To further understand the ATCase/DHOase bifunctional complex formed in Streptomyces, the genes encoding these and other pyrimidine enzymes were identified and characterized. Polymerase chain reaction (PCR) was utilized in this effort. Primers were constructed by selecting conserved regions of pyrimidine genes from known gene and protein sequences of a wide variety of organisms. These sequences were then optimized to Streptomyces codon usage. PCR products were obtained from internal sites within pyrimidine genes and also from primer combinations of different genes. The size, orientation, and partial sequence of the resulting products shows that Streptomyces has a gene organization of pyrR followed by pyrB, pyrC, carA, carB, and pyrF in an operon similar to that found in other Gram-positive bacteria.

The plsX50 mutation is required together with plsX26 (encoding a K_m- defective glycerol 3-phosphate acyltransferase) for the conferral of a glycerol 3- phosphate auxotrophic phenotype. A 4.9 kb segment of DNA complementing the plsX50 mutation have been cloned and sequenced. Six open reading frames (ORF’s) were found with five reading in the same direction and one in the opposite direction relative to the plsX gene. Each ORF encoded a protein, as demonstrated by radiolabeling in maxicells. ORF1 (orfY) encodes a protein of unknown function. ORF2 and ORF3 (rpmF) were sequenced prior to this study and encode a protein called G30k of unknown function and L32, a protein of the large ribosomal subunit, respectively. ORF4 complemented the p/sX50 mutation. ORFS was identified as fabH encoding 3-ketoacyl-ACP synthase III. ORF6 was identified as fabD encoding malonyl-CoA/ACP transacylase. The fabG gene encoding 3- ketoacyl-ACP reductase and the acpP gene encoding acy] carrier protein are located just downstream of the fabD gene. Northern and promoter activity analysis demonstrated that the rpmF-plsX-fabH-fabD-fabG-acpP genes comprise an operon suggesting a coordinate control of the synthesis of a ribosomal protein (L32), PlsX protein, and fatty acid biosynthetic enzymes. However, several features were identified that are likely to be important for differential expression of the individual genes. These include the presence of multiple promoters, an internal terminator (attenuator), differential degradation of transcripts, and differential efficiency of translation initiation. Portions of transcripts arising upstream of rpmF terminate at the attenuator located just downstream of the plsX initiation codon, and some of the transcripts continue into the plsX-jab genes. The fabH-fabD-fabG-acpP genes are also cotranscribed from a promoter located upstream of the fabH gene, within the pisX structural gene. There are additional cotranscripts responsible for the expression of the fabD-fabG-acpP genes. The acpP gene is encoded by several more transcripts. Transcription initiation sites upstream of rpmF were identified by primer extension analysis and the attenuator site was identified by S1 mapping analysis. N-terminal amino acid sequence analysis identified the translation initiation codons of orfY, plsX, fabH, fabD and fabG. Short intergenic distances (15 and 12 bp) found between fabH and fabD and between fabD and fabG implicate translational coupling as a mechanism for coordinate control of fabHDG expression. The p/sX50 mutation was identified as deletion of a single nucleotide from the 6th codon of plsX resulting in a frame shift nonsense mutation. / Ph. D.

The genes encoding malate dehydrogenase (mdh), succinyl-CoA synthetase (sucCD), and subunits of 2-oxoglutarate dehydrogenase (sucAB) constitute an operon in the order mdh-sucCDAB in Sinorhizobium meliloti. Regulation of the operon was studied using beta-galactosidase gene fusions. Expression of the operon was assayed in response to the carbon source provided, and over the growth of the culture. A promoter upstream of the mdh gene was identified, and although the promoter was active in S. meliloti it was not expressed in Escherichia coli. It was demonstrated that the role of 2-oxoglutarate dehydrogenase (OGD) is minimal in symbiosis, as nodules with no OGD activity formed nodules able to fix nitrogen. Alfalfa plants inoculated with strains of S. meliloti carrying extra-chromosomal copies of the mdh gene did not show any increase in shoot dry weight compared to plants inoculated with the wild-type strain.

