. Evidence for the B12-dependent enzyme ethanolamine deaminase in Salmonella. Nature. 1975 Mar 13;254(5496):150–151. doi: 10.1038/254150a0. [DOI] [PubMed] [Google Scholar]
  4. Chen P., Andersson D. I., Roth J. R. The control region of the pdu/cob regulon in Salmonella typhimurium. J Bacteriol. 1994 Sep;176(17):5474–5482. doi: 10.1128/jb.176.17.5474-5482.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Conway T., Ingram L. O. Similarity of Escherichia coli propanediol oxidoreductase (fucO product) and an unusual alcohol dehydrogenase from Zymomonas mobilis and Saccharomyces cerevisiae. J Bacteriol. 1989 Jul;171(7):3754–3759. doi: 10.1128/jb.171.7.3754-3759.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Conway T., Sewell G. W., Osman Y. A., Ingram L. O. Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis. J Bacteriol. 1987 Jun;169(6):2591–2597. doi: 10.1128/jb.169.6.2591-2597.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. English R. S., Lorbach S. C., Qin X., Shively J. M. Isolation and characterization of a carboxysome shell gene from Thiobacillus neapolitanus. Mol Microbiol. 1994 May;12(4):647–654. doi: 10.1111/j.1365-2958.1994.tb01052.x. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Fox D. K., Meadow N. D., Roseman S. Phosphate transfer between acetate kinase and enzyme I of the bacterial phosphotransferase system. J Biol Chem. 1986 Oct 15;261(29):13498–13503. [PubMed] [Google Scholar]
  10. Freter R., Brickner H., Botney M., Cleven D., Aranki A. Mechanisms that control bacterial populations in continuous-flow culture models of mouse large intestinal flora. Infect Immun. 1983 Feb;39(2):676–685. doi: 10.1128/iai.39.2.676-685.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fukuzawa H., Suzuki E., Komukai Y., Miyachi S. A gene homologous to chloroplast carbonic anhydrase (icfA) is essential to photosynthetic carbon dioxide fixation by Synechococcus PCC7942. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4437–4441. doi: 10.1073/pnas.89.10.4437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goodlove P. E., Cunningham P. R., Parker J., Clark D. P. Cloning and sequence analysis of the fermentative alcohol-dehydrogenase-encoding gene of Escherichia coli. Gene. 1989 Dec 21;85(1):209–214. doi: 10.1016/0378-1119(89)90483-6. [DOI] [PubMed] [Google Scholar]
  13. Guiot H. F. Role of competition for substrate in bacterial antagonism in the gut. Infect Immun. 1982 Dec;38(3):887–892. doi: 10.1128/iai.38.3.887-892.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hennecke H. Regulation of bacterial gene expression by metal-protein complexes. Mol Microbiol. 1990 Oct;4(10):1621–1628. doi: 10.1111/j.1365-2958.1990.tb00538.x. [DOI] [PubMed] [Google Scholar]
  15. Hidalgo E., Chen Y. M., Lin E. C., Aguilar J. Molecular cloning and DNA sequencing of the Escherichia coli K-12 ald gene encoding aldehyde dehydrogenase. J Bacteriol. 1991 Oct;173(19):6118–6123. doi: 10.1128/jb.173.19.6118-6123.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Latimer M. T., Ferry J. G. Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila. J Bacteriol. 1993 Nov;175(21):6822–6829. doi: 10.1128/jb.175.21.6822-6829.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Libby S. J., Goebel W., Ludwig A., Buchmeier N., Bowe F., Fang F. C., Guiney D. G., Songer J. G., Heffron F. A cytolysin encoded by Salmonella is required for survival within macrophages. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):489–493. doi: 10.1073/pnas.91.2.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lin E. C., Iuchi S. Regulation of gene expression in fermentative and respiratory systems in Escherichia coli and related bacteria. Annu Rev Genet. 1991;25:361–387. doi: 10.1146/annurev.ge.25.120191.002045. [DOI] [PubMed] [Google Scholar]
  19. MANDELSTAM J. Turnover of protein in growing and non-growing populations of Escherichia coli. Biochem J. 1958 May;69(1):110–119. doi: 10.1042/bj0690110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mason T. G., Richardson G. Escherichia coli and the human gut: some ecological considerations. J Appl Bacteriol. 1981 Aug;51(1):1–16. doi: 10.1111/j.1365-2672.1981.tb00903.x. [DOI] [PubMed] [Google Scholar]
  21. Matin A., Auger E. A., Blum P. H., Schultz J. E. Genetic basis of starvation survival in nondifferentiating bacteria. Annu Rev Microbiol. 1989;43:293–316. doi: 10.1146/annurev.mi.43.100189.001453. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Muto A., Osawa S. The guanine and cytosine content of genomic DNA and bacterial evolution. Proc Natl Acad Sci U S A. 1987 Jan;84(1):166–169. doi: 10.1073/pnas.84.1.166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Neidhardt F. C., Bloch P. L., Smith D. F. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. doi: 10.1128/jb.119.3.736-747.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. O'Toole G. A., Escalante-Semerena J. C. Identification and initial characterization of the eutF locus of Salmonella typhimurium. J Bacteriol. 1991 Aug;173(16):5168–5172. doi: 10.1128/jb.173.16.5168-5172.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Price G. D., Howitt S. M., Harrison K., Badger M. R. Analysis of a genomic DNA region from the cyanobacterium Synechococcus sp. strain PCC7942 involved in carboxysome assembly and function. J Bacteriol. 1993 May;175(10):2871–2879. doi: 10.1128/jb.175.10.2871-2879.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Randle C. L., Albro P. W., Dittmer J. C. The phosphoglyceride composition of Gram-negative bacteria and the changes in composition during growth. Biochim Biophys Acta. 1969;187(2):214–220. doi: 10.1016/0005-2760(69)90030-7. [DOI] [PubMed] [Google Scholar]
