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. 1994 Oct;176(19):6120–6126. doi: 10.1128/jb.176.19.6120-6126.1994

The Calvin cycle enzyme phosphoglycerate kinase of Xanthobacter flavus required for autotrophic CO2 fixation is not encoded by the cbb operon.

W G Meijer 1
PMCID: PMC196832  PMID: 7928974

Abstract

During autotrophic growth of Xanthobacter flavus, energy derived from the oxidation of hydrogen methanol or formate is used to drive the assimilation of CO2 via the Calvin cycle. The genes encoding the Calvin cycle enzymes are organized in the cbb operon, which is expressed only during autotrophic growth. Although it has been established that the transcriptional activator CbbR is required for the expression of the cbb operon, it is unclear whether CbbR is the only factor contributing to the regulation of the cbb operon. This paper describes the isolation of X. flavus mutants which were affected in the regulation of the cbb operon. One of the mutant strains was subject to an enhanced repression of the cbb operon promoter by the gluconeogenic substrate succinate and in addition failed to grow autotrophically. The rate of growth of the X. flavus mutant on succinate-containing medium was lower than that of the wild-type strain, but rates of growth on medium supplemented with gluconate were identical. A genomic library of X. flavus was constructed and was used to complement the mutant strain. The nucleotide sequence of the DNA fragment required to restore autotrophic growth of the X. flavus mutant was determined. One open reading frame that displayed extensive similarities to phosphoglycerate kinase-encoding genes (pgk) was identified. The X. flavus mutant lacked phosphoglycerate kinase activity following growth on gluconate or succinate. Introduction of the pgk gene into the X. flavus mutant partially restored the activity of phosphoglycerate kinase. Induction of the cbb operon of the X. flavus wild-type strain resulted in a simultaneous and parallel increase in the activities of ribulose-1,5-biphosphate carboxylase and phosphoglycerate kinase, whereas the latter activity remained absent in the X. flavus pgk mutant. It is concluded that X. flavus employees a single phosphoglycerate kinase enzyme and this is not encoded within the cbb operon.