En bacteris entèrics, les proteïnes de la família Hha/YmoA estan implicades en la regulació de l'expressió gènica depenent factors ambientals. Ha estat descrit que la proteïna Hha d' Escherichia coli interacciona amb la proteïna H-NS formant un complex amb el DNA responsable de la termoregulació de l'expressió de l'operó hly d' E. coli. Altres proteïnes d'aquesta família, com YmoA i YdgT, també interaccionen amb la proteïna H-NS.La proteïna H-NS d' E. coli és una proteïna associada al nucleoide implicada en la regulació global de l'expressió gènica en funció de les condicions ambientals. H-NS es troba àmpliament distribuida entre bacteris Gram-negatius.El primer objectiu va ser determinar si la participació conjunta de les proteïnes Hha i H-NS era extensible a altres gens regulats per H-NS. Escollirerm l'operó bgl, silenciat per la proteïna H-NS. Mitjançant mesures de l'activitat i de la trasncripció de l'operó vam concloure que les proteïnes Hha i YdgT intervenen en el silenciament de l'operó bgl d'Escherichia coli.Es va analitzar la regulació de l'expressió gènica depenent de les proteïnes Hha i H-NS en diferents condicions d'osmolaritat per identificar les proteïnes diferencialment expressades. S'identificaren les proteïnes HdeA, l'expressió de la qual ja havia estat descrita com a regulada per H-NS; i HtrA, una proteasa sotmesa a osmoregulació. El complex Hha-H-NS és responsable de la repressió de l'expressió d'HtrA a baixa osmolaritat.En la seqüència completa del plàsmid R27 s'han identificat dos gens (orf182 i orf164) que codifiquen dues proteïnes homòlogues a Hha i H-NS, respectivament. Ja que la conjugació d'aquest plàsmid està sotmesa a termoregulació, i donat que les proteïnes Hha i H-NS estan implicades en la regulació de l'expressió gènica depenent de paràmetres ambientals, es va voler determinar: · A) el paper d'aquestes proteïnes, d'origen plasmídic i d'origen cromosòmic, en la regulació de la conjugació de R27,· B) i la intervenció de les proteïnes de codificació plasmídica en la fisiologia de la cèl·lula portadora del plàsmid. Es realitzaren estudis de la freqüència de conjugació del plàsmid, estudis de la transcripció dels gens de transferencia mitjançant microxips i RT-PCR, assaigs de retard en gel amb les proteïnes Hha i H-NS i observació de pilis per MET. Es va concloure que les proteïnes Hha i H-NS, tant de codificació plasmídica com cromosòmica, interven en la termoregulació de la conjugació del plàsmid, reprimint l'expressió dels gens de transferencia a elevada temperatura. En estudis de complementació s'observà que les proteïnes codificades als gens orf182 i orf164, compensen alguns fenotips causats per les mutacions dels gens hha i hns. Es va demostrar també la interacció in vitro de la proteïna H-NS plasmídica (ORF164) amb la proteïna Hha cromosòmica.Finalment, es va realitzar una anàlisi trancriptòmica de l'efecte de les mutacions hns i hha ydgT en el patró d'expressió gènica a Escherichia coli, per determinar quins gens estaven afectats en cada fons genètic i establir la relació entre la regulació per H-NS i per Hha/YdgT. La comparació dels patrons d'expressió entre la soca hns i la soca salvatge, han posat de manifest que un 7% dels gens d'E. coli presenten una alteració significativa de la seva expressió, i que un 68% dels casos corresponen a una sobreexpressió en la soca hns. L'analisi de l'expressió diferencial entre la soca hha ydgT i la soca hns demostra que la regulació de la soca hha ydgT segueix majoritàriment el mateix patró d'expressió que la soca salvatge. Es van identificar 22 gens amb expressió diferencial entre la soca hha ydgT i la soca salvatge, dels quals 18 presenten sobreexpressió en un fons genètic hha ydgT, i 12 coincideixen amb els gens regulats per H-NS. / In enteric bacteria, the proteins of Hha/YmoA family play an important role in regulation of gene expression in response to environmental signals. On another part, the nucleoid-associated protein H-NS is a relevant example of a global modulator. H-NS binds to Hha, and other proteins of Hha/YmoA family, to regulate gene expression depending on environmental conditions.The first goal was to determine if Hha and H-NS participate together regulating genes known as regulated by H-NS, like "bgl" operon silenced by H-NS, or other new genes in different osmolarity conditions. We conclude that Hha play a role in silencing "bgl" operon expression in "E. Coli" and that Hha-H-NS complex is responsible for the HtrA repression at low osmolarity.R27 plasmid code two proteins homologous to Hha and H-NS, respectively. Since R27 conjugation is thermoregulated, and given the fact that Hha and H-NS regulate gene expression depending on environmental parameters, we investigated the role of these proteins, in thermoregulation of R27 conjugation. We conclude that Hha and H-NS, of plasmidic and chromosomic