Abstract
The initial steps in assimilation of sulfate during cysteine biosynthesis entail sulfate uptake and sulfate activation by formation of adenosine 5'-phosphosulfate, conversion to 3'-phosphoadenosine 5'-phosphosulfate, and reduction to sulfite. Mutations in a previously uncharacterized Escherichia coli gene, cysQ, which resulted in a requirement for sulfite or cysteine, were obtained by in vivo insertion of transposons Tn5tac1 and Tn5supF and by in vitro insertion of resistance gene cassettes. cysQ is at chromosomal position 95.7 min (kb 4517 to 4518) and is transcribed divergently from the adjacent cpdB gene. A Tn5tac1 insertion just inside the 3' end of cysQ, with its isopropyl-beta-D-thiogalactopyranoside-inducible tac promoter pointed toward the cysQ promoter, resulted in auxotrophy only when isopropyl-beta-D-thiogalactopyranoside was present; this conditional phenotype was ascribed to collision between converging RNA polymerases or interaction between complementary antisense and cysQ mRNAs. The auxotrophy caused by cysQ null mutations was leaky in some but not all E. coli strains and could be compensated by mutations in unlinked genes. cysQ mutants were prototrophic during anaerobic growth. Mutations in cysQ did not affect the rate of sulfate uptake or the activities of ATP sulfurylase and its protein activator, which together catalyze adenosine 5'-phosphosulfate synthesis. Some mutations that compensated for cysQ null alleles resulted in sulfate transport defects. cysQ is identical to a gene called amtA, which had been thought to be needed for ammonium transport. Computer analyses, detailed elsewhere, revealed significant amino acid sequence homology between cysQ and suhB of E. coli and the gene for mammalian inositol monophosphatase. Previous work had suggested that 3'-phosphoadenoside 5'-phosphosulfate is toxic if allowed to accumulate, and we propose that CysQ helps control the pool of 3'-phosphoadenoside 5'-phosphosulfate, or its use in sulfite synthesis.
Full text
PDF










Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. doi: 10.1128/mr.54.2.130-197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bairoch A. PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids Res. 1991 Apr 25;19 (Suppl):2241–2245. doi: 10.1093/nar/19.suppl.2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barrett E. L., Chang G. W. Cysteine auxotrophs of Salmonella typhimurium which grow without cysteine in a hydrogen/carbon dioxide atmosphere. J Gen Microbiol. 1979 Dec;115(2):513–516. doi: 10.1099/00221287-115-2-513. [DOI] [PubMed] [Google Scholar]
- Beacham I. R., Garrett S. Isolation of Escherichia coli mutants (cpdB) deficient in periplasmic 2':3'-cyclic phosphodiesterase and genetic mapping of the cpdB locus. J Gen Microbiol. 1980 Jul;119(1):31–34. doi: 10.1099/00221287-119-1-31. [DOI] [PubMed] [Google Scholar]
- Berg D. E., Weiss A., Crossland L. Polarity of Tn5 insertion mutations in Escherichia coli. J Bacteriol. 1980 May;142(2):439–446. doi: 10.1128/jb.142.2.439-446.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
- CAMPBELL A. Sensitive mutants of bacteriophage lambda. Virology. 1961 May;14:22–32. doi: 10.1016/0042-6822(61)90128-3. [DOI] [PubMed] [Google Scholar]
- Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
- Chow W. Y., Berg D. E. Tn5tac1, a derivative of transposon Tn5 that generates conditional mutations. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6468–6472. doi: 10.1073/pnas.85.17.6468. [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]
- Csonka L. N., Clark A. J. Deletions generated by the transposon Tn10 in the srl recA region of the Escherichia coli K-12 chromosome. Genetics. 1979 Oct;93(2):321–343. doi: 10.1093/genetics/93.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DREYFUSS J. CHARACTERIZATION OF A SULFATE- AND THIOSULFATE-TRANSPORTING SYSTEM IN SALMONELLA TYPHIMURIUM. J Biol Chem. 1964 Jul;239:2292–2297. [PubMed] [Google Scholar]
