Listeria monocytogenes transporte le cellobiose principalement via le PTS (PEP:carbohydrate phosphotransferase system). La croissance sur cellobiose induit l'expression des opérons celBCA1, celBA2 ainsi que du gène lmrg_01989, qui codent respectivement le composant soluble EIIACel1, le transporteur EIICCel1, le composant soluble EIIBCel1, les protéines EIIBCel2 et EIIACel2, et une seconde EIICCel. La croissance sur glucose réprime fortement l'expression de ces gènes. La délétion de celC1 codant l'EIICCel1 ou des deux gènes, celA1 et celA2, ralentit considérablement la consommation cellobiose. L'expression des trois unités de transcription induite par le cellobiose dépend de CelR. CelR, qui code un régulateur transcriptionnel LevR- like, est situé en aval de l'opéron bicistronique celBA2. CelR est activé par phosphorylation par EI et HPr de l'His550. En revanche, la phosphorylation de l'His823, catalysée par P~EIIBCel1 et P~EIIBCel2, inhibe l'activité de CelR. Le remplacement de l'His823 par une Ala empêchant cette phosphorylation ou la délétion des deux gènes codants les EIIAsCel ou EIIBsCel entraîne l'expression constitutive des trois unités de transcription contrôlées par CelR. Comme le glucose, le cellobiose inhibe fortement l'activité de PrfA, l'activateur des gènes de virulence. Nous avons donc cherché à tester si l'un des composants PTSCel pouvait être impliqué dans la répression de gènes de virulence. Les mutants consommant faiblement le cellobiose, présentaient une levée de la répression des gènes de virulence par le cellobiose, alors que le glucose et les autres sucres-PTS les réprimaient toujours. De manière surprenante, la délétion du gène monocistronique lmrg_00557, qui code un autre composant EIIBCel du PTS, induisait la levée de la répression des gènes de virulence médiée par toutes les sources de carbone mais n'avait aucun effet sur la consommation de glucose ou de cellobiose. Ce gène lmrg_00557 a été appelé vgiB (virulence gene inhibitor B) et la protéine correspondante, qui semble jouer un rôle majeur dans la régulation de l'activité de PrfA, EIIBVir. Cette protéine est phosphorylée par le PEP et les composants PTS EI, HPr et EIIACel2 sur le résidu cystéine-8. La complémentation du mutant ΔvgiB avec l'allèle sauvage, mais également avec l'allèle Cys8Ala, restaurait le mécanisme général de répression des gènes de virulence par les sucres, suggérant ainsi que la forme non phosphorylée de EIIBVir inhibe l'activité de PrfA. / Listeria monocytogenes transports cellobiose mainly via a PEP:carbohydrate phosphotranseferase system (PTS). Growth on cellobiose induces the expression of the celBCA1 and celBA2 operons as well as lmrG01989, which encode the soluble EIIA Cel1 and EIIB Cel1 components, the transporter EIIC Cel1 , the EIIA Cel2 and EIIB Cel2 proteins, and a second EIIC Cel , respectively. Growth on lucose strongly repressed the expression of these genes. Deletion of the EIIC Cel1 –encoding celC1 or of both, celA1 and celA2, significantly slowed cellobiose consumption. The bicistronic operon celBA2 is located downstream from celR, which codes for a LevR-like transcription activator. Expression of the three cellobiose-induced transcription units depends on CelR. The gene encoding CelR is located upstream from the bicistronic operon celBA2. CelR itself is activated via phosphorylation by EI and HPr at His550. In contrast, phosphorylation at His823, which is catalyzed by both, P~EIIB Cel1 and P~EIIB Cel2 , inhibits CelR activity. Preventing this phosphorylation by replacing His823 with Ala or deleting the two EIIA Cel – or EIIB Cel -encoding genes caused constitutive expression of all three CelR-controlled transcription units. Similar to glucose, cellobiose strongly inhibits the activity of the virulence gene activator PrfA. We therefore tested whether one of the PTS Cel components might be involved in virulence gene repression. Mutants, that exhibit slow cellobiose consumption, were relieved from cellobiose-mediated virulence gene repression, whereas glucose and other PTS-sugars still repressed them. Strikingly, deletion of the presumed monocistronic lmrg_00557, which codes for another EIIB Cel -like PTS component, caused a general relief from carbon source-mediated virulence gene repression, but had no effect on cellobiose or glucose consumption. The gene lmrg_00557 was named vgiB (virulence gene inhibitor B) and the encoded protein, which seems to play a major role in PrfA regulation, was called EIIB Vir . It becomes phosphorylated by PEP and the PTS components enzyme I, HPr and EIIA Cel2 at cysteine-8. Complementation of the ΔvgiB mutant with wild-type vgiB, but also with the Cys8Ala allele restored general virulence gene repression, thus suggesting that it is the unphosphorylated form of EIIB Vir , which inhibits the activity of PrfA.