  28. Ratzkin B., Roth J. Cluster of genes controlling proline degradation in Salmonella typhimurium. J Bacteriol. 1978 Feb;133(2):744–754. doi: 10.1128/jb.133.2.744-754.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Reith M., Munholland J. An hsp70 homolog is encoded on the plastid genome of the red alga, Porphyra umbilicalis. FEBS Lett. 1991 Dec 2;294(1-2):116–120. doi: 10.1016/0014-5793(91)81355-c. [DOI] [PubMed] [Google Scholar]
  30. Repaske R., Clayton M. A. Control of Escherichia coli growth by CO2. J Bacteriol. 1978 Sep;135(3):1162–1164. doi: 10.1128/jb.135.3.1162-1164.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Roof D. M., Roth J. R. Autogenous regulation of ethanolamine utilization by a transcriptional activator of the eut operon in Salmonella typhimurium. J Bacteriol. 1992 Oct;174(20):6634–6643. doi: 10.1128/jb.174.20.6634-6643.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Roof D. M., Roth J. R. Ethanolamine utilization in Salmonella typhimurium. J Bacteriol. 1988 Sep;170(9):3855–3863. doi: 10.1128/jb.170.9.3855-3863.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Roof D. M., Roth J. R. Functions required for vitamin B12-dependent ethanolamine utilization in Salmonella typhimurium. J Bacteriol. 1989 Jun;171(6):3316–3323. doi: 10.1128/jb.171.6.3316-3323.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Roszak D. B., Colwell R. R. Survival strategies of bacteria in the natural environment. Microbiol Rev. 1987 Sep;51(3):365–379. doi: 10.1128/mr.51.3.365-379.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sanderson K. E., Roth J. R. Linkage map of Salmonella typhimurium, edition VII. Microbiol Rev. 1988 Dec;52(4):485–532. doi: 10.1128/mr.52.4.485-532.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Scarlett F. A., Turner J. M. Microbial metabolism of amino alcohols. Ethanolamine catabolism mediated by coenzyme B12-dependent ethanolamine ammonia-lyase in Escherichia coli and Klebsiella aerogenes. J Gen Microbiol. 1976 Jul;95(1):173–176. doi: 10.1099/00221287-95-1-173. [DOI] [PubMed] [Google Scholar]
  38. Sheppard D. E., Roth J. R. A rationale for autoinduction of a transcriptional activator: ethanolamine ammonia-lyase (EutBC) and the operon activator (EutR) compete for adenosyl-cobalamin in Salmonella typhimurium. J Bacteriol. 1994 Mar;176(5):1287–1296. doi: 10.1128/jb.176.5.1287-1296.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sprenger G. A. Two open reading frames adjacent to the Escherichia coli K-12 transketolase (tkt) gene show high similarity to the mannitol phosphotransferase system enzymes from Escherichia coli and various gram-positive bacteria. Biochim Biophys Acta. 1993 Aug 20;1158(1):103–106. doi: 10.1016/0304-4165(93)90103-f. [DOI] [PubMed] [Google Scholar]
  40. Stojiljkovic I., Bäumler A. J., Hantke K. Fur regulon in gram-negative bacteria. Identification and characterization of new iron-regulated Escherichia coli genes by a fur titration assay. J Mol Biol. 1994 Feb 18;236(2):531–545. doi: 10.1006/jmbi.1994.1163. [DOI] [PubMed] [Google Scholar]
  41. Stojiljkovic I., Hantke K. Transport of haemin across the cytoplasmic membrane through a haemin-specific periplasmic binding-protein-dependent transport system in Yersinia enterocolitica. Mol Microbiol. 1994 Aug;13(4):719–732. doi: 10.1111/j.1365-2958.1994.tb00465.x. [DOI] [PubMed] [Google Scholar]
  42. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  43. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  44. Walker H. H. CARBON DIOXIDE AS A FACTOR AFFECTING LAG IN BACTERIAL GROWTH. Science. 1932 Dec 23;76(1982):602–604. doi: 10.1126/science.76.1982.602. [DOI] [PubMed] [Google Scholar]
  45. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Williamson V. M., Paquin C. E. Homology of Saccharomyces cerevisiae ADH4 to an iron-activated alcohol dehydrogenase from Zymomonas mobilis. Mol Gen Genet. 1987 Sep;209(2):374–381. doi: 10.1007/BF00329668. [DOI] [PubMed] [Google Scholar]
  47. Youngleson J. S., Jones W. A., Jones D. T., Woods D. R. Molecular analysis and nucleotide sequence of the adh1 gene encoding an NADPH-dependent butanol dehydrogenase in the Gram-positive anaerobe Clostridium acetobutylicum. Gene. 1989 May 30;78(2):355–364. doi: 10.1016/0378-1119(89)90238-2. [DOI] [PubMed] [Google Scholar]
  48. de Vries G. E., Arfman N., Terpstra P., Dijkhuizen L. Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene. J Bacteriol. 1992 Aug;174(16):5346–5353. doi: 10.1128/jb.174.16.5346-5353.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]