Suvendra Kumar Ray

Mutants of Xanthomonas oryzae pv. oryzae deficient in general secretory pathway are virulence deficient and unable to secrete xylanase

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, a serious disease of rice. A virulence- and xylanase-deficient mutant of Xoo was isolated following ethyl methane sulfonate (EMS) mutagenesis. A cosmid clone that restored virulence and xylanase secretion was obtained from a genomic library by functional complementation. Transposon mutagenesis and marker exchange studies revealed genes on the cloned DNA that were required for xylanase production and virulence. Sequence analysis with transposon-specific primers revealed that these genes were homologues of xps F and xps D, which encode components of a protein secretion system in Xanthomonas campestris pv. campestris. Enzyme assays showed xylanase accumulation in the periplasmic space and cytoplasm of the xps F mutant and the complementing clone restored transport to the extracellular space.

A Study in Entire Chromosomes of Violations of the Intra-strand Parity of Complementary Nucleotides (Chargaff's Second Parity Rule)

Chargaffs rule of intra-strand parity (ISP) between complementary mono/oligonucleotides in chromosomes is well established in the scientific literature. Although a large numbers of papers have been published citing works and discussions on ISP in the genomic era, scientists are yet to find all the factors responsible for such a universal phenomenon in the chromosomes. In the present work, we have tried to address the issue from a new perspective, which is a parallel feature to ISP. The compositional abundance values of mono/oligonucleotides were determined in all non-overlapping sub-chromosomal regions of specific size. Also the frequency distributions of the mono/oligonucleotides among the regions were compared using the Kolmogorov–Smirnov test. Interestingly, the frequency distributions between the complementary mono/oligonucleotides revealed statistical similarity, which we named as intra-strand frequency distribution parity (ISFDP). ISFDP was observed as a general feature in chromosomes of bacteria, archaea and eukaryotes. Violation of ISFDP was also observed in several chromosomes. Chromosomes of different strains belonging a species in bacteria/archaea (Haemophilus influenza, Xylella fastidiosa etc.) and chromosomes of a eukaryote are found to be different among each other with respect to ISFDP violation. ISFDP correlates weakly with ISP in chromosomes suggesting that the latter one is not entirely responsible for the former. Asymmetry of replication topography and composition of forward-encoded sequences between the strands in chromosomes are found to be insufficient to explain the ISFDP feature in all chromosomes. This suggests that multiple factors in chromosomes are responsible for establishing ISFDP.

Transfer RNA Gene Numbers may not be Completely Responsible for the Codon Usage Bias in Asparagine, Isoleucine, Phenylalanine, and Tyrosine in the High Expression Genes in Bacteria

It is generally believed that the effect of translational selection on codon usage bias is related to the number of transfer RNA genes in bacteria, which is more with respect to the high expression genes than the whole genome. Keeping this in the background, we analyzed codon usage bias with respect to asparagine, isoleucine, phenylalanine, and tyrosine amino acids. Analysis was done in seventeen bacteria with the available gene expression data and information about the tRNA gene number. In most of the bacteria, it was observed that codon usage bias and tRNA gene number were not in agreement, which was unexpected. We extended the study further to 199 bacteria, limiting to the codon usage bias in the two highly expressed genes rpoB and rpoC which encode the RNA polymerase subunits β and β′, respectively. In concordance with the result in the high expression genes, codon usage bias in rpoB and rpoC genes was also found to not be in agreement with tRNA gene number in many of these bacteria. Our study indicates that tRNA gene numbers may not be the sole determining factor for translational selection of codon usage bias in bacterial genomes.It is generally believed that the effect of translational selection on codon usage bias is related to the number of transfer RNA genes in bacteria, which is more with respect to the high expression genes than the whole genome. Keeping this in the background, we analyzed codon usage bias with respect to asparagine, isoleucine, phenylalanine, and tyrosine amino acids. Analysis was done in seventeen bacteria with the available gene expression data and information about the tRNA gene number. In most of the bacteria, it was observed that codon usage bias and tRNA gene number were not in agreement, which was unexpected. We extended the study further to 199 bacteria, limiting to the codon usage bias in the two highly expressed genes rpoB and rpoC which encode the RNA polymerase subunits β and β′, respectively. In concordance with the result in the high expression genes, codon usage bias in rpoB and rpoC genes was also found to not be in agreement with tRNA gene number in many of these bacteria. Our study indicates that tRNA gene numbers may not be the sole determining factor for translational selection of codon usage bias in bacterial genomes.

rpoN1, but not rpoN2, is required for twitching motility, natural competence, growth on nitrate, and virulence of Ralstonia solanacearum

The plant pathogen Ralstonia solanacearum has two genes encoding for the sigma factor σ54: rpoN1, located in the chromosome and rpoN2, located in a distinct “megaplasmid” replicon. In this study, individual mutants as well as a double mutant of rpoN were created in R. solanacearum strain GMI1000 in order to determine the extent of functional overlap between these two genes. By virulence assay we observed that rpoN1 is required for virulence whereas rpoN2 is not. In addition rpoN1 controls other important functions such twitching motility, natural transformation and growth on nitrate, unlike rpoN2. The rpoN1 and rpoN2 genes have different expression pattern, the expression of rpoN1 being constitutive whereas rpoN2 expression is induced in minimal medium and in the presence of plant cells. Moreover, the expression of rpoN2 is dependent upon rpoN1. Our work therefore reveals that the two rpoN genes are not functionally redundant in R. solanacearum. A list of potential σ54 targets was identified in the R. solanacearum genome and suggests that multiple traits are under the control of these regulators. Based on these findings, we provide a model describing the functional connection between RpoN1 and the PehR pathogenicity regulator and their dual role in the control of several R. solanacearum virulence determinants.

