No description available.

The studies reported in this thesis describe the work done in the area of translation initiation where a previously unknown role of multiple copies of initiator tRNA in E. coli has been reported. Also the role of SsrA resume codon in resumption of translation, until not clearly known has been reported here. Chapter -1 discusses the relevant literature in understanding translation and initiator tRNA selection on the ribosome during initiation. It also discusses the literature pertaining to the aspect of release of stalled ribosomal complexes by SsrA. This is followed by the next chapter (chapter- 2) which discusses the materials and methods used throughout the study. Chapter- 3 describes the studies leading to the role of multiple copies of initiator tRNA in E. coli in governing the fidelity of initiator tRNA selection on the P site of the ribosome. This is followed by Chapter-4 which describes the role of the resume codon of the SsrA in governing the efficiency of trans-translation in releasing the stalled ribosomal complexes. The summaries of the chapters 3 and chapter 4 are briefly described below. i) Role of conserved 3GC base pairs of initiator tRNA in the initiator-elongator tRNA discrimination. Translation initiation is the first step in the very important and highly conserved biological process of protein biosynthesis. The process involves many steps, a wide array of protein factors at each specialized step and a large ribonucleoprotein particle; the ribosome to decode the information of the mRNA template into biologically active proteins. The process of initiation is still unclear largely due to fewer reports of available structural data. One of the very interesting questions that people have been trying to address is how the initiator tRNA is selected on the P- site of the ribosome and what is the importance of the conserved three GC base pairs in the anticodon stem of the initiator tRNA. Here in this study, I have studied this question by using the classical genetic technique of generating and characterizing the mutant initiator tRNA defective at the step of initiation. I have identified and analyzed the suppressors which are capable of rescuing this defect in initiation. The study involves two such E. coli suppressor strains (named D4 and D27). These suppressors can initiate translation from a reporter CAT mRNA with amber codon, independent of the presence of the three consecutive GC base pairs in the anticodon stem of initiator tRNAs. Mapping of the mutations revealed that the mutants are defective in expression of the tRNA1fMet (metZVW) gene locus which encodes the initiator tRNA. Both the suppressors (D4 and D27) also allow initiation with elongator tRNA species in E. coli. Taken together, the results show that E. coli when deficient in the initiator tRNA concentration can lead to initiation with elongator tRNA species. ii) The Role of SsrA/tmRNA in ribosome recycling and rescue. Occasionally during the process of translation, the ribosomes stall on the mRNA before the polypeptide synthesis is complete. This situation is detrimental to the organism because of the sequestration of the tRNAs as ‘peptidyl tRNAs’ and the ribosomes. In E. coli one of the pathways to rescue stalled ribosomes involves disassembly of these stalled complexes to release peptidyl tRNAs which are then recycled by peptidyl tRNA hydrolase (Pth), an essiential enzyme in E. coli. The other pathway which is not essential in E. coli but is conserved in all prokaryotes involves SsrA or tmRNA (transfer messenger RNA). The tmRNA is charged with alanine and recognizes the stalled ribosomal complexes and acts as tRNA to bind the A-site. It also functions as mRNA by adding a undecapeptide (which is actually a tag for degradation by cellular proteases) to the existing polypeptide and there is normal resumption of the translation. In most sequences of SsrA ORF, the first codon of the ORF, called as resume codon, is conserved. I wanted to understand the importance of the conservation of the resume codon. Towards this end I randomly mutated the resume codon and studied the effect of the altered resume codon in the rescue of stalled ribosomal complexes. The effect of over-expression of these mutants was investigated in the rescue of the Pthts defect since it is known that the overexpression of SsrA rescues the temperature sensitive phenotype of the Pthts strain and so causes less accumulation of peptidyl–tRNA in E. coli .The effect for these mutants has also been studied by the growth of hybrid λimmP22 phages. I also used AGA minigene system to study the effect of various mutants which has been shown to sequester tRNAArg (UCU) in the ribosomal P-site, translation of this minigene causes toxicity to E. coli. I have tried to study the effect of the SsrA mutants in rescue of toxicity caused by the minigene. Overall, the observations indicate that the conservation of the resume codon is important in E. coli and having mutated resume codon probably leads to deficient trans-translation during one or the other growth conditions.

