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Model for regulation of cit operons by citi and citrate

Citrate is present in milk at concentrations of 8 to 9 mM and is cometabolised with sugars by strains of lactic acid bacteria (LAB), including Weisella paramesenteroides. The breakdown of citrate results in production of carbon dioxide (responsible for the texture of some cheeses) and production of the flavour compound diacetyl, which is essential for the quality of dairy products such as butter, buttermilk, and cottage cheese. Therefore, to improve and control production of aroma compounds by different LAB, it is important to understand the molecular mechanism which regulate the citrate metabolic pathways.

The dairy Weissella paramesenteroides citrate fermenting J1 strain isolated from an Argentinean cheese has been the subject of this study. We have previously characterised its citMCDEFGRP (cit) operon, encoding the citrate permease and the two enzymes (citrate lyase and oxaloacetate decarboxylase), which catalyse conversion of citrate into pyruvate. The expression of both the cit operon and the upstream divergent gene citI are induced by the presence of citrate in the medium. In this work DNA protein interaction studies as well as in vitro transcription reveal that CitI, encoded by citI, recognises two A+T rich operator sites located between citI and citM and that the DNA-binding affinity of CitI is increased by citrate. Subsequently, this citrate-signal-propagation leads to activation of the cit operon through an enhanced recruitment of RNA polymerase (RNAP) to its promoters. Our results indicate that control of CitI by the cellular pools of citrate provides a mechanism for sensing the availability of citrate and adjust the expression of the cit operon accordingly.

The results presented in this work suggest a scenario for the transcriptional activation of the Pcit and PcitI promoters in W. paramesenteroides. In the absence of citrate, CitI would bind to the operator sites stimulating the RNA polymerase to form complexes on both promoters. This would result in low expression levels of the citrate fermentation enzymes as well as of the citrate permease P, which will catalyse the uptake of citrate when this compound becomes available in the environment. Once transported inside the cell, citrate would bind to CitI enhancing the regulator affinity for its DNA operators and resulting in increased RNAP recruitment at Pcit and PcitI. This would result in transcriptional activation from both promoters. Activation of Pcit results in coordinated synthesis of the citrate fermentation enzymes and breakdown of citrate. Thus, our model predicts that a small increase in CitI transcription could account for the large increase of the cit mRNA detected in W. paramesenteroides growing in a citrate-supplemented medium.

In W. paramesenteroides, Leuconostoc mesenteroides, Oenococcus oeni and Lactobacillus plantarum, citI is placed in opposite orientation with respect to the cit operon. In addition, in these bacteria two consensus CitI binding operators could be found in the intergenic region between citI and citM. These facts suggest a similar mechanism of transcriptional activation by CitI in conjunction with citrate in all these bacteria. Thus, CitI seems to play a pivotal role in citrate sensing and in the transcriptional stimulation of the operons involved in the transport and metabolism of citrate in LAB.

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