origin, downregulate plasmid conjugation at high temperature. As well, we studied the intervention of Hha and H-NS R27 encoded proteins in the physiology of R27 host cells. We observed that these proteins compensate some of the phenotypes caused by hha and hns mutations.Finally, a transcriptomic analysis of the effect of the mutations "hns" and "hha ydgT" in "E. coli"i was carried out to establish the relationship between H-NS and Hha/YdgT regulation. The result showed that 7% of genes were affected by "hns" mutation and that 68% of these cases corresponds to an overexpression in hns strain. The analysis of "hha ydgT" strain demonstrates that its regulation follows mostly the same pattern of expression than the wild type strain, because "hha ydgT" mutations only affected 22 genes.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-13T18:17:32Z No. of bitstreams: 1 resumo.pdf: 17263 bytes, checksum: 8e51a65fe7d8eecb448411acb64cf05b (MD5) / Made available in DSpace on 2017-06-13T18:17:32Z (GMT). No. of bitstreams: 1 resumo.pdf: 17263 bytes, checksum: 8e51a65fe7d8eecb448411acb64cf05b (MD5) Previous issue date: 2003-02-20 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / A estirpe probiótica Lactobacillus UFV H2b20, previamente classificada como Lactobacillus acidophilus por suas características de fermentação de açúcares, apresentou-se mais semelhante à espécie Lactobacillus delbrueckii, quanto à seqüência de rDNA 16S, o que levou ao questionamento acerca da identidade da linhagem. Para o esclarecimento da real classificação da linhagem, o método de hibridização DNA-DNA foi empregado. A linhagem apresentou 75,2% e 77,4% de reassociação com L. delbrueckii subsp. lactis (ATCC 12315) e L. delbrueckii subsp. delbrueckii (ATCC 9649), respectivamente. Dado que a homologia de 70% ou mais, por esse método, tem sido usada como padrão para agrupamento de bactérias em uma mesma espécie, sugere-se, aqui, que Lactobacillus UFV H2b20 seja, daqui para frente, denominado L. delbrueckii UFV H2b20. Identificada a linhagem, outro objetivo do trabalho era desenvolver um protocolo para detecção in situ de L. delbrueckii. Uma sonda de 26 nucleotídeos (SA) foi construída e testada com outras espécies de Lactobacillus relacionadas geneticamente entre si. Estes estudos demonstraram que a seqüência de assinatura (SA) estava presente em L. delbrueckii UFV H2b20, L. delbrueckii UFV H2b21, L. delbrueckii subsp. delbrueckii e L. delbrueckii subsp. lactis, o que indica ser ela eficaz para ser usada como sonda para rRNA 16S espécie-específica pelo método de FISH. A hipótese de existência de polimorfismos, levantada em trabalhos prévios no rDNA 16S da linhagem, foi confirmada após as análises dos segmentos de DNA clonados e selecionados do banco genômico construído para a linhagem L. delbrueckii UFV H2b20. As seqüências analisadas demonstraram, também, presença de segmentos correspondentes a quatro genes codificadores de rRNA 16S distintos, e seis segmentos distintos para uma mesma região de rRNA 23S, indicando seis operons putativos. Há evidência de, pelo menos, um operon putativo completo seguido de região codificadora de seis tRNAs. Não se detectou região espaçadora longa entre rDNA 16 e 23S. / Lactobacillus UFV H2b20, a probiotic strain, previously identified as Lactobacillus acidophilus due to its sugar fermentation pattern, was found to be more closely related to Lactobacillus delbrueckii regarding its 16S rDNA sequence. It was demonstrated by DNA-DNA hybridization that this strain presented 75.2% and 77.4% of reassociation with L. delbrueckii subsp. lactis ATCC 12315 and L. delbrueckii subsp. delbrueckii ATCC 9649, respectively. These results place Lactobacillus UFV H2b20 within the L. delbrueckii species, for 70% reassociation as measured by the method used has been a standard to cluster bacteria within the same species. A protocol for in situ detection of L. delbrueckii was developed by means of Fluorescent in situ Hybridization, FISH. A probe consisting of 26 nucleotides labeled with rhodamine was designed based on the signature sequence within the rDNA, and was tested against genetically related Lactobacillus species. A species- specific method was obtained capable of discriminating L. delbrueckii strains from other Lactobacillus species. Previous studies raised the hypothesis of polymorphism among the copies of 16S rDNA in L. delbrueckii UFV H2b20. This was confirmed by sequence analysis of rDNA from a gene library of this strain cloned in phage lambda and subcloned in pBluescript. Sequence analyses of cloned fragments demonstrated the presence of at least four distinct genes encoding 16S rRNAs. Distinct fragments containing 23S rRNA related genes indicated six putative rrn operons. One complete putative rrn operon displays a region encoding 6 different tRNAs. Long spacer regions between 16S and 23S rDNA were not detected.

