Catabolite repression of the citST two-component system in Bacillus subtilis

In Bacillus subtilis, expression of the citrate transporter CitM is under strict control. Transcription of the citM gene is induced by citrate in the medium mediated by the CitS-CitT two-component system and repressed by rapidly degraded carbon sources mediated by carbon catabolite repression (CCR). In this study, we demonstrate that citST genes are part of a bicistronic operon. The promoter region was localized in a stretch of 58 base pairs upstream of the citS gene by deletion experiments. Transcription of the operon was repressed in the presence of glucose by the general transcription factor CcpA. A distal consensus cre site in the citS-coding sequence was implicated in the mechanism of repression. Furthermore, this repression was relieved in Bacillus subtilis mutants deficient in CcpA or Hpr/Crh, components essential to CCR. Thus, we demonstrate that CCR represses the expression of the citST operon, which is responsible for the induction of citM, through the cre site located 1326 bp from transcriptional start site of citST

CcpA represses the expression of the divergent cit operons of Enterococcus faecalis through multiple cre sites

<p>Abstract</p> <p>Background</p> <p>In <it>Enterococcus faecalis </it>the genes encoding the enzymes involved in citrate metabolism are organized in two divergent operons, <it>citHO </it>and <it>oadHDB-citCDEFX-oadA-citMG </it>(<it>citCL </it>locus). Expression of both operons is specifically activated by adding citrate to the medium. This activation is mediated by binding of the GntR-like transcriptional regulator (CitO) to the <it>cis</it>-acting sequences located in the <it>cit </it>intergenic region. Early studies indicated that citrate and glucose could not be co-metabolized suggesting some form of catabolite repression, however the molecular mechanism remained unknown.</p> <p>Results</p> <p>In this study, we observed that the <it>citHO </it>promoter is repressed in the presence of sugars transported by the Phosphoenolpyruvate:carbohydrate Phosphotranserase System (PTS sugars). This result strongly suggested that Carbon Catabolic Repression (CCR) impedes the expression of the activator CitO and the subsequent induction of the <it>cit </it>pathway. In fact, we demonstrate that CCR is acting on both promoters. It is partially relieved in a <it>ccpA</it>-deficient <it>E. faecalis </it>strain indicating that a CcpA-independent mechanism is also involved in regulation of the two operons. Furthermore, sequence analysis of the <it>citH</it>/<it>oadH </it>intergenic region revealed the presence of three putative catabolite responsive elements (<it>cre</it>). We found that they are all active and able to bind the CcpA/P-Ser-HPr complex, which downregulates the expression of the <it>cit </it>operons. Systematic mutation of the CcpA/P-Ser-HPr binding sites revealed that <it>cre1 </it>and <it>cre2 </it>contribute to <it>citHO </it>repression, while <it>cre3 </it>is involved in CCR of <it>citCL</it></p> <p>Conclusion</p> <p>In conclusion, our study establishes that expression of the <it>cit </it>operons in <it>E. faecalis </it>is controlled by CCR via CcpA-dependent and -independent mechanisms.</p

Citrate metabolism and aroma compound production in lactic acid bacteria

24 p.-8 fig.-1 tab.The main activity of lactic acid bacteria (LAB) during fermentation is the catabolism of sugars present in food, producing lactic acid by homoor heterofermentative pathways. In addition, these microorganisms also have the capability to metabolize other substrates, such as citrate. Citrate is present in fruit juices, milk and vegetables and is also added as a preservative to foods. Citrate fermentation by LAB leads to the production of 4-carbon compounds, mainly diacetyl, acetoin and butanediol, which have aromatic properties. One of these compounds, diacetyl is responsible for the buttery aroma of dairy products such as butter, acid cream and cottage cheese. In addition, it is an important component of the flavour of different kinds of chesses and yoghurt. Moreover, the CO2 produced as a consequence of citrate metabolism contributes to the formation of "eyes" (holes) in Gouda, Danbo and other cheeses. Thus, the utilization of citrate in milk by LAB has a very positive effect on the quality of the end products. Therefore, the interest of the dairy industry in controlling citrate utilization by LAB has promoted research into the proteins and effectors controlling its metabolic pathway. In this chapter we summarize the current knowledge of citrate utilization by LAB. The transport of citrate and its metabolism to pyruvate, as well as further conversion to aroma compounds, is described and, the differences in the co-metabolism of citrate with glucose between homo or heterofermentative bacteria is discussed. In addition, the molecular mechanisms controlling expression of genes responsible for transport and conversion of citrate into pyruvate are presented, as are their correlation with the physiological function of citrate metabolism. To date, two different models of regulation have been described which are unique to LAB. In Lactococcus lactis, a specific transcriptional activation of the promoters controlling the cit operons takes place at low pH to provide an adaptative response to acidic stress. In Weissella paramesenteroides, the CitI transcriptional regulator functions as a citrate-activated switch allowing the cell to optimize the generation of metabolic energy. CitI, its operators and citrate transport and metabolic operons are highly conserved in several LAB. Therefore, this mechanism of sensing and response to citrate appears to have been conserved and propogated during the evolution of LABThis work was supported by the European Union grants QLK1-CT-2002-02388and KBBE-CT-2007-211441, the Spanish Ministry of Education grant AGL2006-1193-C05-01 and the Agencia Nacional de Promoción Científica y Tecnológica grant PICT 15-38025Peer reviewedPostprin

