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. 1997 Aug;179(16):5118–5125. doi: 10.1128/jb.179.16.5118-5125.1997

Identification and analysis of a gene encoding L-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase involved in the 1,3-diaminopropane production pathway in Acinetobacter baumannii.

H Ikai 1, S Yamamoto 1
PMCID: PMC179370  PMID: 9260954

Abstract

The ca. 2.2-kbp region upstream of the ddc gene encoding L-2,4-diaminobutyrate decarboxylase in Acinetobacter baumannii was sequenced and found to contain another open reading frame of 1,338 nucleotides encoding a protein with a deduced molecular mass of 47,423 Da. Analysis of the homologies observed from the deduced amino acid sequence indicated that the gene product is an enzyme belonging to subgroup II of the aminotransferases. This was first verified when examination of the crude extracts from Escherichia coli transformants led to detection of a novel aminotransferase activity catalyzing the following reversible reactions: L-2,4-diaminobutyric acid + 2-ketoglutaric acid<-->L-glutamic acid + L-aspartic beta-semialdehyde. Further confirmation was obtained when the gene was overexpressed in E. coli and the corresponding protein was purified to homogeneity. It catalyzed the same reactions and its N-terminal amino acid sequence was consistent with that deduced from the nucleotide sequence. Therefore, the gene and its product were named dat and L-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase (DABA AT), respectively. Feeding experiments of A. baumannii with L-[U-14C]aspartic acid resulted in the incorporation of the label into 1,3-diaminopropane. Apparent homologs of dat and DABA AT were detected in other Acinetobacter species by PCR amplification and Western blotting. These results indicate that the dat gene (as well as the ddc gene) participates in the synthesis of 1,3-diaminopropane, the only diamine found in this genus. However, the biological role, if one exists, of 1,3-diaminopropane synthesis is unknown.