No description available.

En la adaptación de las bacterias ante cambios ambientales juegan un importante papel las proteínas asociadas al nucleoide. Estas proteínas presentan una doble función: la estructuración del nucleoide bacteriano y otros procesos relacionados con el ADN como es la regulación de la expresión génica. Una de las proteínas asociadas al nucleoide mejor caracterizada es la proteína H-NS, que se encuentra ampliamente distribuída en bacterias G(-). Una de las características de H-NS es su capacidad de formar dímeros con miembros de la familia de proteínas Hha/YmoA, formando de esta manera complejos represores que intervienen en la regulación de determinados operones, como es el caso del operón hly de E. coli. En este trabajo, y en colaboración con el grupo de RMN de biomoléculas dirigido por el doctor Miquel Pons, se han realizado una serie de trabajos con la intención de conocer mejor las características estructurales de la proteína Hha y su interacción con H-NS, en los que se ha puesto de manifiesto que, aún sin ser unos de los residuos aminoacídicos más afectados en la interacción de Hha con H-NS, la cisteína en posición 18 juega un importante papel en el equilibrio conformacional de Hha cuando interacciona con H-NS. La sustitución de esta cisteína por una isoleucina da lugar a una proteína Hha mutante incapaz de complementar la mutación hha y que provoca una reducción en la tasa de crecimiento de E. coli en condiciones de baja osmolaridad. Este efecto podría venir explicado por la mayor resistencia a la fuerza iónica del medio de la interacción de esta proteína Hha mutante con la proteína H-NS, lo que podría provocar la desregulación de algún gen/es esenciales en condiciones de baja osmolaridad. Una segunda parte de esta Tesis Doctoral está dedicada al gen hns de Y. enterocolitica. Estudios previos realizados en nuestro grupo de investigación pusieron de manifiesto la esencialidad del gen hns en esta bacteria, ya que únicamente es posible obtener mutantes hns en Y. enterocolitica en presencia de algún miembro funcional de la familia de proteínas H-NS. En esta Tesis, nos centramos en el sistema que permite obtener un mutante hns en presencia de la proteína StpA, paráloga a H-NS en E. coli. Los resultados obtenidos permitieron comprobar que la presencia de StpA en Y. enterocolitica provoca drásticas alteraciones en su patrón de expresión proteico, efecto no observado en otras bacterias entéricas, y pusieron de manifiesto la importancia de las proteínas H-NS e YmoA en esta bacteria. La proteína YmoA, que interviene en la termorregulación de la expresión de factores de virulencia en Y. enterocolitica, es homologa a la proteína Hha de E. coli, y también es capaz de interaccionar con H-NS. El análisis de estos resultados ha puesto de manifiesto, por un lado, que la presencia de StpA en Y. enterocolitica simula un incremento no fisiológico en los niveles de proteína H-NS, y por otro lado, la importancia no solo de la presencia de las proteínas H-NS e YmoA en Y. enterocolitica, sino también la importancia de sus niveles relativos, ya que un incremento en los niveles de H-NS o una disminución en los de YmoA provocan una drástica alteración en el patrón de expresión proteico de Y. enterocolitica. / "Hha and YmoA family of proteins: structural studies and regulatory role in Y. enterocolitica"TEXT:H-NS protein is a nucleoid-associated protein, widely dstribute in Gram - bacteria. It has a dual function: nucleoid structure protein and others processes related to DNA like gene expression regulation. This protein is able to both oligomerize and heterodimerize with other proteins. One of the partners of these heterodimers are members of Hha/YmoA family of proteins.In the current work, we carry out the production of both Hha and H-NS proteins in order to perform structural studies by RMN. These studies show that cysteine at position 18 in Hha protein is not one of the most affected aminoacids for Hha/H-NS interaction. However, its location between two α-helix domains suggested that it might be an important residue for Hha conformation during its interaction with H-NS. Our results demonstrated that cys 18 substitution by ile, which hypothetically gives rise to a more open structure, genetates a non-functional protein. In addition, we could observe a toxic effect under low osmolarity condition in E. coli. Finally, a higher