Métabolisme et régulation de processus cellulaires chez Bacillus subtilis
Mots clés : sucres, métabolisme, phosphorylation, Bacillus subtilis
The assimilation of carbon substrates plays a crucial role in providing energy and precursors required for survival and multiplication of bacteria. It is at the crossroads of all cellular processes. However, direct connections between carbon metabolism and other cell functions are not well understood. Recent results obtained from the bacterium Bacillus subtilis seem to directly link glycolysis and DNA replication or nutritional conditions and cell size. Our goal is to study the relationship between carbon metabolism and key cellular processes in the model bacterium B. subtilis. For that purpose, we tried to unravel new roles for proteins involved in carbon utilization. We also focused on genes of unknown function that are physically clustered with genes implicated in carbon metabolism.
RECENT RESULTS OF THE LAB
I. New functions for proteins involved in carbon metabolism ? (Pompeo et al. 2007)
The HPr protein, involved in carbohydrate uptake, possesses a key regulatory role in several bacteria. In order to unravel new functions of HPr, we tried to identify new partners of this protein by the TAP method. Among the co-purified proteins, we identified GapA, a glyceraldehyde-3-P dehydrogenase. We validated this interaction by several biochemical experiments. In order to understand the role of these interactions, we measured GapA activity in the presence of HPr, Crh or the phosphorylated forms of these proteins. No inhibition of GapA activity was detected in the presence of HPr or Crh or in the presence of P-His-HPr. By contrast, the presence of the seryl-phosphorylated proteins led to an inhibition of GapA activity. Our results suggest novel levels of regulation of a key step in glycolysis in B. subtilis.
II. Characterization of proteins of unknown function
The B. subtilis crh gene is part of the yvclJKL-crh-yvcN operon. Apart from crh, the function of the proteins encoded by this operon has not been identified. The yvcJ and yvcK genes are conserved in many bacteria suggesting that they may play a key role in bacterial physiology. Thus, we decided to characterize the function of these genes using several approaches in genetics and biochemistry, and cell biology.
1. Characterization of YvcJ and its implication in competence in Bacillus subtilis (Luciano et al. 2009)
yvcJ and yhbJ encode two homologous proteins in B. subtilis and E. coli, respectively. In several Proteobacteria, yhbJ is associated with genes related to sugar transport while in most Gram-positive bacteria, yvcJ is clustered with yvcK. We showed that both YvcJ from B. subtilis and YhbJ from E. coli bind and hydrolyze nucleotides. The cellular function of yvcJ was investigated. In contrast to results recently obtained for E. coli, which indicated that yhbJ mutants strongly overproduced glucosamine-6-phosphate synthase (GlmS), glmS expression was quite similar in a yvcJ mutant and a wild-type strain. However, in mutants defective in yvcJ, the cell fraction that expressed competence were reduced. Thus, transformation efficiency was affected. Furthermore, our data indicated that YvcJ positively controls the expression of late competence genes. Our results showed that even if YvcJ and YhbJ are homologous and belong to the same family of P-loop-containing proteins, they seem to play different biological functions in B. subtilis and in E. coli.
2. The YvcK protein is required for morphogenesis via localization of PBP1 under gluconeogenic growth conditions in B. subtilis (Foulquier et al. 2011)
The YvcK protein was previously shown to be dispensable when B. subtilis cells were grown on glycolytic carbon sources but essential for growth and normal shape on gluconeogenic carbon sources (Görke et al. 2005). We observed that YvcK is localized as a helical-like pattern in the cell . (movie S1)
This localization seems independent of the actin-like protein MreB. Surprisingly, YvcK overproduction restored a normal morphology in an mreB mutant strain when bacteria were grown on PAB medium. Reciprocally, an additional copy of mreB restored a normal growth and morphology in a yvcK mutant strain when bacteria were grown on a gluconeogenic carbon source such as gluconate. Furthermore, as already observed in the case of an mreB mutant, the deletion of the gene encoding the penicillin-binding protein PBP1 restored growth and normal shape in a yvcK mutant cultivated on gluconeogenic carbon sources. PBP1 is delocalized in an mreB mutant grown in the absence of magnesium and in a yvcK mutant grown on gluconate medium. Interestingly, PBP1 proper localization can be rescued by YvcK overproduction. Therefore, in gluconeogenic growth conditions, YvcK is required for the correct localization of PBP1 and hence for displaying a normal rod shape.