Helicobacter pylori is one of the most potential and successful human pathogen which colonizes atleast 50% of world population. One of the important characteristics of this pathogen is the degree of allelic diversity and genetic variability which helps it to adapt and colonize. Phase variation is one of the mechanisms used by Helicobacter pylori to generate variation, where presence of homopolymeric nucleotide or dinucleotide repeats in an ORF make it prone to frequent length changes as a consequence of slipped strand mispairing mediated mutagenesis. An important feature of H. pylori biology is the presence of a large number of Restriction-Modification (R-M) systems in its genome. Till date, seven strains have been completely sequenced and each have more than 20 R-M systems. The presence of homopolymeric nucleotide or dinucleotide repeats in many of R-M systems make them an interesting subject for investigation. Here, we show that hp0051 which codes for a C5 cytosine methyltransferase from H. pylori is a hypermutable gene, which undergoes random mutations. In addition it exhibits phase variation due to presence of a dinucleotide (AG) repeat which results in a truncated protein. hp0051 homologs were amplified and sequenced from different clinical isolates of H. pylori. Sequence analysis showed that hp0051 homologs from 23 clinical isolates are different from each other suggesting a hypervariable nature of the sequence. It was observed that when over expressed in E. coli hp0051 undergoes random mutations. These mutations give rise to different variants of HP0051 with different biochemical properties. Different variants of HP0051 were biochemically characterized. All variants recognize 5´-CCTC-3´ and methylate the first cytosine. A few of the isoforms exhibit degeneracy in the recognition site as they recognize 5´- CNCC-3´ (N being any nucleotide) and methylates third cytosine. Molecular modelling studies suggest that HP0051 has two domains, one large domain having catalytic and AdoMet binding motifs and small domain having target recognition domain. DNA sequencing, peptide finger mapping, MALDI MS-MS and CD have been used to establish the differences between the different isoforms of HP0051. Interestingly when a mutant protein which lacks methylation activity was expressed in E.coli random mutations were not observed. To understand the role of methylation in the occurrence of random mutations, microarray analysis was done. Microarray analysis have shown that the overexpression of HP0051 results in the down regulation of deoxyadenine methyltransferase (dam) in E.coli. Microarray data were further authenticated by RT PCR analysis. dam plays a vital role in mismatch repair pathway and down regulation of dam results in enhanced mutation rates. A large number of clinical isolates were analysed for the presence of hp0051 and hp0051 was found to be present in 83% of strains obtained from patients compared to 25 % of strains from healthy volunteers. Single colonies obtained from the same patient were analysed and it was found that variation in hp0051 exists within a patient also. Deletion of an orphan C5 cytosine methyltransferase, hp0051 in H. pylori strains 26695, SS1 and 98.4 has a significant effect on the expression of number of genes belonging to motility, adhesion and virulence. 98.4∆hp0051 mutant strain has a different LPS profile and is able to induce high IL-8 production compared to wild-type. H. pylori strain 26695∆hp0051 is more motile than the wild- type. hp0051 from strain 26695 is able to complement mutant SS1 and 98.4 strains. This study highlights the possible significance of cytosine methylation in the physiology of H. pylori. hp0050 is a N6 DNA adenine methyltransferase which overlaps with the hp0051 ORF .hp0050 was cloned, over expressed and purified to near homogeneity. It recognizes the sequence 5´GRRG 3´ (where R is A or G) and most intriguingly methylates both adenines when R is A (5´GAAG 3´). Kinetic analysis suggest a non processive (repeated hit) mechanism of methylation in which HP0050 methyltransferase methylates one adenine at a time in sequence 5´GAAG 3´. Interestingly, HP0050 homologs from two clinical strains PG227 and 128 methylate only 5´GAGG 3´ compared to 5´GRRG 3´ in strain 26695. HP0050 MTase is highly conserved as it is present in more than 90% of strains. Inactivation of hp0050 in strain PG227 resulted in poor growth suggesting its important role in the physiology of Helicobacter pylori. Collectively, these findings provide impetus for exploring the role(s) of this conserved DNA methyltransferase in the cellular processes of Helicobacter pylori. In one of the clinical isolate it was found that hp0051 and hp0050 can code for a single polypeptide due to an insertion mutation. This mutant ( hp0050 and hp0051 fusion ) was cloned, overexpressed and purified. It was found that fusion protein is able to methylate both adenine and cytosine in the cognate sequence. Similarly, hp1369 - hp1370 is a phase variable type III MTase and it belongs to ɛ group of MTases based on the arrangement of motifs. It has a poly G repeat in its ORF and any change in the number of repeats can result in a functional (full length) or non functional (truncated) protein. Within strain 26695, it has 10 G repeat which results in a truncated protein. Addition of a single nucleotide by site directed mutagenesis in the repeat results in a full length functional protein. HP1369_HP1370 fusion protein recognizes and methylates 5´ TCAGC 3´. DNA methyltransferases are known to play a critical role in gene regulation, cell cycle regulation and pathogenesity in a number of pathogens. H. pylori genome is rich in DNA methyltransferases and this study shows that these methyltransferases exhibit unique features like phase - variation and polymorphism .We propose that high degree of variation that exists in these methyltransferases could play a vital role in enhancing the ability of H. pylori to adapt its host.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

As enzimas lipolíticas vêm atraindo atenção no mercado global devido ao enorme potencial biotecnológico, como: na formulação de detergentes; na indústria de couro; produção de cosméticos, fármacos, aromas, biodiesel, etc. O objetivo deste trabalho foi prospectar genes codificadores de enzimas lipolíticas em biblioteca metagenômica de um consórcio microbiano degradador de óleo diesel. A seleção foi feita pela atividade lipolítica através do cultivo dos clones em placa de petri e a avaliação foi pela observação de halo ao redor da colônia, sendo positiva para 30 clones dentre os quais dois se destacaram. Estes dois clones foram selecionados e subclonados. Os DNAs das sub-bibliotecas foram sequenciados, gerando um contig completo para cada clone. Através do ORF Finder foi identificado cinco ORFs de esterase/lipase, dentre as quais uma alcançou 58% de identidade com uma bactéria não cultivável. As árvores filogenéticas indicam que duas ORFs são similares à família IV das enzimas lipolíticas, enquanto que as outras três ORFs à família V. / Lipolytic enzymes have been attracting global market attention because they show enormous biotechnological potential. The present work was done as an attempt to find genes which codify lipolytic enzymes in a metagenomic library composed of diesel oil degradation microbe consortia. Clones were selected according to lipolytic activity and were then evaluated after cultivation in Petri dishes by observation of halo formation around the colonies. 30 clones produced halo formations and were identified as positives, two of which showed prominent results. These two were then selected and sub cloned. DNA from the sub libraries was sequenced, generating a complete contig for each clone. Using the ORF Finder five esterase/lipase ORFs were identified, with one of these attaining 58% of identity to a non cultivatable bacteria species. Assessment of the cladograms showed that two ORFs were similar to lipolytic enzyme family IV, while the other three ORFs were similar to family V.