Proteome analysis of a Lactococcus lactis strain overexpressing gapA suggests that the gene product is an auxiliary glyceraldehyde 3-phosphate dehydrogenase

Willemoës, Martin; Kilstrup, Mogens; Roepstorff, Peter; Hammer, Karin

doi:10.1002/1615-9861(200208)2:8<1041::aid-prot1041>3.0.co;2-7

Cited by 20 publications

(16 citation statements)

References 14 publications

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“…This does not appear to be the case for the two GAPDH proteins in L. lactis. Rather, it looks like gapA is an auxiliary GAPDH, expressed only under certain stress conditions, and that gapB is the main source of GAPDH activity in L. lactis growing under standard laboratory conditions (41). Our finding that lowering the expression of gapB almost abolishes GAPDH activity in L. lactis would fit this interpretation.…”

Section: Discussionsupporting

confidence: 71%

“…Since two gap genes (gapA and gapB) have been identified in L. lactis, it was also of interest to study whether the GAPDH activity encoded by gapA had control over the glycolytic flux. The gapA gene was recently found to encode an auxiliary GAPDH which is expressed only under certain stress conditions in L. lactis (41). We analyzed two strains with increased expression of gapA, but the resulting increase in GAPDH activity had no effect on the glycolytic flux in growing cells of MG1363 (Fig.…”

Section: Construction Of Strains With Modulated Gapdh Activitiesmentioning

confidence: 99%

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Glyceraldehyde-3-Phosphate Dehydrogenase Has No Control over Glycolytic Flux in Lactococcus lactis MG1363

2003

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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has previously been suggested to have almost absolute control over the glycolytic flux in Lactococcus lactis (B. Poolman, B. Bosman, J. Kiers, and W. N. Konings, J. Bacteriol. 169:5887-5890, 1987). Those studies were based on inhibitor titrations with iodoacetate, which specifically inhibits GAPDH, and the data suggested that it should be possible to increase the glycolytic flux by overproducing GAPDH activity. To test this hypothesis, we constructed a series of mutants with GAPDH activities from 14 to 210% of that of the reference strain MG1363. We found that the glycolytic flux was unchanged in the mutants overproducing GAPDH. Also, a decrease in the GAPDH activity had very little effect on the growth rate and the glycolytic flux until 25% activity was reached. Below this activity level, the glycolytic flux decreased proportionally with decreasing GAPDH activity. These data show that GAPDH activity has no control over the glycolytic flux (flux control coefficient ‫؍‬ 0.0) at the wild-type enzyme level and that the enzyme is present in excess capacity by a factor of 3 to 4. The early experiments by Poolman and coworkers were performed with cells resuspended in buffer, i.e., nongrowing cells, and we therefore analyzed the control by GAPDH under similar conditions. We found that the glycolytic flux in resting cells was even more insensitive to changes in the GAPDH activity; in this case GAPDH was also present in a large excess and had no control over the glycolytic flux.Lactic acid bacteria (LAB) are used extensively for the fermentation of food products, where the resulting acidification preserves the food and contributes to the texture and organoleptic quality. The major fermentation product of LAB is lactic acid, but depending on the actual LAB species and the conditions for growth, other products can be formed, which may contribute to the flavor of the fermented products. The regulation of by-products formed by LAB sugar metabolism has been studied extensively, mainly with Lactococcus lactis as the model organism. The work has focused on identification of key metabolites and mechanisms involved in regulating the switch between fermentation modes (7,8,12,13,14,15,18,28,30,39,40).Much less work has been performed with respect to investigating the control of the glycolytic flux in L. lactis. Andersen and colleagues used modulation of gene expression to show that lactate dehydrogenase has no control on the glycolytic flux in L. lactis MG1363 (1). Phosphofructokinase (PFK), on the other hand, appeared to be present in a very low excess, and even a small reduction of PFK activity resulted in an almost proportional decrease in the glycolytic flux (2). However, recent studies showed that PFK has no control over the glycolytic flux either (25; B. J. Koebmann et al., unpublished data). Poolman and coworkers used inhibition with iodoacetate to investigate the control by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in nongrowing cells of L. lactis subsp. cremoris strai...

