The chb operon constitutes the genes essential for utilization of chitooligosaccharides in Escherichia coli and related species. The six genes of the operon code for a transcriptional regulator (ChbR) of the operon, a permease (ChbBCA), a monodeacetylase (ChbG), and a phospho-beta-glucosidase (ChbF). In the absence of the substrate, the operon is maintained in a transcriptionally repressed state, while presence of the substrate leads to transcriptional activation. Regulation of the chb operon is brought about by the concerted action of three proteins, the negative regulator NagC coded by the nag operon, the dual function regulator ChbR coded by the chb operon and the universal regulatory protein CRP. Mutations that lead to alterations in the regulation of the operon can facilitate utilization of cellobiose, in addition to chitooligosaccharides by E. coli. The studies presented in Chapter II were aimed at understanding the evolution of cellobiose utilization in Shigella sonnei, which is phylogenetically very close to E. coli. Cel+ mutants were isolated from a Cel- wild type S. sonnei strain. Interestingly, Cel+ mutants arose relatively faster on MacConkey cellobiose agar from the S. sonnei wild type strain compared to E. coli. Similar to E. coli, the Cel+ phenotype in S. sonnei mutants was linked to the chb operon. Deletion of the phospho-β-glucosidase gene, chbF also resulted in loss of the Cel+ phenotype, indicating that ChbF is responsible for hydrolysis of cellobiose in these mutants. Previous work from the lab has shown that acquisition of two classes of mutations is necessary and sufficient to give rise to Cel+ mutants in E. coli. The first class of mutations either within the nagC locus or at the NagC binding site within the chb promoter, lead to NagC derepression. The second class consisting of gain-of-function mutations in chbR enable the recognition of cellobiose as an inducer by ChbR and subsequent activation of the operon. However, in S. sonnei a single mutational event of an IS element insertion resulted in acquisition of this phenotype. Depending on the type and location of the insertion, the mutants were grouped as Type I, and Type II. In Type I mutants an 1S600 insertion between the inherent -10 and -35 elements within the chb promoter leads to ChbR-independent constitutive activation of the operon, while in Type II mutants, an IS2/600 insertion at -113/-114, leads to ChbR-dependent, cellobiose-inducible expression of the operon. The results presented also indicate that in addition to relieving NagC mediated repression, the insertion in Type II mutants also leads to increase in basal transcription from the chb promoter. Constitutive expression of the chb operon also results in utilization of the aromatic β-glucosides salicin and arbutin, in addition to cellobiose in Type I mutants, which indicates the promiscuous nature of permease and hydrolysis enzyme of the chb operon. This part of the thesis essentially demonstrates the different trajectories taken for the evolution of new metabolic function under conditions of nutrient stress by two closely related species. It emphasizes the significance of the strain background, namely the diversity of transposable elements in the acquisition of the novel function. The second part of this research investigation, detailed in Chapter III deals with experiments to characterize the eukaryotic orthologs of the last gene of the chb operon. The chbG gene of E. coli codes for a monodeacetylase of chitooligosaccharides like chitobiose and chitotriose. The protein belongs to a highly conserved, but less explored family of proteins called YdjC, whose orthologs are present in many prokaryotes and eukaryotes including mammals. The human YDJC locus located on chromosome 22 is linked to a variety of inflammatory diseases and the transcript levels are relatively high in stem cells and a few cancer cells. In silico analysis suggested that the mammalian YdjC orthologs possess sequence and structural similarity with the prokaryotic counterpart. The full length mouse YdjC ortholog, which is 85% identical to the human ortholog was cloned into a bacterial vector and expressed in a chbG deletion strain of E. coli. The mouse YdjC ortholog could neither promote growth of the strain on chitobiose nor induce transcription from the chb promoter. The purified mouse YdjC ortholog could not deacetylate chitobiose in vitro as well, suggesting that the mouse ortholog failed to complement the function of the E. coli counterpart, ChbG under the conditions tested in this study. In order to characterize the mammalian YdjC orthologs more elaborately, further experimentation was performed in mammalian cell lines. The results indicate that YdjC is expressed in mammalian cell lines of different tissue origin and the expression was seen throughout the cell. Overexpression of mouse Ydjc in a few mammalian cells also resulted in increased proliferation and migration, indicating a direct or indirect role of this protein in cell growth/proliferation. The mammalian orthologs of ChbG therefore appear to have related but distinct activities and substrates compared to the bacterial counterpart that need to be elucidated further.

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.