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Selected References

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

  1. Alefounder P. R., Perham R. N. Identification, molecular cloning and sequence analysis of a gene cluster encoding the class II fructose 1,6-bisphosphate aldolase, 3-phosphoglycerate kinase and a putative second glyceraldehyde 3-phosphate dehydrogenase of Escherichia coli. Mol Microbiol. 1989 Jun;3(6):723–732. doi: 10.1111/j.1365-2958.1989.tb00221.x. [DOI] [PubMed] [Google Scholar]
  2. Andersen K., Wilke-Douglas M. Genetic and physical mapping and expression in Pseudomonas aeruginosa of the chromosomally encoded ribulose bisphosphate carboxylase genes of Alcaligenes eutrophus. J Bacteriol. 1987 May;169(5):1997–2004. doi: 10.1128/jb.169.5.1997-2004.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Chen J. H., Gibson J. L., McCue L. A., Tabita F. R. Identification, expression, and deduced primary structure of transketolase and other enzymes encoded within the form II CO2 fixation operon of Rhodobacter sphaeroides. J Biol Chem. 1991 Oct 25;266(30):20447–20452. [PubMed] [Google Scholar]
  6. Conway T., Ingram L. O. Phosphoglycerate kinase gene from Zymomonas mobilis: cloning, sequencing, and localization within the gap operon. J Bacteriol. 1988 Apr;170(4):1926–1933. doi: 10.1128/jb.170.4.1926-1933.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cooper R. A. Metabolism of methylglyoxal in microorganisms. Annu Rev Microbiol. 1984;38:49–68. doi: 10.1146/annurev.mi.38.100184.000405. [DOI] [PubMed] [Google Scholar]
  8. Davies G. J., Littlechild J. A., Watson H. C., Hall L. Sequence and expression of the gene encoding 3-phosphoglycerate kinase from Bacillus stearothermophilus. Gene. 1991 Dec 20;109(1):39–45. doi: 10.1016/0378-1119(91)90586-z. [DOI] [PubMed] [Google Scholar]
  9. Dijkhuizen L., Harder W. Current views on the regulation of autotrophic carbon dioxide fixation via the Calvin cycle in bacteria. Antonie Van Leeuwenhoek. 1984;50(5-6):473–487. doi: 10.1007/BF02386221. [DOI] [PubMed] [Google Scholar]
  10. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gibson J. L., Falcone D. L., Tabita F. R. Nucleotide sequence, transcriptional analysis, and expression of genes encoded within the form I CO2 fixation operon of Rhodobacter sphaeroides. J Biol Chem. 1991 Aug 5;266(22):14646–14653. [PubMed] [Google Scholar]
  12. Gibson J. L., Tabita F. R. Different molecular forms of D-ribulose-1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides. J Biol Chem. 1977 Feb 10;252(3):943–949. [PubMed] [Google Scholar]
  13. Gibson J. L., Tabita F. R. Localization and mapping of CO2 fixation genes within two gene clusters in Rhodobacter sphaeroides. J Bacteriol. 1988 May;170(5):2153–2158. doi: 10.1128/jb.170.5.2153-2158.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gibson J. L., Tabita F. R. Organization of phosphoribulokinase and ribulose bisphosphate carboxylase/oxygenase genes in Rhodopseudomonas (Rhodobacter) sphaeroides. J Bacteriol. 1987 Aug;169(8):3685–3690. doi: 10.1128/jb.169.8.3685-3690.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hallenbeck P. L., Kaplan S. Cloning of the gene for phosphoribulokinase activity from Rhodobacter sphaeroides and its expression in Escherichia coli. J Bacteriol. 1987 Aug;169(8):3669–3678. doi: 10.1128/jb.169.8.3669-3678.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  17. Im D. S., Friedrich C. G. Fluoride, hydrogen, and formate activate ribulosebisphosphate carboxylase formation in Alcaligenes eutrophus. J Bacteriol. 1983 May;154(2):803–808. doi: 10.1128/jb.154.2.803-808.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Irani M. H., Maitra P. K. Properties of Escherichia coli mutants deficient in enzymes of glycolysis. J Bacteriol. 1977 Nov;132(2):398–410. doi: 10.1128/jb.132.2.398-410.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Keen N. T., Tamaki S., Kobayashi D., Trollinger D. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene. 1988 Oct 15;70(1):191–197. doi: 10.1016/0378-1119(88)90117-5. [DOI] [PubMed] [Google Scholar]
  20. Knauf V. C., Nester E. W. Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid. 1982 Jul;8(1):45–54. doi: 10.1016/0147-619x(82)90040-3. [DOI] [PubMed] [Google Scholar]
  21. Kossmann J., Klintworth R., Bowien B. Sequence analysis of the chromosomal and plasmid genes encoding phosphoribulokinase from Alcaligenes eutrophus. Gene. 1989 Dec 21;85(1):247–252. doi: 10.1016/0378-1119(89)90490-3. [DOI] [PubMed] [Google Scholar]
