Transcriptome, Proteome, and Metabolite Analyses of a Lactate Dehydrogenase-Negative Mutant of Enterococcus faecalis V583

Abstract

A constructed lactate dehydrogenase (LDH)-negative mutant of Enterococcus faecalis V583 grows at the same rate as the wild type but ferments glucose to ethanol, formate, and acetoin. Microarray analysis showed that LDH deficiency had profound transcriptional effects: 43 genes in the mutant were found to be upregulated, and 45 were found to be downregulated. Most of the upregulated genes encode enzymes of energy metabolism or transport. By two-dimensional (2D) gel analysis, 45 differentially expressed proteins were identified. A comparison of transcriptomic and proteomic data suggested that for several proteins the level of expression is regulated beyond the level of transcription. Pyruvate catabolic genes, including the truncated ldh gene, showed highly increased transcription in the mutant. These genes, along with a number of other differentially expressed genes, are preceded by sequences with homology to binding sites for the global redox-sensing repressor, Rex, of Staphylococcus aureus. The data indicate that the genes are transcriptionally regulated by the NADH/ NAD ratio and that this ratio plays an important role in the regulatory network controlling energy metabolism in E. faecalis.Lactic acid bacteria (LAB) are widely used for production of lactic acid in fermented food. During the fermentation process, pyruvate is converted to lactate in addition to a number of minor metabolites, such as acetic acid, acetaldehyde, ethanol, acetoin, and acetate. However, under certain conditions, these bacteria shift from homolactic to heterolactic (or mixed-acid) fermentation, with formate, acetate, acetoin, ethanol, and CO 2 as end products. In Lactococcus lactis, mixed-acid fermentation has been shown to take place at low grow rates under microaerobic conditions (11), under true carbon-limited conditions, and while growing at low pH on carbon sources other than glucose (15,20).Mixed-acid fermentation was also seen after removing the lactate dehydrogenase (LDH) activity in Enterococcus faecalis V583 (12). This bacterium has two ldh genes, but ldh-1 is the main contributor to lactate production. A mutant with deletions in both ldh genes (the ⌬ldh1.2 mutant) was constructed and shown to direct its carbon flow from pyruvate away from lactate toward formate, acetoin, and alcohol production (12). Alternative carbon fluxes in different knockout mutants have also been reported for Lactococcus lactis (22).The mechanism of the shift from homolactic to mixed-acid fermentation is still not fully understood. During transformation of pyruvate to lactate, LDH regenerates NAD ϩ from NADH formed during glycolysis. When pyruvate is converted to acetyl-coenzyme A (acetyl-CoA) by either pyruvate formate lyase (PFL) or pyruvate dehydrogenase (PDH), reduction of acetyl-CoA to ethanol regenerates NAD ϩ from NADH and is an alternative to lactate formation in redox balancing. The carbon flux is biochemically regulated (4, 5). Fructose-1,6-bisphosphate is an allosteric activator of lactate production, and dihydroxyacetone phosphate and D-glyce...

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“…3D). It is suggested that formic acid-producing strains could convert to heterolactic (or mixed-acid) fermentation under certain conditions as reported by Mehmeti et al (2011). In addition, the diverse physiological and biochemical properties represent the degree of phenotypic diversity existing within the species.…”

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“…Comparative proteomic and transcriptomic and metabolic analyses showed that the pyruvate catabolic genes, including the truncated ldh gene, showed highly increased transcription in the mutant., and that protein expression was regulated beyond the level of transcription, as suggested by Mehmeti and colleagues (2011), who also demonstrated that these genes, along with a number of other differentially expressed genes, are preceded by sequences with homology to binding sites for the global redox-sensing repressor, Rex, of Staphylococcus aureus. The results of this research demonstrates that the NADH/NAD ratio is involved in the transcriptional regulation of these genes, thus having a critical role in the regulatory network controlling energy metabolism in E. faecalis (Mehmeti et al, 2011).…”

“…This has already been demonstrated in L. lactis, where enzyme levels have been found to be regulated in response to growth conditions, and correlations between metabolic and transcriptomic or proteomic data have been established (Dressaire et al, 2009;Mehmeti et al, 2011). In this latest research, Mehmeti and colleagues (2011) compared a constructed lactate dehydrogenase (LDH)-negative mutant of E. faecalis V583 (ldh1.2) with its wild type via metabolic, transcriptomic, and proteomic analyses.…”

“…Mixed-acid fermentation has also been observed in E. faecalis V583, but only after removal of the lactate dehydrogenase (LDH) activity (Jonsson, et al, 2009)). This emerging pathogen has two ldh genes, but it is the ldh-1 gene which is the main contributor to lactate production (Mehmeti et al, 2011).…”

“…During homolactic fermentation in LAB, pyruvate is converted to lactate in addition to a number of minor metabolites, such as acetic acid, acetaldehyde, ethanol, acetoin, and acetate, but, under certain environmental conditions, metabolism switches to heterolactic fermentation (mixedacid producing formate, acetate, acetoin, ethanol, and CO 2 as the final products (Mehmeti et al, 2011). An example of such a situation was observed in Lactococcus lactis, where mixedacid fermentation has been shown to occur at low growth rates under microaerobic conditions (Jensen et al, 2001;Mehmeti et al, 2011), under true carbon-limited conditions, and while growing at low pH on carbon sources other than glucose (Melchiorsen et al, 2002;Mehmeti et al, 2011)). Mixed-acid fermentation has also been observed in E. faecalis V583, but only after removal of the lactate dehydrogenase (LDH) activity (Jonsson, et al, 2009)).…”