Abstract
The Cgl1427 gene was previously found to be relevant to the microaerobic growth of Corynebacterium glutamicum (Ikeda et al. Biosci Biotechnol Biochem 73:2806–2808, 2009). In the present work, Cgl1427 was identified as a cytidylate kinase gene (cmk) by homology analysis of its deduced amino acid sequence with that of other bacterial cytidylate kinases (CMP kinases) and on the basis of findings that deletion of Cgl1427 results in loss of CMP kinase activity. Deletion of the cmk gene significantly impaired the growth of C. glutamicum in oxygen-limiting static culture, and the impaired growth was restored by introducing a plasmid containing the cmk gene, suggesting that this gene plays an important role in the microaerobic growth of C. glutamicum. On the other hand, in the main culture with aerobic shaking, a prolonged lag phase was observed in the cmk disruptant, despite an unchanged growth rate, compared to the behavior of the wild-type strain. The prolongation was observed when using seed culture grown to later growth stages in which oxygen limitation occurred, but it was not observed when using seed culture grown to an earlier growth stage in which oxygen remained relatively plentiful. Since nucleotide biosynthesis in C. glutamicum requires oxygen, we hypothesized that the ability of the cmk disruptant to synthesize nucleotides was influenced by oxygen limitation in the later growth stages of the seed culture, which caused the prolongation of the lag phase in the following shaken culture. To verify this hypothesis, a plasmid containing genes encoding all components of a homologous ribonucleotide reductase, a key enzyme for nucleotide synthesis that requires oxygen for its reaction, was introduced into the cmk disruptant, which significantly ameliorated the lag phase prolongation. Furthermore, this experimental setup almost completely restored the growth of the cmk disruptant in the oxygen-limiting static culture. These results indicate that CMP kinase plays an important role in normal nucleotide biosynthesis under an oxygen-limiting environment.
Similar content being viewed by others
References
Blondin C, Serina L, Wiesmuller L, Gilles AM, Barzu O (1994) Improved spectrophotometric assay of nucleoside monophosphate kinase activity using the pyruvate kinase/lactate dehydrogenase coupling system. Anal Biochem 220:219–221
Brejning J, Jespersen L, Arneborg N (2003) Genome-wide translational changes during the lag phase of Saccharomyces cerevisiae. Arch Microbiol 179:278–294
Chien LJ, Chen HT, Yang PF, Lee CK (2006) Enhancement of cellulose pellicle production by constitutively expressing Vitreoscilla hemoglobin in Acetobacter xylinum. Biotechnol Prog 22:1598–1603
Climploy K, Tassotto ML, Mathews CK (2000) Ribonucleotide reductase, a possible agent in deoxyribonucleotide pool asymmetries induced by hypoxia. J Biol Chem 275:39267–39271
Cotruvo JA Jr, Stubbe J (2010) An active dimanganese(III)-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase. Biochemistry 49:1297–1309
Favaro R, Deho G (2003) Polynucleotide phosphorylase-deficient mutants of Pseudomonas putida. J Bacteriol 185:5279–5286
Folch-Mallol JL, Martinez LM, Casas SJ, Yang R, Martinez-Anaya C, Lopez L, Hernandez A, Nieto-Sotelo J (2004) New roles for CDC25 in growth control, galactose regulation and cellular differentiation in Saccharomyces cerevisiae. Microbiology 150:2865–2879
Fricke J, Neuhard J, Kelln RA, Pedersen S (1995) The cmk gene encoding cytidine monophosphate kinase is located in the rpsA operon and is required for normal replication rate in Escherichia coli. J Bacteriol 177:517–523
Hofer A, Crona M, Logan DT, Sjoberg BM (2012) DNA building blocks: keeping control of manufacture. Crit Rev Biochem Mol Biol 47:50–63
Horng YT, Chang KC, Chien CC, Wei YH, SunYM Soo PC (2010) Enhanced polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose P-BAD promoter in Escherichia coli. Lett Appl Microbiol 50:158–167
Ikeda M (2003) Amino acid production processes. In: Faurie R, Thommel J (eds) Microbial production of l-amino acids. Advances in biochemical engineering/biotechnology, vol 79. Springer, Berlin, pp 1–35
Ikeda M, Baba M, Tsukamoto N, Komatsu T, Mitsuhashi S, Takeno S (2009) Elucidation of genes relevant to the microaerobic growth of Corynebacterium glutamicum. Biosci Biotechnol Biochem 73:2806–2808
Ikeda M, Nakagawa S (2003) The Corynebacterium glutamicum genome: features and impacts on biotechnological processes. Appl Microbiol Biotechnol 62:99–109
