Escherichia coli has the ability to respond to stress such as starvation in a very efficient manner. Under conditions of starvation wherein a novel substrate is provided as a sole nutritional source, Spontaneous mutants arise in a population of E.Coli that are able to utilize this novel carbon Many generic systems, upon mutational activation, have been shown to allow E.coli to Grow on novel substrates. . Wildtype E.coli is not able to utilize cellobiose, a disaccharide of glucose, as a carbon source. However after prolonged incubation with cellobiose as a sole carbon source, spontaneous Cel+ mutants can be isolated. The Cel+ derivatives have mutations in the chb operon involved in the utilization of N-N-diacetylchitobiose, a disaccharide of N-acetyl glucosamine. The chb operon of E.coli is comprised of six ORFs (chbBCARFG) with a ~200bp regulatory region (chbOP); chbBCA encode the IIB, IIC and IIA domains of the PTS-dependent permease respectively, chbR encodes for a dual function activator/repressor, chbF encodes the phopho-chitobiase and chbG codes for a protein of unknown function. It has been shown that the three proteins ChbR, CAP and NagC regulate the expression of the chb operon. ChbR along with CAP activates the chb operon in the presence of chitobiose. In the absence of the inducer, ChbR, along with NagC, represses the chb operon. Activation of the chb operon allowing utilization of cellobiose was earlier shown to occur either via insertion of IS1, IS2 or IS5 into the regulatory region (chbOP) upstream of the transcription start site or by base substitutions in chbR. Comparison of the chb operon sequence obtained from various Cel+ mutants with E.coli K12 genome sequence showed many differences. These differences were clustered in both the permease (chbBCA) as well as the enzyme (chbF) of the chb operon, suggesting that mutations are needed in all the ORFs of this operon in order to alter the specificity of E.coli towards utilization of cellobiose. The main objective of this thesis is to elucidate the mechanism of mutational activation of the chb operon of E.coli to allow utilization of cellobiose. These studies have shown that two classes of mutations, those that abrogate repression by NagC and those that alter the regulation by ChbR, together are necessary and sufficient to confer a Cel+ phenotype to E.coli. These studies also show that the wildtype permease and phospho-â -glucosidase are able to recognize and cleave cellobiose. Initial experiments were designed to study the role of independent mutational events of either insertion within the regulatory region or loss-of-function of chbR in conferring E.coli a Cel+ phenotype. The single mutational event of either the insertion within the regulatory region chbOP that disrupts the strong NagC binding site (mimicking an IS element) or knockout of chbR did not confer on E.c oli a Cel+ phenotype. However the presence of the artificial insertion within chbOP accelerated the process of obtaining Cel+ mutants suggesting a positive role for insertion elements. The apparent inability of the chbR knockout strain to mutate to Cel+ suggested that chbR is essential for acquisition of a Cel+ phenotype. Reporter gene assays showed that the presence of an insertion within chbOP enhances the promoter activity marginally. The role of chbR as a repressor was further ascertained by increased promoter activity seen from wildtype chbOP-lacZ fusion in a chbR knockout strain. A marginal enhancement in promoter activity in the presence of cellobiose in a strain carrying a wildtype chbR as compared to chbR knockout strain suggested an additional positive role of chbR. The inability of cellobiose to produce an inducing signal necessary for activation by wildtype ChbR protein suggested that gain-of-function mutations within chbR locus might play a crucial role in acquisition of cellobiose utilization phenotype by E.coli. The chbR clones obtained from various Cel+ mutants could activate transcription from the chb promoter at a higher level in the presence of cellobiose. However this activation was seen only in a strain carrying disruptions of the chromosomal nagC and chbR loci. These transformants also showed a Cel+ phenotype on the MacConkey cellobiose medium suggesting that the wildtype permease and enzyme upon induction could recognise, transport and cleave cellobiose, respectively. This was confirmed by cloning the wildtype genes encoding the permease and phospho-â -glucosidase under a heterologous promoter (Plac). The wildtype E.coli strain transformed with a plasmid carrying the genes could utilize cellobiose efficiently. Large scale isolation of Cel+ mutants was undertaken. Variation in the ability of cellobiose utilization was observed among the different mutants. Several Cel+ mutants retained the ability to utilize chitobiose. Cel+ mutants lacking insertions within chbOP contained a loss-of-function mutation at the nagC locus. The sequencing of the chbR locus from Cel+ mutant strains showed a single basepair change at the DNA level translating into a single amino acid change when compared to the Cel- counterpart. Nucleotide sequence of chbR obtained from two Cel+ natural isolates of E.coli also showed a single base mutation. The chbR clones from the two mutants, when transformed into a strain carrying disruptions at the chromosomal nagC and chbR loci, conferred it a Cel+ phenotype. Initial characterization of one of the mutant ChbR (N238S) was carried out. Reporter assays in a strain containing a wildtype copy of chbR at the genomic locus and a disruption of nagC showed that the wildtype ChbR is dominant over the mutant ChbR (N238S). The biochemical investigations of the wildtype and mutant ChbR (N238S) were undertaken. Wildtype ChbR showed non-specific binding to chbOP that could not be competed out by excess cold DNA. DNaseI protection assays confirmed that wildtype ChbR formed a relatively nonspecific complex with chbOP as compared to mutant ChbR (N238S). Finally DNaseI footprinting experiments showed that mutant ChbR (N238S) binds the specific direct repeat within chbOP better than the wildtype protein. These results indicated that mutant ChbR (N238S) has lost its ability to repress transcription by its inability to bind chbOP non-specifically. In addition, the mutant ChbR (N238S) has acquired the ability to activate transcription in the presence of cellobiose. This could be partly mediated via enhanced binding of the mutant ChbR (N238S) to the specific DNA binding site within chbOP in contrast to its wildtype counterpart. To conclude, this work has shown that acquisitive evolution of E.coli towards utilization of cellobiose in laboratory conditions alters the regulation of the chb operon and allows it to acquire new metabolic capability for utilizing cellobiose under selective pressure.