Selection on GGU and CGU codons in the high expression genes in bacteria.

The fourfold degenerate site (FDS) in coding sequences is important for studying the effect of any selection pressure on codon usage bias (CUB) because nucleotide substitution per se is not under any such pressure at the site due to the unaltered amino acid sequence in a protein. We estimated the frequency variation of nucleotides at the FDS across the eight family boxes (FBs) defined as Um(g), the unevenness measure of a gene g. The study was made in 545 species of bacteria. In many bacteria, the Um(g) correlated strongly with Nc′—a measure of the CUB. Analysis of the strongly correlated bacteria revealed that the U-ending codons (GGU, CGU) were preferred to the G-ending codons (GGG, CGG) in Gly and Arg FBs even in the genomes with G+C % higher than 65.0. Further evidence suggested that these codons can be used as a good indicator of selection pressure on CUB in genomes with higher G+C %.

Cotranslational protein folding reveals the selective use of synonymous codons along the coding sequence of a low expression gene.

Due to the degeneracy in the genetic code, many amino acids are encoded by more than one codon, called synonymous codons. The usage of synonymous codons in a coding sequence is not random, a phenomenon known as codon usage bias (CUB) that occurs in all genomes. It is believed that synonymous codons are not equivalent with respect to their coding efficiency during translation. This phenomenon has been demonstrated by the difference between the high expression genes (HEG) and low expression genes (LEG) within a genome with respect to the compositional abundance values of different synonymous codons (Ermolaeva 2001; Hershberg and Petrov 2008; Plotkin and Kudla 2011). Optimal codons, whose frequency is higher in HEGs than in LEGs or whole genome, are believed to be translated more rapidly and accurately than the other synonymous codons or nonoptimal codons (Sharp et al. 2005; Ran and Higgs 2010; Satapathy et al. 2014). As greater numbers of proteins are synthesized by the HEGs (Ghaemmaghami et al. 2003; dos Reis et al. 2003; Ishihama et al. 2008; Hiraoka et al. 2009), selection pressure on the coding sequence of these genes is believed to be higher for efficient translation to occur. Therefore, synonymous codons that are more efficient in translation are found in higher frequency in these genes in comparison to the LEGs (Satapathy et al. 2012). Thus, the translational selection pressure is considered to be the major determining factor for CUB in HEGs. In contrast, translational selection pressure is believed to be weaker in LEGs as a fewer number of protein molecules are synthesized by these genes. Therefore, CUB in LEGs are predominantly determined by mutation pressures such as genomeG+C composition (Muto and Osawa 1987; Palidwor et al. 2010) and strand

Higher tRNA diversity in thermophilic bacteria: A possible adaptation to growth at high temperature

We have done a comparative study of tRNA diversity and total tRNA genes among different strains of bacteria with respect to the optimum growth temperature of the cells. Our observation suggests that higher tRNA diversity usually occurs in thermophiles in comparison to non-thermophiles. Among psychrophiles total tRNA was observed to be more than two-fold higher than in the non-psychrophiles. Though tRNA diversity and total tRNA have recently been shown to be affected by an organisms genomic GC% and growth rate, this work is the first report on growth temperature affecting these features in bacteria. This work extends the list of molecular features undergoing adaptation due to growth temperature and supports the view that growth temperature acts as a selecting factor at the molecular level during evolution.

Strand‐specific mutational bias influences codon usage of weakly expressed genes in Escherichia coli

According to the selection‐mutation‐drift theory of molecular evolution, mutation predominates in determining codon usage bias (CUB) in weakly expressed genes (WEG) whereas selection predominates in determining CUB in highly expressed genes (HEG). Strand‐specific mutational bias causes compositional asymmetry of the nucleotides between leading and lagging strands (LaS) in bacterial chromosomes. Keeping in view the aforementioned points, CUB between the strands were compared in Escherichia coli chromosome. In comparison with HEG, codon usage of WEG was observed to be more biased toward strands: G ending codons were significantly more in leading strands than in LaS and the reverse was true for the C ending codons. In case of WEG, the GC3 skews were found to be significantly different between the strands. This suggests that strand‐specific mutational bias influences codon usage of WEG to a greater extent than that of HEG. The differential effect of strand‐specific mutational bias in E. coli might be attributed to stronger purifying selection in the HEG than the WEG. The observation here in E. coli supports the SMD theory of molecular evolution.

Codon degeneracy and amino acid abundance influence the measures of codon usage bias: improved Nc (N̂c) and ENCprime (N̂′c) measures

Effective number of codons ( N^c ) and its variant N^′c (effective number of codons prime) are the two widely used methods for measuring unequal usage of synonymous codons in coding sequences, known as the codon usage bias (CUB). The mathematical formula used in calculating N^c and N^′c values is giving inappropriate measures of CUB in case of low abundance of amino acids. In addition, the magnitude of error also varies according to codon degeneracy. In this study, a modified formula for N^c and N^′c has been developed to measure the CUB more accurately. Online implementations of the modified formula are available in the web portal at http://agnigarh.tezu.ernet.in/~ssankar/cub.php.

Synonymous codons influencing gene expression in organisms

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