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

The sequence of the genome from the Lactococcus lactis subspecies lactis strain IL1403 shows the presence of two reading frames, gapA and gapB, putatively encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Previous proteomic analysis of the L. lactis subspecies cremoris strain MG1363 has revealed two neighbouring protein spots, GapBI and GapBII, with amino terminal sequences identical to the product of gapA from the L. lactis subspecies cremoris strain LM0230 and that of the two IL1403 sequences. In order to assign the two protein spots to their respective genes we constructed an L. lactis strain that overexpessed the gapA gene derived from MG1363 upon nisin induction. Compared to the wild-type, the overexpressing strain had a 3.4-fold elevated level of specific GAPDH activity when grown in the presence of nisin. In both MG1363 and the gapA overexpressing strain the GAPDH activity was specific for NAD. No NADP dependent activity was detected. Proteome analysis of the gapA overexpressing strain revealed two new protein spots, GapAI and GapAII, not previously detected in proteome analysis of MG1363. Results from mass spectrometry analysis of GapA and GapB and comparison with the deduced protein sequences for the GAPDH isozymes from the genome sequence of strain IL1403 allowed us to assign GapA and GapB to their apparent IL1403 homologues encoded by gapA and gapB, respectively. Furthermore, we suggest that a homologue of a gapB product, represented by GapB, is the main source of GAPDH activity in L. lactis during normal growth.     

Cloning and chromosomal mapping of nuclear genes encoding chloroplast and cytosolic glyceraldehyde-3-phosphate-dehydrogenase from Arabidopsis thaliana

Both cDNA and genomic clones for the nuclear genes encoding chloroplast (cp) (gapA and gapB) and cytosolic (gapC) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Arabidopsis thaliana have been isolated and characterized. Genomic Southern-blot analyses indicate that there is only one copy of each gapA, gapB and gapC gene in A. thaliana. Comparison of the deduced amino acid (aa) sequences shows that the A and B subunits are highly similar (80% positional aa identity), while there is less similarity between the cp and cytosolic subunits (45% aa identity). These relationships are consistent with the idea that the cp and cytosolic GAPDHs evolved from different lineages, as suggested in our previous study of tobacco GAPDHs [Shih et al., Cell 47 (1986) 73-80]. In addition, the chromosomal locations for the three gap genes were determined by restriction fragment length polymorphism mapping; the three gap genes are not closely linked, gapA (55.8 cM) and gapC (0.0 cM) are on chromosome 3, and gapB (51.3 cM) is on chromosome 1.     

Proteomic analysis of spontaneous mutants of Lactococcus lactis: Involvement of GAPDH and arginine deiminase pathway in H2O2 resistance

Lactococcus lactis, one of the most commonly used dairy starters, is often subjected to oxidative stress in cheese manufacturing. A comparative proteomic analysis was performed to identify the molecular modifications responsible for the robustness of three spontaneous H(2)O(2)-resistant (SpOx) strains. In the parental strain, glyceraldehyde-3-phosphate deshydrogenase (GAPDH) activity is ensured by GapB and the second GAPDH GapA is not produced in standard growth conditions. We showed that GapA was overproduced in the highly resistant SpOx2 and SpOx3 mutants. Its overproduction in the MG1363 strain led to an increased H(2)O(2) resistance of exponential growing cells. Upon H(2)O(2) exposure, GapB was fully inactivated by oxidation in the parental strain. In SpOx mutants, it partly remained in the reduced form sustaining partially GAPDH activity. The analysis of gapA disruption in these SpOx strains indicated that additional unraveled mechanisms likely contribute to the resistance phenotype. In the SpOx1 mutant, the arginine deiminase pathway was found to be upregulated and disruption of arcA or arcB genes abolished H(2)O(2) resistance. We concluded that arginine consumption was directly responsible for the SpOx1 phenotype. Finally, these results suggest that sustaining energy supply is a major way of leading to oxidative stress resistance in L. lactis.     

