Characterization of a Corynebacterium glutamicum Lactate Utilization Operon Induced during Temperature-Triggered Glutamate Production

ABSTRACT Gene expression changes of glutamate-producing Corynebacterium glutamicum were identified in transcriptome comparisons by DNA microarray analysis. During glutamate production induced by a temperature shift, C. glutamicum strain 2262 showed significantly higher mRNA levels of the NCgl2816 and NCgl2817 genes than its non-glutamate-producing derivative 2262NP. Reverse transcription-PCR analysis showed that the two genes together constitute an operon. NCgl2816 putatively codes for a lactate permease, while NCgl2817 was demonstrated to encode quinone-dependent l-lactate dehydrogenase, which was named LldD. C. glutamicum LldD displayed Michaelis-Menten kinetics for the substrate l-lactate with a Km of about 0.51 mM. The specific activity of LldD was about 10-fold higher during growth on l-lactate or on an l-lactate-glucose mixture than during growth on glucose, d-lactate, or pyruvate, while the specific activity of quinone-dependent d-lactate dehydrogenase differed little with the carbon source. RNA levels of NCgl2816 and lldD were about 18-fold higher during growth on l-lactate than on pyruvate. Disruption of the NCgl2816-lldD operon resulted in loss of the ability to utilize l-lactate as the sole carbon source. Expression of lldD restored l-lactate utilization, indicating that the function of the permease gene NCgl2816 is dispensable, while LldD is essential, for growth of C. glutamicum on l-lactate.

[1]  S. Udaka,et al.  STUDIES ON THE AMINO ACID FERMENTATION , 1957 .

[2]  S. Udaka SCREENING METHOD FOR MICROORGANISMS ACCUMULATING METABOLITES AND ITS USE IN THE ISOLATION OF MICROCOCCUS GLUTAMICUS , 1960, Journal of bacteriology.

[3]  F. Lara,et al.  The lactic dehydrogenase of Propionibacterium pentosaceum. , 1960, The Biochemical journal.

[4]  I. Shiio,et al.  Cellular permeability and extracellular formation of glutamic acid in Brevibacterium flavum. , 1963, Journal of biochemistry.

[5]  K. Aida,et al.  Studies on Amino Acid Fermentation , 1965 .

[6]  K. Takinami Control of L-glutamic acid fermentation by biotin and fatty acid , 1968 .

[7]  G. G. Pritchard An NAD + -independent L-lactate dehydrogenase from Rhizopus oryzae. , 1971, Biochimica et biophysica acta.

[8]  D. Hanahan Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

[9]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[10]  C. Hoischen,et al.  Membrane alteration is necessary but not sufficient for effective glutamate secretion in Corynebacterium glutamicum , 1990, Journal of bacteriology.

[11]  F. S. Mathews,et al.  Molecular structure of flavocytochrome b2 at 2.4 A resolution. , 1990, Journal of molecular biology.

[12]  C. Hoischen,et al.  Carrier-mediated glutamate secretion by Corynebacterium glutamicum under biotin limitation. , 1992, Biochimica et biophysica acta.

[13]  E. Lin,et al.  Three overlapping lct genes involved in L-lactate utilization by Escherichia coli , 1993, Journal of bacteriology.

[14]  H. Sahm,et al.  Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon , 1993, Journal of bacteriology.

[15]  Bernhard J. Eikmanns,et al.  Phosphoenolpyruvate carboxylase in Corynebacterium glutamicum is dispensable for growth and lysine production , 1993 .

[16]  V. Massey,et al.  Lactate monooxygenase. I. Expression of the mycobacterial gene in Escherichia coli and site-directed mutagenesis of lysine 266. , 1994, The Journal of biological chemistry.

[17]  J. Kalinowski,et al.  Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. , 1994, Gene.

[18]  H. Sahm,et al.  Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase. , 1994, Microbiology.

[19]  Michael A. Quail,et al.  Purification, characterization and mode of action of PdhR, the transcriptional repressor of the pdhR–aceEF–Ipd operon of Escherichia coli , 1995, Molecular microbiology.

[20]  M. Quail,et al.  Purification, characterization and mode of action of PdhR, the transcriptional repressor of the pdhR-aceEF-lpd operon of Escherichia coli. , 1995, Molecular microbiology.

[21]  C. Lambert,et al.  Triggering Glutamate Excretion in Corynebacterium glutamicum by Modulating the Membrane State with Local Anesthetics and Osmotic Gradients , 1995, Applied and environmental microbiology.

[22]  A. D. de Graaf,et al.  Response of the central metabolism of Corynebacterium glutamicum to different flux burdens. , 1997, Biotechnology and bioengineering.