- Diehl R. E., Whiting P., Potter J., Gee N., Ragan C. I., Linemeyer D., Schoepfer R., Bennett C., Dixon R. A. Cloning and expression of bovine brain inositol monophosphatase. J Biol Chem. 1990 Apr 15;265(11):5946–5949. [PubMed] [Google Scholar]
- Fabiny J. M., Jayakumar A., Chinault A. C., Barnes E. M., Jr Ammonium transport in Escherichia coli: localization and nucleotide sequence of the amtA gene. J Gen Microbiol. 1991 Apr;137(4):983–989. doi: 10.1099/00221287-137-4-983. [DOI] [PubMed] [Google Scholar]
- Gillespie D., Demerec M., Itikawa H. Appearance of double mutants in aged cultures of Salmonella typhimurium cysteine-requiring strains. Genetics. 1968 Aug;59(4):433–442. doi: 10.1093/genetics/59.4.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guyer M. S., Reed R. R., Steitz J. A., Low K. B. Identification of a sex-factor-affinity site in E. coli as gamma delta. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):135–140. doi: 10.1101/sqb.1981.045.01.022. [DOI] [PubMed] [Google Scholar]
- Hryniewicz M. M., Kredich N. M. The cysP promoter of Salmonella typhimurium: characterization of two binding sites for CysB protein, studies of in vivo transcription initiation, and demonstration of the anti-inducer effects of thiosulfate. J Bacteriol. 1991 Sep;173(18):5876–5886. doi: 10.1128/jb.173.18.5876-5886.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hryniewicz M., Sirko A., Pałucha A., Böck A., Hulanicka D. Sulfate and thiosulfate transport in Escherichia coli K-12: identification of a gene encoding a novel protein involved in thiosulfate binding. J Bacteriol. 1990 Jun;172(6):3358–3366. doi: 10.1128/jb.172.6.3358-3366.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huang C. J., Barrett E. L. Identification and cloning of genes involved in anaerobic sulfite reduction by Salmonella typhimurium. J Bacteriol. 1990 Jul;172(7):4100–4102. doi: 10.1128/jb.172.7.4100-4102.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jayakumar A., Hwang S. J., Fabiny J. M., Chinault A. C., Barnes E. M., Jr Isolation of an ammonium or methylammonium ion transport mutant of Escherichia coli and complementation by the cloned gene. J Bacteriol. 1989 Feb;171(2):996–1001. doi: 10.1128/jb.171.2.996-1001.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jayakumar A., Rudd K. E., Fabiny J. M., Barnes E. M., Jr Localization of the Escherichia coli amtA gene to 95.8 minutes. J Bacteriol. 1991 Mar;173(5):1572–1573. doi: 10.1128/jb.173.5.1572-1573.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
- Kredich N. M., Foote L. J., Hulanicka M. D. Studies on the mechanism of inhibition of Salmonella typhimurium by 1,2,4-triazole. J Biol Chem. 1975 Sep 25;250(18):7324–7331. [PubMed] [Google Scholar]
- Kulakauskas S., Wikström P. M., Berg D. E. Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome. J Bacteriol. 1991 Apr;173(8):2633–2638. doi: 10.1128/jb.173.8.2633-2638.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kurnit D. M., Seed B. Improved genetic selection for screening bacteriophage libraries by homologous recombination in vivo. Proc Natl Acad Sci U S A. 1990 Apr;87(8):3166–3169. doi: 10.1073/pnas.87.8.3166. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lahti R., Pitkäranta T., Valve E., Ilta I., Kukko-Kalske E., Heinonen J. Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12. J Bacteriol. 1988 Dec;170(12):5901–5907. doi: 10.1128/jb.170.12.5901-5907.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leyh T. S., Taylor J. C., Markham G. D. The sulfate activation locus of Escherichia coli K12: cloning, genetic, and enzymatic characterization. J Biol Chem. 1988 Feb 15;263(5):2409–2416. [PubMed] [Google Scholar]
- Liu J., Beacham I. R. Transcription and regulation of the cpdB gene in Escherichia coli K12 and Salmonella typhimurium LT2: evidence for modulation of constitutive promoters by cyclic AMP-CRP complex. Mol Gen Genet. 1990 Jun;222(1):161–165. doi: 10.1007/BF00283039. [DOI] [PubMed] [Google Scholar]