stability in H-NS/Hha interaction could be observed.The second part of this work deals with Y. enterocolitica hns gene. hns mutants can only be obtained in this bacteria in presence of some functional members of this protein family. This fact is an evidence of the essenciality of hns in Y. enterocolitica. For instance, an hns mutant can be isolated in presence of stpa gene, which is an hns paralogous gene in E.coli, but not present in Y. enterocolitica. Our results demonstrate that drastic effects in the protein expression pattern of Y. enterocolitica are produced in presence of StpA. The analysis of the previous results gave evidence of the importance of YmoA and H-NS proteins in Y. enterocolitica. Not only the presence of the proteins but also their relative levels turned out to be important, since both an increase in H-NS levels and a decrease in YmoA levels gave rise to a dramatic change in Y. enterocolitica protein expression pattern.

The vast majority of valuable agronomic traits are encoded polygenetically. Chloroplast genetic engineering offers an alternate approach to multigene engineering by allowing the insertion of entire pathways in a single transformation event, while being an environmentally friendly approach. Stable integration into the chloroplast genome and transcription of the phaA gene coding for β-ketothiolase was confirmed by Southern and northern blots. Coomassie-stained gel and western blots confirmed hyperexpression of β-ketothiolase in leaves and anthers, with high enzyme activity. The transgenic lines were normal except for the male sterile phenotype, lacking pollen. Scanning electron microscopy revealed a collapsed morphology of the pollen grains. Transgenic lines followed an accelerated anther developmental pattern, affecting their development and maturation, resulting in aberrant tissue patterns. Abnormal thickening of the outer wall, enlarged endothecium and vacuolation, decreased the inner space of the locules, affecting pollen grain and resulted in the irregular shape and collapsed phenotype. Reversibility of the male sterility phenotype was achieved by exposing the plants to continuous illumination, producing viable pollen and copious amounts of seeds. This is the first report of engineered cytoplasmic male sterility and offers a new tool for transgene containment for both nuclear and organelle genomes. Detailed characterization of transcriptional, posttranscriptional and translational processes of heterologous operons expressed via the chloroplast genome is reported here. Northern blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons, revealed that in most cases, only polycistronic mRNA was produced or polycistrons were the most abundant form and that they were not processed into monocistrons. Despite such lack of processing, abundant foreign protein accumulation was detected in these transgenic lines. Interestingly, a stable secondary structure formed from a heterologous bacterial intergenic sequence was recognized and efficiently processed, indicating that the chloroplast posttranscriptional machinery can indeed recognize sequences that are not of chloroplast origin, retaining its prokaryotic ancestral features. Processed and unprocessed heterologous polycistrons were quite stable even in the absence of 3'UTRs and were efficiently translated. Unlike native 5'UTRs, heterologous secondary structures or 5'UTRs showed efficient translational enhancement independent of any cellular control. Finally, we observed abundant read-through transcription in the presence of chloroplast 3'UTRs. Such read-through transcripts were efficiently processed at introns present within native operons. Addressing questions about polycistrons, as well as the sequences required for their processing and transcript stability are essential for future approaches in metabolic engineering. Finally, we have shown phytoremediation of mercury by engineering the mer operon via the chloroplast genome under the regulation of chloroplast native and heterologous 5'UTRs. These transgenenic plants hyperexpress were able to translate MerA and MerB enzymes to levels detectable by coomassie stained gel. The knowledge acquired from these studies offer guidelines for engineering multigene pathways via the chloroplast genome.

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 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.