[23]  S. Delaunay,et al.  An improved temperature-triggered process for glutamate production with Corynebacterium glutamicum , 1999 .

[24]  E. Kimura,et al.  Glutamate Overproduction in Corynebacterium glutamicum Triggered by a Decrease in the Level of a Complex Comprising DtsR and a Biotin-containing Subunit. , 1999, Bioscience, biotechnology, and biochemistry.

[25]  J. Badia,et al.  The gene yghK linked to the glc operon of Escherichia coli encodes a permease for glycolate that is structurally and functionally similar to L-lactate permease. , 2001, Microbiology.

[26]  H. Sahm,et al.  Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum. , 2001, Journal of molecular microbiology and biotechnology.

[27]  S. Atkinson,et al.  Elevated Intact Parathyroid Hormone Is Associated with Reduced Biochemical Markers of Bone Formation and Resorption Measured in Blood in Infant Piglets Receiving Oral Dexamethasone for 15 Days , 2001, Neonatology.

[28]  H. Sahm,et al.  L-glutamate efflux with Corynebacterium glutamicum: why is penicillin treatment or Tween addition doing the same? , 2001, Journal of molecular microbiology and biotechnology.

[29]  R. Benz,et al.  Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane. , 2001, Microbiology.

[30]  J. Aguilar,et al.  Transport of L-Lactate, D-Lactate, and glycolate by the LldP and GlcA membrane carriers of Escherichia coli. , 2002, Biochemical and biophysical research communications.

[31]  J. Kalinowski,et al.  Efficient Electrotransformation of Corynebacterium diphtheriae with a Mini-Replicon Derived from the Corynebacterium glutamicum Plasmid pGA1 , 2002, Current Microbiology.

[32]  H. Sahm,et al.  The Phosphate Starvation Stimulon of Corynebacterium glutamicum Determined by DNA Microarray Analyses , 2003, Journal of bacteriology.

[33]  A. Goesmann,et al.  The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins. , 2003, Journal of biotechnology.

[34]  M. Bott,et al.  The respiratory chain of Corynebacterium glutamicum. , 2003, Journal of biotechnology.

[35]  H. Sahm,et al.  Global Expression Profiling and Physiological Characterization of Corynebacterium glutamicum Grown in the Presence of l-Valine , 2003, Applied and Environmental Microbiology.

[36]  Volker F Wendisch,et al.  Acetate metabolism and its regulation in Corynebacterium glutamicum. , 2003, Journal of biotechnology.

[37]  V. Wendisch Genome-wide expression analysis in Corynebacterium glutamicum using DNA microarrays. , 2003, Journal of biotechnology.

[38]  D. Uy,et al.  Instability of glutamate production by Corynebacterium glutamicum 2262 in continuous culture using the temperature-triggered process. , 2003, Journal of biotechnology.

[39]  T. Hermann Industrial production of amino acids by coryneform bacteria. , 2003, Journal of biotechnology.

[40]  V. Wendisch,et al.  Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays , 2004, Applied biochemistry and biotechnology.

[41]  M. Inui,et al.  Metabolic Analysis of Corynebacterium glutamicum during Lactate and Succinate Productions under Oxygen Deprivation Conditions , 2004, Journal of Molecular Microbiology and Biotechnology.

[42]  K. Matsushita,et al.  Effect of NADH dehydrogenase-disruption and over-expression on respiration-related metabolism in Corynebacterium glutamicum KY9714 , 2004, Applied Microbiology and Biotechnology.

[43]  P. Daran-Lapujade,et al.  Glutamate as an inhibitor of phosphoenolpyruvate carboxylase activity in Corynebacterium glutamicum , 2004, Journal of Industrial Microbiology and Biotechnology.

[44]  B. Eikmanns,et al.  RamB, a Novel Transcriptional Regulator of Genes Involved in Acetate Metabolism of Corynebacterium glutamicum , 2004, Journal of bacteriology.

[45]  S. Udaka,et al.  Studies on the amino acid fermentation. Part 1. Production of L-glutamic acid by various microorganisms. , 2004, The Journal of general and applied microbiology.

[46]  V. Wendisch,et al.  Identification of AcnR, a TetR-type Repressor of the Aconitase Gene acn in Corynebacterium glutamicum* , 2005, Journal of Biological Chemistry.

[47]  G. Besra,et al.  Ethambutol, a cell wall inhibitor of Mycobacterium tuberculosis, elicits L-glutamate efflux of Corynebacterium glutamicum. , 2005, Microbiology.

[48]  F. Fischer,et al.  Mapping the membrane proteome of Corynebacterium glutamicum , 2005, Proteomics.

[49]  Genomic analyses of transporter proteins in Corynebacterium glutamicum and Corynebacterium efficiens , 2005 .