Characterization of a Corynebacterium glutamicum lactate utilization operon induced during temperature-triggered glutamate production - PubMed (original) (raw)

Characterization of a Corynebacterium glutamicum lactate utilization operon induced during temperature-triggered glutamate production

Corinna Stansen et al. Appl Environ Microbiol. 2005 Oct.

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 K(m) 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.

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Figures

FIG. 1.

FIG. 1.

Continuous culture of glutamate-producing C. glutamicum 2262 (A and B) and its non-glutamate-producing mutant 2262NP (C and D) at 39°C and a dilution rate of 0.05 h−1. (A and C) Optical densities at 570 nm (▴); (B and D) intracellular (×) and extracellular (⧫) glutamate concentrations. Arrows indicate the time when both the continuous supplementation of medium and the temperature increase from 33 to 39°C were imposed. The pH was maintained at 7.

FIG. 2.

FIG. 2.

l

-Lactate concentrations in supernatants (○) and specific activities of quinone-dependent

l

-lactate dehydrogenase (▪) in crude extracts of glutamate-producing C. glutamicum 2262 (A) and of its non-glutamate-producing mutant 2262NP (B) during continuous culture. Samples analyzed were taken from the continuous cultures described in the legend to Fig. 1.

FIG. 3.

FIG. 3.

Substrate dependence of C. glutamicum WT(pEKEx3-lldD) quinone-dependent

l

-lactate dehydrogenase. Shown are specific activities of quinone-dependent

l

-lactate dehydrogenase in crude extracts from C. glutamicum WT(pEKEx3-lldD) with varying concentrations of the substrate

l

-lactate. (Inset) Double-reciprocal plot of the data.

FIG. 4.

FIG. 4.

Transcriptional organization of the NCgl2816_-lldD_ locus in C. glutamicum analyzed by RT-PCR. (A) Scheme showing the NCgl2816_-lldD_ locus in C. glutamicum and the RT-PCRs used to determine cotranscription of NCgl2816 and lldD. RNA from wild-type C. glutamicum was transcribed into cDNA with two different primers in the two separate reverse transcriptase reactions cDNA-A and cDNA-B. Subsequently, these cDNAs were used as templates for the PCRs labeled 1 to 6. (B) Results from the RT-PCR analyses described above. The lower DNA fragment visible in lanes 1 to 6 represents dnaE, and RT-PCR of dnaE served as a positive control in all reactions. The upper bands correspond to the products of PCRs 1 to 6, diagramed in panel A. Lanes 7 to 12 represent control reactions confirming the absence of DNA in the RNA preparation. The reactions were identical to PCRs 1 to 6 (for which results are shown in lanes 1 to 6, respectively) except that reverse transcriptase was omitted in reactions cDNA-A and cDNA-B.

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