The genome of Escherichia coli harbors several catabolic operons involved in the utilization of a wide variety of natural compounds as carbon sources. The chitobiose (chu) operons of E.coli Is involved in the utilization of chitobiose(disaccharide of N-acety1-D-glucosamine) and cellbiose (disaccharide of glucose) derived from the two most abundant naturally occurring carbon sources on earth, chitin and cellulose respectively. The operon consists of the chbBCARFG genes coding for transport, regulation and hydrolysis functions required to utilize these compounds; the chuyBCA genes code for a multi-subuni PTS transporter ; the chuR codes for a dual function repressor/activator of the operon; the chbF codes for a phospho-glucosidase and the chbG codes for a protein of unknown function. The chu operon Is regulated by three transcription factors; NagC, a key regulator of the nag genes involved in amino sugar metabolism; ChbR, a dual function operon-specific regulator; and CRP_cAMP. The operon is repressed by NagC and ChbR in the absence of catabolic substrate. In the presence of chitobiose, expression is induced by the abrogation of NagC-mediated repression by GlcNAc-6-P generated by the hydrolysis of chitobiose-6-P and subsequent activation of transcription by ChbR and CPR-cAMP. Wild type E.coli connot utilize cellbiose due to the inability of cellbiose to induce expression from the operon. The simultaneous presence of a loss of function mutation in nagC and a gain –of-function mutation in chbR is necessary and sufficient to allow cellbiose to induce expression and confer on E.coli the ability to utilize cellbiose. The activation step by ChbR and CPR-cAMP requires an inducer that is recognized by ChbR. The chemical identity of the inducer and the mechanism of transcriptional activation by ChbR and CPR-cAMP are not understood. The studies described in the chapter 2 shows that chbG is essential for the utilization of the acetylated sugars chitobiose and chitotriose while it is dispensable for the sugars lacking the acety1group such as cellobiose and chitosan dimer, a disaccharide of N-glucosamine. ChbG is produced as a cytosolic protein and removes one acety1 group from chitobiose and chitotriose thus shows a mono-decetylase activity. Taken together, the observing suggest that ChbG deacetylates chitobiose-6-P and chitotriose-6-P producing the mono-decetylated from of the sugars. The deacetylateion is necessary for their recognition both as inducers by ChbR to activate transcription along with CRP-cAMP and as substractes by phosop-glucosidase ChbF. Cellobiose positive(Cel+) mutants carrying nagC delection and different gain-of-function mutations in chbR are independent of chbG for induction by chitobiose suggesting that the mutations in ChbR can allow it to recognize the acetylated form of chitobiose-6-P. Despite normal induction, the mutants to grow on chitobiose without chbG are consistant with the requirement of deacetylation for hydrolysis by ChbF. The prediction active site of chbG was validated by demonstrating the loss of chbG function upon alanine substitution of the putative metal binding residues. Vibro cholerace ChbG can complement the function of E.coli ChbG indicating that ChbG is conserved in both the organisms. The studies presented in chapter 3 address the mechanism of transcriptional activation of the chb operon by ChbR and CPR-cAMP. ChbR and CPR-cAMP function in a synergistic manner in response to the induction signal. The synergy is not because of their cooperative binding to the DNA. The role of CRP as a class I activator via the known mechanism involving interaction between the Activation region1 (AR1) and the C-terminal domain of the alpha subunit of RNA polymerase (CTD) was not crucial for the chb operon. A direct interaction between the two activators in virto was observed. Based on these results and the close spacing of the synergy is due to interaction between the two regulators bound to DNA that is enhanced in the presence of the inducer, binding about an optimal confirmation in ChbR required to interact with RNA polymerase. ChbR contacts different residues in the subunit in response to cellbiose and chitobiose; whereas it utilizes the known residues in the presence cellbiose, it appears to require different and unknown residues for induction in the presence of chitobiose. In conclusion, the studies reported in chapter 2 and 3 provide an understanding of the regulation of the chitin oligosaccharides utilization in E.coli at different levels. The broad implications of these studies and possible future directions are discussed in chapter 4. ChbG is an evolutionary conserved protein found in both prokaryotes and enkayotes including humans. ChbG homologs have been implicated in inflammatory bowel disorders in humans and development in metazoans. Therefore, the studies on chbG described in this thesis have been broader significance.