Implications of the expression of Enterococcus faecalis citrate fermentation genes during infection.

Citrate is an ubiquitous compound in nature. However, citrate fermentation is present only in a few pathogenic or nonpathogenic microorganisms. The citrate fermentation pathway includes a citrate transporter, a citrate lyase complex, an oxaloacetate decarboxylase and a regulatory system. Enterococcus faecalis is commonly present in the gastro-intestinal microbiota of warm-blooded animals and insect guts. These bacteria can also cause infection and disease in immunocompromised individuals. In the present study, we performed whole genome analysis in Enterococcus strains finding that the complete citrate pathway is present in all of the E. faecalis strains isolated from such diverse habitats as animals, hospitals, water, milk, plants, insects, cheese, etc. These results indicate the importance of this metabolic preservation for persistence and growth of E. faecalis in different niches. We also analyzed the role of citrate metabolism in the E. faecalis pathogenicity. We found that an E. faecalis citrate fermentation-deficient strain was less pathogenic for Galleria mellonella larvae than the wild type. Furthermore, strains with deletions in the oxaloacetate decarboxylase subunits or in the α-acetolactate synthase resulted also less virulent than the wild type strain. We also observed that citrate promoters are induced in blood, urine and also in the hemolymph of G. mellonella. In addition, we showed that citrate fermentation allows E. faecalis to grow better in blood, urine and G. mellonella. The results presented here clearly indicate that citrate fermentation plays an important role in E. faecalis opportunistic pathogenic behavior

Catabolite repression of the citST two-component system in Bacillus subtilis

In Bacillus subtilis, expression of the citrate transporter CitM is under strict control. Transcription of the citM gene is induced by citrate in the medium mediated by the CitS–CitT two-component system and repressed by rapidly degraded carbon sources mediated by carbon catabolite repression (CCR). In this study, we demonstrate that citST genes are part of a bicistronic operon. The promoter region was localized in a stretch of 58 base pairs upstream of the citS gene by deletion experiments. Transcription of the operon was repressed in the presence of glucose by the general transcription factor CcpA. A distal consensus cre site in the citS-coding sequence was implicated in the mechanism of repression. Furthermore, this repression was relieved in Bacillus subtilis mutants deficient in CcpA or Hpr/Crh, components essential to CCR. Thus, we demonstrate that CCR represses the expression of the citST operon, which is responsible for the induction of citM, through the cre site located 1326 bp from transcriptional start site of citST.

Strains, plasmids and oligonucleotides used in this study.

<p>Strains, plasmids and oligonucleotides used in this study.</p

Ammonium production and final pH values of <i>E. faecalis</i> strains grown in the presence or absence of agmatine.

<p>Ammonium production and final pH values of <i>E. faecalis</i> strains grown in the presence or absence of agmatine.</p

CcpA interaction with the <i>aguR-aguBDAC</i> intergenic region.

<p>For band shift assays, <i>agu</i> or <i>agu^mut</i> amplicons (2.69 nM each) were incubated with increasing concentrations of CcpA (0.025–0.7 mM), 5 mM of P-Ser-HPr and 20 mM FBP. The arrow indicates position of the retarded complex (C). Consensus, wild type and mutated sequence of <i>cre</i> sites are indicated.</p

Analysis of CcpA and PTS^Man effects on expression of the <i>aguBDAC</i> operon.

<p>β-galactosidase activity of P<i>aguB</i>-<i>lacZ</i> transcriptional fusion in wild type (JH2-2), <i>ccpA</i>⁻ (CL14), <i>mpt</i>⁻ (JH98) and <i>ccpA</i>⁻<i>mpt</i>⁻ (CL98) strains. Cells were grown in LBA with or without 30 mM glucose (Glu), lactose (Lac), maltose (Mal) or fructose (Fru). Error bars represent standard deviation of at least triplicate measurements.</p