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.

The genetic systems involved in the utilisation of aromatic β-glucosides in E. coli consist of the bgl, asc, and chb operons and the locus bglA encoding phospho-β-glucosidase A. The bgl and asc operons are known as cryptic or silent systems since their expression is not sufficient for utilisation of these sugars in wild type strains of E. coli. Their transcriptional activation by different classes of mutations confers a Bgl+ phenotype to the mutant. The maintenance of cryptic genes without accumulating deleterious mutation in spite of being silent is an evolutionary puzzle. Several observations have suggested the possibility that these genes may be expressed under specific physiological conditions conferring a fitness advantage to the organism. The main aim of this study was to investigate the possible role of aromatic β-glucoside catabolic systems of E. coli in combating nutrient stress and microaerobic growth conditions. The results presented in Chapter 2 address the evolution of aromatic β-glucoside catabolic systems when exposed to a novel β-glucoside as the sole substrate. The results indicate that the bgl opeon, the primary system involved in the utilisation of the aromatic β-glucosides arbutin and salicin, is also involved in esculin utilisation. In the absence of bglB encoding the enzyme phospho-β-glucosidase B, activation of the silent asc operon enables esculin utilisation. The bglA gene encoding phospho-β-glucosidase A specific for arbutin, can undergo successive mutations to evolve the ability to hydrolyse esculin and salicin sequentially when bglB and ascB are absent. The Esc+ and Sal+ mutants retain their arbutin+ phenotype, indicating that the mutations enhance the promiscuity of the enzyme. Sequencing data indicate that the first step Esc+ mutant carries a four base insertion within the promoter of the bglA gene that results in enhanced transcription of bglA. RT-PCR studies confirm that both the steady-state levels as well as the half-life of the bglA mRNA are enhanced in the mutant. This is further corroborated by the observation that overexpression of wild type bglA in the parent strain using a multicopy plasmid confers an Esc+ phenotype. The second step Sal+ mutant carries a point mutation within bglA ORF, a thymine to guanine transversion at position 583 (T583G) of the bglA gene, resulting in an amino acid change from cysteine to glycine at position 195 (C195G) of the BglA ORF close to the active site. Presence of a plasmid carrying the T583G mutation, introduced by site-directed mutagenesis, results in a Sal+ phenotype, confirming the role of the transversion in conferring the Sal+ phenotype. Based on docking studies, the positioning of salicin into the substrate binding site of the mutant BglA enzyme is different compared to wild type BglA due to the loss of stearic hindrance for the binding of salicin when C195 is replaced by the smaller amino acid glycine in the mutant protein. These observations indicate that under conditions of nutrient deprivation, exposure to novel substrates can result in the evolution of new metabolic capabilities by the sequential modification of a pre-existing genetic system. In the case of one novel substrate, the mutation results in the overexpression of the hydrolytic enzyme, while in the case of the second substrate, a mutation close to its active site increases its substrate specificity. Results presented in Chapter 3 specifically deal with the involvement of the bgl operon under low levels of oxygen. Earlier observations have shown that there is a 22 fold enhancement in the expression of the bgl operon under anaerobic condition. The present results provide evidence that bgl expression has a physiological role under low levels of oxygen and in addition suggest a possible mechanism for the overexpression of the bgl operon that involves the ArcAB two component system known to mediate regulation under microaerobic and static conditions. Transcription studies using a lacZ reporter fused to the wild type bgl promoter show that there is enhanced transcription from the bgl promoter under microaerobic and static conditions in the presence of arcA encoding the response regulator compared to that in its absence. The positive effect of arcA on the expression of the bgl operon is dispensable in the absence of H-NS since presence or absence of arcA does not change the expression of the bgl operon in an hns-null background, implying that the involvement of ArcA is via antagonizing H-NS. Competition experiments indicate that there is growth advantage associated with the activated allele of the bgl operon under low levels of oxygen since Bgl+ strains carrying the activated allele of the bgl operon as well as strains expressing BglG constitutively can out-compete wild-type strains. Presence of the wild type arcA allele results in a strong growth advantage compared to its absence under static conditions but not aerobic condition. The bgl operon seems to be one of the possible downstream targets of ArcA under static condition since absence of the bgl operon results in a modest reduction of the growth advantage (GASP) phenotype conferred by arcA. The up-regulation of the bgl operon is likely to enable the cells to scavenge available nutrients from their niche more efficiently. These experiments also show that the GASP phenotype associated with BglG constitutive strains under static conditions involves downstream genes that are different from oppA known to be one of the downstream targets during aerobic growth. It is possible that under low level of oxygen, the bgl operon is regulating a different set of downstream genes involving a different mechanism. In summary, the results of this investigation show that the aromatic β-glucoside catabolic systems in E. coli play a role in the generation of new metabolic capabilities via mutations in pre-existing genetic systems as well as through changes in gene expression patterns. The mechanisms outlined in this study are likely to be of broader significance applicable to microbial evolution under stress in general.