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Selected References

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  1. BLACK S., WRIGHT N. G. Aspartic beta-semialdehyde dehydrogenase and aspartic beta-semialdehyde. J Biol Chem. 1955 Mar;213(1):39–50. [PubMed] [Google Scholar]
  2. Bartsch K., von Johnn-Marteville A., Schulz A. Molecular analysis of two genes of the Escherichia coli gab cluster: nucleotide sequence of the glutamate:succinic semialdehyde transaminase gene (gabT) and characterization of the succinic semialdehyde dehydrogenase gene (gabD). J Bacteriol. 1990 Dec;172(12):7035–7042. doi: 10.1128/jb.172.12.7035-7042.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baumann P. Isolation of Acinetobacter from soil and water. J Bacteriol. 1968 Jul;96(1):39–42. doi: 10.1128/jb.96.1.39-42.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benson D. A., Boguski M., Lipman D. J., Ostell J. GenBank. Nucleic Acids Res. 1996 Jan 1;24(1):1–5. doi: 10.1093/nar/24.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bergogne-Bérézin E., Towner K. J. Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin Microbiol Rev. 1996 Apr;9(2):148–165. doi: 10.1128/cmr.9.2.148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bowman W. H., Tabor C. W., Tabor H. Spermidine biosynthesis. Purification and properties of propylamine transferase from Escherichia coli. J Biol Chem. 1973 Apr 10;248(7):2480–2486. [PubMed] [Google Scholar]
  8. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  9. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  10. Floriano B., Herrero A., Flores E. Analysis of expression of the argC and argD genes in the cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol. 1994 Oct;176(20):6397–6401. doi: 10.1128/jb.176.20.6397-6401.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Groth I., Schumann P., Weiss N., Martin K., Rainey F. A. Agrococcus jenensis gen. nov., sp. nov., a new genus of actinomycetes with diaminobutyric acid in the cell wall. Int J Syst Bacteriol. 1996 Jan;46(1):234–239. doi: 10.1099/00207713-46-1-234. [DOI] [PubMed] [Google Scholar]
  12. Harley C. B., Reynolds R. P. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. doi: 10.1093/nar/15.5.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harrison L., Teplow D. B., Rinaldi M., Strobel G. Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity. J Gen Microbiol. 1991 Dec;137(12):2857–2865. doi: 10.1099/00221287-137-12-2857. [DOI] [PubMed] [Google Scholar]
  14. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Henriksen S. D. Moraxella, neisseria, branhamella, and acinetobacter. Annu Rev Microbiol. 1976;30:63–83. doi: 10.1146/annurev.mi.30.100176.000431. [DOI] [PubMed] [Google Scholar]
  16. Ikai H., Yamamoto S. Cloning and expression in Escherichia coli of the gene encoding a novel L-2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii. FEMS Microbiol Lett. 1994 Dec 1;124(2):225–228. doi: 10.1016/0378-1097(94)90253-4. [DOI] [PubMed] [Google Scholar]
  17. Ikai H., Yamamoto S. Sequence analysis of the gene encoding a novel L-2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii: similarity to the group II amino acid decarboxylases. Arch Microbiol. 1996 Aug;166(2):128–131. doi: 10.1007/s002030050366. [DOI] [PubMed] [Google Scholar]
  18. Kirsch J. F., Eichele G., Ford G. C., Vincent M. G., Jansonius J. N., Gehring H., Christen P. Mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure. J Mol Biol. 1984 Apr 15;174(3):497–525. doi: 10.1016/0022-2836(84)90333-4. [DOI] [PubMed] [Google Scholar]
  19. Kok R. G., van Thor J. J., Nugteren-Roodzant I. M., Brouwer M. B., Egmond M. R., Nudel C. B., Vosman B., Hellingwerf K. J. Characterization of the extracellular lipase, LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structural gene. Mol Microbiol. 1995 Mar;15(5):803–818. doi: 10.1111/j.1365-2958.1995.tb02351.x. [DOI] [PubMed] [Google Scholar]
  20. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Mehta P. K., Hale T. I., Christen P. Aminotransferases: demonstration of homology and division into evolutionary subgroups. Eur J Biochem. 1993 Jun 1;214(2):549–561. doi: 10.1111/j.1432-1033.1993.tb17953.x. [DOI] [PubMed] [Google Scholar]
  24. Morris D. R., Jorstad C. M. Growth and macromolecular composition of a mutant of Escherichia coli during polyamine limitation. J Bacteriol. 1973 Jan;113(1):271–277. doi: 10.1128/jb.113.1.271-277.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Murray M. G., Thompson W. F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 1980 Oct 10;8(19):4321–4325. doi: 10.1093/nar/8.19.4321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nigam S. N., Ressler C. Biosynthesis of 2,4-diaminobutyric acid from L-[3H]homoserine and DL-[1-14C]aspartic acid in Lathyrus sylvestris W. Biochemistry. 1966 Nov;5(11):3426–3431. doi: 10.1021/bi00875a006. [DOI] [PubMed] [Google Scholar]
  27. PAULUS H., GRAY E. THE BIOSYNTHESIS OF POLYMYXIN B BY GROWING CULTURES OF BACILLUS POLYMYXA. J Biol Chem. 1964 Mar;239:865–871. [PubMed] [Google Scholar]
  28. PERKINS H. R., CUMMINS C. S. CHEMICAL STRUCTURE OF BACTERIAL CELL WALLS. ORNITHINE AND 2,4-DIAMINOBUTYRIC ACID AS COMPONENTS OF THE CELL WALLS OF PLANT PATHOGENIC CORYNEBACTERIA. Nature. 1964 Mar 14;201:1105–1107. doi: 10.1038/2011105a0. [DOI] [PubMed] [Google Scholar]
  29. Rao D. R., Hariharan K., Vijayalakshmi K. R. A study of the metabolism of L-alpha gamma-diaminobutyric acid in a Xanthomonas species. Biochem J. 1969 Aug;114(1):107–115. doi: 10.1042/bj1140107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Seifert H., Baginski R., Schulze A., Pulverer G. Antimicrobial susceptibility of Acinetobacter species. Antimicrob Agents Chemother. 1993 Apr;37(4):750–753. doi: 10.1128/aac.37.4.750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Struelens M. J., Carlier E., Maes N., Serruys E., Quint W. G., van Belkum A. Nosocomial colonization and infection with multiresistant Acinetobacter baumannii: outbreak delineation using DNA macrorestriction analysis and PCR-fingerprinting. J Hosp Infect. 1993 Sep;25(1):15–32. doi: 10.1016/0195-6701(93)90005-k. [DOI] [PubMed] [Google Scholar]
  32. Tabor C. W., Tabor H. Polyamines in microorganisms. Microbiol Rev. 1985 Mar;49(1):81–99. doi: 10.1128/mr.49.1.81-99.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tankovic J., Legrand P., De Gatines G., Chemineau V., Brun-Buisson C., Duval J. Characterization of a hospital outbreak of imipenem-resistant Acinetobacter baumannii by phenotypic and genotypic typing methods. J Clin Microbiol. 1994 Nov;32(11):2677–2681. doi: 10.1128/jcm.32.11.2677-2681.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  35. Yamamoto S., Hamanaka K., Suemoto Y., Ono B., Shinoda S. Evidence for the presence of a novel biosynthetic pathway for norspermidine in Vibrio. Can J Microbiol. 1986 Feb;32(2):99–103. doi: 10.1139/m86-021. [DOI] [PubMed] [Google Scholar]
  36. Yamamoto S., Kiyama S., Watanabe Y., Makita M. Practical gas-liquid chromatographic method for the determination of amino acids in human serum. J Chromatogr. 1982 Dec 10;233:39–50. doi: 10.1016/s0378-4347(00)81729-6. [DOI] [PubMed] [Google Scholar]
  37. Yamamoto S., Mutoh N., Ikai H., Nagasaka M. Occurrence of a novel L-2,4-diaminobutyrate decarboxylase activity in some species of Enterobacteriaceae, and purification and characterization of the enzymes of Enterobacter aerogenes and Serratia marcescens. Biol Pharm Bull. 1996 Oct;19(10):1298–1303. doi: 10.1248/bpb.19.1298. [DOI] [PubMed] [Google Scholar]
  38. Yamamoto S., Okujo N., Sakakibara Y. Isolation and structure elucidation of acinetobactin, a novel siderophore from Acinetobacter baumannii. Arch Microbiol. 1994;162(4):249–254. doi: 10.1007/BF00301846. [DOI] [PubMed] [Google Scholar]
  39. Yamamoto S., Tsuzaki Y., Kishi R., Nakao H. Occurrence of L-2,4-diaminobutyrate decarboxylase activity in Acinetobacter. Chem Pharm Bull (Tokyo) 1991 Sep;39(9):2451–2453. doi: 10.1248/cpb.39.2451. [DOI] [PubMed] [Google Scholar]
  40. Yamamoto S., Tsuzaki Y., Tougou K., Shinoda S. Purification and characterization of L-2,4-diaminobutyrate decarboxylase from Acinetobacter calcoaceticus. J Gen Microbiol. 1992 Jul;138(7):1461–1465. doi: 10.1099/00221287-138-7-1461. [DOI] [PubMed] [Google Scholar]

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