Kinoshita S, Nakayama K (1978) Amino acids. In: Rose AH (ed) Primary products of metabolism. Academic, London, pp 209–261
Koistinen KM, Plumed-Ferrer C, Lehesranta SJ, Karenlampi SO, von Wright A (2007) Comparison of growth-phase-dependent cytosolic proteomes of two Lactobacillus plantarum strains used in food and feed fermentations. FEMS Microbiol Lett 273:12–21
Kolberg M, Strand KR, Graff P, Andersson KK (2004) Structure, function, and mechanism of ribonucleotide reductases. Biochim Biophys Acta 1699:1–34
Larsen N, Boye M, Siegumfeldt H, Jakobsen M (2006) Differential expression of proteins and genes in the lag phase of Lactococcus lactis subsp. lactis grown in synthetic medium and reconstituted skim milk. Appl Environ Microbiol 72:1173–1179
Li M, Wu J, Lin J, Wei D (2010) Expression of Vitreoscilla hemoglobin enhances cell growth and dihydroxyacetone production in Gluconobacter oxydans. Curr Microbiol 61:370–375
Liu Q, Zhang J, Wei XX, Ouyang SP, Wu Q, Chen GQ (2008) Microbial production of l-glutamate and l-glutamine by recombinant Corynebacterium glutamicum harboring Vitreoscilla hemoglobin gene vgb. Appl Microbiol Biotechnol 77:1297–1304
Liu Y, Gong G, Xie L, Yuan N, Zhu C, Zhu B, Hu Y (2010) Improvement of cephalosporin C production by recombinant DNA integration in Acremonium chrysogenum. Mol Biotechnol 44:101–109
Mitsuhashi S, Ohnishi J, Hayashi M, Ikeda M (2004) A gene homologous to β-type carbonic anhydrase is essential for the growth Corynebacterium glutamicum under atmospheric conditions. Appl Microbiol Biotechnol 63:592–601
Oehlmann W, Auling G (1999) Ribonucleotide reductase (RNR) of Corynebacterium glutamicum ATCC 13032—genetic characterization of a second class IV enzyme. Microbiology 145:1595–1604
Ohnishi J, Mitsuhashi S, Hayashi M, Ando S, Yokoi H, Ochiai K, Ikeda M (2002) A novel methodology employing Corynebacterium glutamicum genome information to generate a new l-lysine-producing mutant. Appl Microbiol Biotechnol 58:217–223
Oka T (1999) Amino acids, production processes. In: Flickinger MC, Drew SW (eds) Encyclopedia of bioprocess technology: fermentation, biocatalysis, and bioseparation. Wiley, New York, pp 89–100
Priscila G, Fernandez FJ, Absalon AE, Suarez MR, Sainoz M, Barrios-Gonzalez J, Mejia A (2008) Expression of the bacterial hemoglobin gene from Vitreoscilla stercoraria increases rifamycin B production in Amycolatopsis mediterranei. J Biosci Bioeng 106:493–497
Saito H, Miura K (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619–629
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor, Cold Spring Harbor Laboratory
Schultz CP, Ylisastigui-Pons L, Serina L, Sakamoto H, Mantsch HH, Neuhard J, Barzu O, Gilles AM (1997) Structural and catalytic properties of CMP kinase from Bacillus subtilis: a comparative analysis with the homologous enzyme from Escherichia coli. Arch Biochem Biophys 340:144–153
Sjoberg BM (2010) A never-ending story. Science 329:1475–1476
Takeno S, Ohnishi J, Komatsu T, Masaki T, Sen K, Ikeda M (2007) Anaerobic growth and potential for amino acid production by nitrate respiration in Corynebacterium glutamicum. Appl Microbiol Biotechnol 75:1173–1182
Thum C, Schneider CZ, Palma MS, Santos DS, Basso LA (2009) The Rv1712 locus from Mycobacterium tuberculosis H37Rv codes for a functional CMP kinase that preferentially phosphorylates dCMP. J Bacteriol 191:2884–2887
Yukawa H, Omumasaba CA, Nonaka H, KosP Okai N, Suzuki N, Suda M, Tsuge Y, Watanabe J, Ikeda Y, Vertes AA, Inui M (2007) Comparative analysis of the Corynebacterium glutamicum group and complete genome sequence of strain R. Microbiology 153:1042–1058
Zhu H, Sun S, Zhang S (2011) Enhanced production of total flavones and exopolysaccharides via Vitreoscilla hemoglobin biosynthesis in Phellinus igniarius. Bioresour Technol 102:1747–1751
van der Rest ME, Lange C, Molenaar D (1999) A heat shock following electroporation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl Microbiol Biotechnol 52:541–545
Acknowledgments
We thank Y. Ueda for encouraging support of our work and also S. Hashimoto, S. Koizumi, and T. Ogawa for their useful discussion.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Takeno, S., Shirakura, D., Tsukamoto, N. et al. Significance of the Cgl1427 gene encoding cytidylate kinase in microaerobic growth of Corynebacterium glutamicum . Appl Microbiol Biotechnol 97, 1259–1267 (2013). https://doi.org/10.1007/s00253-012-4275-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-012-4275-x