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Section: Discussionsupporting

confidence: 71%

Section: Construction Of Strains With Modulated Gapdh Activitiesmentioning

confidence: 99%

Glyceraldehyde-3-Phosphate Dehydrogenase Has No Control over Glycolytic Flux in Lactococcus lactis MG1363

2003

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“…These proteins were classified into two main groups, corresponding to those involved in carbon metabolism and nitrogen metabolism, plus one group comprising four unclassified proteins. L. lactis encodes a second glyceraldehyde dehydrogenase, GapA (5), which is poorly, if at all, produced and migrates at distinct positions on two-dimensional gels (56). GapB appeared as two major isoforms, a and b, in agreement with previous results (25, 56) ( Fig.…”

Section: Resultssupporting

confidence: 53%

“…The amounts of the a isoform are strongly decreased, whereas the amounts of the b isoform remain unchanged. GapB appears to be the major source of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity in L. lactis (56). To our knowledge, this is the first demonstration that GAPDH isoform levels vary during growth.…”

Section: Resultsmentioning

confidence: 79%

Proteome Analyses of Heme-Dependent Respiration inLactococcus lactis: Involvement of the Proteolytic System

Vido

Bars²,

Mistou³

et al. 2004

J Bacteriol

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Sugar fermentation was long considered the sole means of energy metabolism available to lactic acid bacteria. We recently showed that metabolism of Lactococcus lactis shifts progressively from fermentation to respiration during growth when oxygen and heme are available. To provide insights into this phenomenon, we compared the proteomic profiles of L. lactis under fermentative and respiratory growth conditions in rich medium. We identified 21 proteins whose levels differed significantly between these conditions. Two major groups of proteins were distinguished, one involved in carbon metabolism and the second in nitrogen metabolism. Unexpectedly, enzymes of the proteolytic system (PepO1 and PepC) which are repressed in rich medium in fermentation growth were induced under respiratory conditions despite the availability of free amino acids. A triple mutant (dtpT dtpP oppA) deficient in oligopeptide transport displayed normal respiration, showing that increased proteolytic activity is not an absolute requirement for respiratory metabolism. Transcriptional analysis confirmed that pepO1 is induced under respiration-permissive conditions. This induction was independent of CodY, the major regulator of proteolytic functions in L. lactis. We also observed that pepO1 induction is redox sensitive. In a codY mutant, pepO1 expression was increased twofold in aeration and eightfold in respiration-permissive conditions compared to static conditions. These observations suggest that new regulators activate proteolysis in L. lactis, which help to maintain the energetic needs of L. lactis during respiration.

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“…GAPDH protein, as is seen in some gram-positive bacteria (13,42). The GAPDH protein has been reported to exist in several forms with a conserved molecular weight but different pIs (4,42).…”

Section: Discussionmentioning

confidence: 99%

Development of a Listeria monocytogenes EGDe Partial Proteome Reference Map and Comparison with the Protein Profiles of Food Isolates

Ramnath

Rechinger²,

Jänsch

et al. 2003

Appl Environ Microbiol

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A partially annotated proteome reference map of the food pathogen Listeria monocytogenes was developed for exponentially growing cells under standardized, optimal conditions by using the sequenced strain EGDe (serotype 1/2a) as a model organism. The map was developed by using a reproducible total protein extraction and two-dimensional (2-D) polyacrylamide gel electrophoresis analysis procedure, and it contained 33 identified proteins representing the four main protein functional classes. In order to facilitate analysis of membrane proteins, a protein compartmentalization procedure was assessed. The method used provided partial fractionation of membrane and cytosolic proteins. The total protein 2-D profiles of three serotype 1/2a strains and one serotype 1/2b strain isolated from food were compared to the L. monocytogenes EGDe proteome. An average of 13% of the major protein spots in the food strain proteomes were not matched in the strain EGDe proteome. The variation was greater for the less intense spots, and on average 28% of these spots were not matched. Two of the proteins identified in L. monocytogenes EGDe were missing in one or more of the food isolates. These two proteins were proteins involved in the main glycolytic pathway and in metabolism of coenzymes and prosthetic groups. The two corresponding genes were found by PCR amplification to be present in the four food isolates. Our results show that the L. monocytogenes EGDe reference map is a valuable starting point for analyses of strains having various origins and could be useful for analyzing the proteomes of different isolates of this pathogen.

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Proteome analysis of a Lactococcus lactis strain overexpressing gapA suggests that the gene product is an auxiliary glyceraldehyde 3-phosphate dehydrogenase

Cited by 20 publications

References 14 publications

Glyceraldehyde-3-Phosphate Dehydrogenase Has No Control over Glycolytic Flux in Lactococcus lactis MG1363

Glyceraldehyde-3-Phosphate Dehydrogenase Has No Control over Glycolytic Flux in Lactococcus lactis MG1363

Proteome Analyses of Heme-Dependent Respiration inLactococcus lactis: Involvement of the Proteolytic System

Development of a Listeria monocytogenes EGDe Partial Proteome Reference Map and Comparison with the Protein Profiles of Food Isolates

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