- Liu J., Burns D. M., Beacham I. R. Isolation and sequence analysis of the gene (cpdB) encoding periplasmic 2',3'-cyclic phosphodiesterase. J Bacteriol. 1986 Mar;165(3):1002–1010. doi: 10.1128/jb.165.3.1002-1010.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- MacNeil T., MacNeil D., Tyler B. Fine-structure deletion map and complementation analysis of the glnA-glnL-glnG region in Escherichia coli. J Bacteriol. 1982 Jun;150(3):1302–1313. doi: 10.1128/jb.150.3.1302-1313.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
- Neuwald A. F., York J. D., Majerus P. W. Diverse proteins homologous to inositol monophosphatase. FEBS Lett. 1991 Dec 2;294(1-2):16–18. doi: 10.1016/0014-5793(91)81332-3. [DOI] [PubMed] [Google Scholar]
- Pardee A. B., Prestidge L. S., Whipple M. B., Dreyfuss J. A binding site for sulfate and its relation to sulfate transport into Salmonella typhimurium. J Biol Chem. 1966 Sep 10;241(17):3962–3969. [PubMed] [Google Scholar]
- Parra F., Britton P., Castle C., Jones-Mortimer M. C., Kornberg H. L. Two separate genes involved in sulphate transport in Escherichia coli K12. J Gen Microbiol. 1983 Feb;129(2):357–358. doi: 10.1099/00221287-129-2-357. [DOI] [PubMed] [Google Scholar]
- 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]
- Phadnis S. H., Huang H. V., Berg D. E. Tn5supF, a 264-base-pair transposon derived from Tn5 for insertion mutagenesis and sequencing DNAs cloned in phage lambda. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5908–5912. doi: 10.1073/pnas.86.15.5908. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Phadnis S. H., Kulakauskas S., Krishnan B. R., Hiemstra J., Berg D. E. Transposon Tn5supF-based reverse genetic method for mutational analysis of Escherichia coli with DNAs cloned in lambda phage. J Bacteriol. 1991 Jan;173(2):896–899. doi: 10.1128/jb.173.2.896-899.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Russel M., Model P., Holmgren A. Thioredoxin or glutaredoxin in Escherichia coli is essential for sulfate reduction but not for deoxyribonucleotide synthesis. J Bacteriol. 1990 Apr;172(4):1923–1929. doi: 10.1128/jb.172.4.1923-1929.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Satishchandran C., Markham G. D. Adenosine-5'-phosphosulfate kinase from Escherichia coli K12. Purification, characterization, and identification of a phosphorylated enzyme intermediate. J Biol Chem. 1989 Sep 5;264(25):15012–15021. [PubMed] [Google Scholar]
- Singer M., Baker T. A., Schnitzler G., Deischel S. M., Goel M., Dove W., Jaacks K. J., Grossman A. D., Erickson J. W., Gross C. A. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol Rev. 1989 Mar;53(1):1–24. doi: 10.1128/mr.53.1.1-24.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sirko A., Hryniewicz M., Hulanicka D., Böck A. Sulfate and thiosulfate transport in Escherichia coli K-12: nucleotide sequence and expression of the cysTWAM gene cluster. J Bacteriol. 1990 Jun;172(6):3351–3357. doi: 10.1128/jb.172.6.3351-3357.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tsang M. L. Assimilatory sulfate reduction in Escherichia coli: identification of the alternate cofactor for adenosine 3'-phosphate 5'-phosphosulfate reductase as glutaredoxin. J Bacteriol. 1981 Jun;146(3):1059–1066. doi: 10.1128/jb.146.3.1059-1066.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tsang M. L., Schiff J. A. Sulfate-reducing pathway in Escherichia coli involving bound intermediates. J Bacteriol. 1976 Mar;125(3):923–933. doi: 10.1128/jb.125.3.923-933.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tyler B. Regulation of the assimilation of nitrogen compounds. Annu Rev Biochem. 1978;47:1127–1162. doi: 10.1146/annurev.bi.47.070178.005403. [DOI] [PubMed] [Google Scholar]
- 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]
- Wanner B. L. Novel regulatory mutants of the phosphate regulon in Escherichia coli K-12. J Mol Biol. 1986 Sep 5;191(1):39–58. doi: 10.1016/0022-2836(86)90421-3. [DOI] [PubMed] [Google Scholar]
- Yano R., Nagai H., Shiba K., Yura T. A mutation that enhances synthesis of sigma 32 and suppresses temperature-sensitive growth of the rpoH15 mutant of Escherichia coli. J Bacteriol. 1990 Apr;172(4):2124–2130. doi: 10.1128/jb.172.4.2124-2130.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]