Synthesis of oxaloacetate in Bacillus subtilis mutants lacking the 2-ketoglutarate dehydrogenase enzymatic complex.

Bacillus subtilis mutants deficient in the 2-ketoglutarate dehydrogenase enzymatic complex required aspartate for growth at wild-type rates on carbon sources for which synthesis of the degradative enzymes is sensitive to catabolite repression (e.g., poor carbon sources), but did not require aspartate for growth on carbon sources which exert catabolite repression (e.g., good carbon sources). Measurement of metabolite pools in a mutant lacking the 2-ketoglutarate dehydrogenase active complex showed that the aspartate requirement for growth on poor carbon sources resulted from a deficiency in intracellular oxaloacetate pools even through pyruvate carboxylase was present at levels corresponding to those in wild-type cells. The oxaloacetate deficiency most likely resulted from the inability of the mutant to regenerate oxaloacetate from citrate due to the enzymatic block in the tricarboxylic acid cycle. Mutants in the enzymes of the dicarboxylic acid half of the citric acid cycle similarly required aspartate for wild-type growth in minimal medium. These results suggested that the complete turning of the tricarboxylic acid cycle is involved in the maintainance of oxaloacetate levels in B. subtilis. The ability of the mutants lacking the 2-ketoglutarate dehydrogenase enzymatic complex to grow at wild-type rates on media containing good carbon sources in the absence of exogenous aspartate is not understood.     

Isolation and properties of a Bacillus subtilis mutant unable to produce fructose-bisphosphatase.

A Bacillus subtilis mutation (gene symbol fdpA1), producing a deficiency of D-fructose-1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11, fructose-bisphosphatase), was isolated and genetically purified. An fdpA1-containing mutant did not produce cross-reacting material. It grew on any carbon source that allowed growth of the standard strain except myo-inositol and D-gluconate. Because the mutant could grow on D-fructose, glycerol, or L-malate as the sole carbon source, B. subtilis can produce fructose-6-phosphate and the derived cell wall precursors from these carbon sources in the absence of fructose-bisphosphatase. In other words, during gluconeogenesis B. subtilis must be able to bypass this reaction. Fructose-bisphosphatase is also not needed for the sporulation of B., subtilis. The fdpA1 mutation has the pleiotropic consequence that mutants carrying it cannot produce inositol dehydrogenase (EC 1.1.1.18) and gluconate kinase (EC 2.7.1.12) under conditions that normally induce these enzymes.     

Conversion of methionine to cysteine in Bacillus subtilis and its regulation

Bacillus subtilis can use methionine as the sole sulfur source, indicating an efficient conversion of methionine to cysteine. To characterize this pathway, the enzymatic activities of CysK, YrhA and YrhB purified in Escherichia coli were tested. Both CysK and YrhA have an O-acetylserine-thiol-lyase activity, but YrhA was 75-fold less active than CysK. An atypical cystathionine beta-synthase activity using O-acetylserine and homocysteine as substrates was observed for YrhA but not for CysK. The YrhB protein had both cystathionine lyase and homocysteine gamma-lyase activities in vitro. Due to their activity, we propose that YrhA and YrhB should be renamed MccA and MccB for methionine-to-cysteine conversion. Mutants inactivated for cysK or yrhB grew similarly to the wild-type strain in the presence of methionine. In contrast, the growth of an DeltayrhA mutant or a luxS mutant, inactivated for the S-ribosyl-homocysteinase step of the S-adenosylmethionine recycling pathway, was strongly reduced with methionine, whereas a DeltayrhA DeltacysK or cysE mutant did not grow at all under the same conditions. The yrhB and yrhA genes form an operon together with yrrT, mtnN, and yrhC. The expression of the yrrT operon was repressed in the presence of sulfate or cysteine. Both purified CysK and CymR, the global repressor of cysteine metabolism, were required to observe the formation of a protein-DNA complex with the yrrT promoter region in gel-shift experiments. The addition of O-acetyl-serine prevented the formation of this protein-DNA complex.