  22. Kusian B., Yoo J. G., Bednarski R., Bowien B. The Calvin cycle enzyme pentose-5-phosphate 3-epimerase is encoded within the cfx operons of the chemoautotroph Alcaligenes eutrophus. J Bacteriol. 1992 Nov;174(22):7337–7344. doi: 10.1128/jb.174.22.7337-7344.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lehmicke L. G., Lidstrom M. E. Organization of genes necessary for growth of the hydrogen-methanol autotroph Xanthobacter sp. strain H4-14 on hydrogen and carbon dioxide. J Bacteriol. 1985 Jun;162(3):1244–1249. doi: 10.1128/jb.162.3.1244-1249.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Levering P. R., van Dijken J. P., Veenhius M., Harder W. Arthrobacter P1, a fast growing versatile methylotroph with amine oxidase as a key enzyme in the metabolism of methylated amines. Arch Microbiol. 1981 Mar;129(1):72–80. doi: 10.1007/BF00417184. [DOI] [PubMed] [Google Scholar]
  25. Longstaff M., Raines C. A., McMorrow E. M., Bradbeer J. W., Dyer T. A. Wheat phosphoglycerate kinase: evidence for recombination between the genes for the chloroplastic and cytosolic enzymes. Nucleic Acids Res. 1989 Aug 25;17(16):6569–6580. doi: 10.1093/nar/17.16.6569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Meijer W. G., Arnberg A. C., Enequist H. G., Terpstra P., Lidstrom M. E., Dijkhuizen L. Identification and organization of carbon dioxide fixation genes in Xanthobacter flavus H4-14. Mol Gen Genet. 1991 Feb;225(2):320–330. doi: 10.1007/BF00269865. [DOI] [PubMed] [Google Scholar]
  27. Meijer W. G., Croes L. M., Jenni B., Lehmicke L. G., Lidstrom M. E., Dijkhuizen L. Characterization of Xanthobacter strains H4-14 and 25a and enzyme profiles after growth under autotrophic and heterotrophic conditions. Arch Microbiol. 1990;153(4):360–367. doi: 10.1007/BF00249006. [DOI] [PubMed] [Google Scholar]
  28. Meijer W. G., Dijkhuizen L. Regulation of autotrophic metabolism in Pseudomonas oxalaticus OX1 wild-type and an isocitrate-lyase-deficient mutant. J Gen Microbiol. 1988 Dec;134(12):3231–3237. doi: 10.1099/00221287-134-12-3231. [DOI] [PubMed] [Google Scholar]
  29. Meijer W. G., Enequist H. G., Terpstra P., Dijkhuizen L. Nucleotide sequences of the genes encoding fructosebisphosphatase and phosphoribulokinase from Xanthobacter flavus H4-14. J Gen Microbiol. 1990 Nov;136(11):2225–2230. doi: 10.1099/00221287-136-11-2225. [DOI] [PubMed] [Google Scholar]
  30. Muller E. D., Chory J., Kaplan S. Cloning and characterization of the gene product of the form II ribulose-1,5-bisphosphate carboxylase gene of Rhodopseudomonas sphaeroides. J Bacteriol. 1985 Jan;161(1):469–472. doi: 10.1128/jb.161.1.469-472.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  32. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  33. Quivey R. G., Jr, Tabita F. R. Cloning and expression in Escherichia coli of the form II ribulose 1,5-bisphosphate carboxylase/oxygenase gene from Rhodopseudomonas sphaeroides. Gene. 1984 Nov;31(1-3):91–101. doi: 10.1016/0378-1119(84)90198-7. [DOI] [PubMed] [Google Scholar]
  34. Reutz I., Schobert P., Bowien B. Effect of phosphoglycerate mutase deficiency on heterotrophic and autotrophic carbon metabolism of Alcaligenes eutrophus. J Bacteriol. 1982 Jul;151(1):8–14. doi: 10.1128/jb.151.1.8-14.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schäferjohann J., Yoo J. G., Kusian B., Bowien B. The cbb operons of the facultative chemoautotroph Alcaligenes eutrophus encode phosphoglycolate phosphatase. J Bacteriol. 1993 Nov;175(22):7329–7340. doi: 10.1128/jb.175.22.7329-7340.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tabita F. R., Gibson J. L., Bowien B., Dijkhuizen L., Meijer W. G. Uniform designation for genes of the Calvin-Benson-Bassham reductive pentose phosphate pathway of bacteria. FEMS Microbiol Lett. 1992 Dec 1;78(2-3):107–110. doi: 10.1111/j.1574-6968.1992.tb05551.x. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Zimmermann J., Voss H., Schwager C., Stegemann J., Erfle H., Stucky K., Kristensen T., Ansorge W. A simplified protocol for fast plasmid DNA sequencing. Nucleic Acids Res. 1990 Feb 25;18(4):1067–1067. doi: 10.1093/nar/18.4.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. van den Bergh E. R., Dijkhuizen L., Meijer W. G. CbbR, a LysR-type transcriptional activator, is required for expression of the autotrophic CO2 fixation enzymes of Xanthobacter flavus. J Bacteriol. 1993 Oct;175(19):6097–6104. doi: 10.1128/jb.175.19.6097-6104.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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