RESULTS:
1 - 6 of 6 for "Alexander Bolotin"
Long inverted repeats around the chromosome replication terminus in the model strain Bacillus thuringiensis serovar israelensis BGSC 4Q7
Bacillus thuringiensis serovar israelensis is the most widely used natural biopesticide against mosquito larvae worldwide. Its lineage has been actively studied and a plasmid-free strain B . thuringiensis serovar israelensis BGSC 4Q7 (4Q7) has been produced. Previous sequencing of the genome of this strain has revealed the persistent presence of a 235 kb extrachromosomal element pBtic235 which has been shown to be an inducible prophage although three putative chromosomal prophages have been lost. Moreover a 492 kb region potentially including the standard replication terminus has also been deleted in the 4Q7 strain indicating an absence of essential genes in this area. We reanalysed the genome coverage distribution of reads for the previously sequenced variant strain and sequenced two independently maintained samples of the 4Q7 strain. A 553 kb area close to the 492 kb deletion was found to be duplicated. This duplication presumably restored the equal sizes of the replichores and a balanced functioning of replication termination. An analysis of genome assembly graphs revealed a transient association of the host chromosome with the pBtic235 element. This association may play a functional role in the replication of the bacterial chromosome and the termination of this process in particular. The genome-restructuring events detected may modify the genetic status of cytotoxic or haemolytic toxins potentially influencing strain virulence. Twelve of the single-nucleotide variants identified in 4Q7 were probably due to the procedure used for strain construction or were present in the precursor of this strain. No sequence variants were found in pBtic235 but the distribution of the corresponding 4Q7 reads indicates a significant difference from counterparts in natural B. thuringiensis serovar israelensis strains suggesting a duplication or over-replication in 4Q7. Thus the 4Q7 strain is not a pure plasmid-less offshoot but a highly genetically modified derivative of its natural ancestor. In addition to potentially influencing virulence genome-restructuring events can modify the replication termination machinery. These findings have potential implications for the conclusions of virulence studies on 4Q7 as a model but they also raise interesting fundamental questions about the functioning of the Bacillus genome.
A genome-wide survey of short coding sequences in streptococci
Identification of short genes that encode peptides of fewer than 60 aa is challenging both experimentally and in silico. As a consequence the universe of these short coding sequences (CDSs) remains largely unknown although some are acknowledged to play important roles in cell–cell communication particularly in Gram-positive bacteria. This paper reports a thorough search for short CDSs across streptococcal genomes. Our bioinformatic approach relied on a combination of advanced intrinsic and extrinsic methods. In the first step intrinsic sequence information (nucleotide composition and presence of RBSs) served to identify new short putative CDSs (spCDSs) and to eliminate the differences between annotation policies. In the second step pseudogene fragments and false predictions were filtered out. The last step consisted of screening the remaining spCDSs for lines of extrinsic evidence involving sequence and gene-context comparisons. A total of 789 spCDSs across 20 complete genomes (19 Streptococcus and one Enterococcus) received the support of at least one line of extrinsic evidence which corresponds to an average of 20 short CDSs per million base pairs. Most of these had no known function and a significant fraction (31 %) are not even annotated as hypothetical genes in GenBank records. As an illustration of the value of this list we describe a new family of CDSs encoding very short hydrophobic peptides (20–23 aa) situated just upstream of some of the positive transcriptional regulators of the Rgg family. The expression of seven other short CDSs from Streptococcus thermophilus CNRZ1066 that encode peptides ranging in length from 41 to 56 aa was confirmed by real-time quantitative RT-PCR and revealed a variety of expression patterns. Finally one peptide from this list encoded by a gene that is not annotated in GenBank was identified in a cell-envelope-enriched fraction of S. thermophilus CNRZ1066.
Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin
Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs) composed of 25–50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species named here as cas1B cas5 and cas6 and also revealed a certain number of spacers that have homology with extant genes most frequently derived from phages but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements and hypothesize that they provide the cell immunity against phage infection and more generally foreign DNA expression by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.
Characterization of AcmB, an N-acetylglucosaminidase autolysin from Lactococcus lactis
A gene encoding a putative peptidoglycan hydrolase named acmB which is a paralogue of the major autolysin acmA gene was identified in the Lactococcus lactis genome sequence. The acmB gene is transcribed in L. lactis MG1363 and its expression is modulated during cellular growth. The encoded AcmB protein has a modular structure with three domains: an N-terminal domain especially rich in Ser Thr Pro and Asn residues resembling a cell-wall-associated domain; a central domain homologous to the Enterococcus hirae muramidase catalytic domain; and a C-terminal domain of unknown function. A recombinant AcmB derivative devoid of its N-terminal domain was expressed in Escherichia coli. It exhibited hydrolysing activity on the peptidoglycan of several Gram-positive bacteria including L. lactis. Though showing sequence similarity with enterococcal muramidase AcmB has N-acetylglucosaminidase specificity. The acmB gene was inactivated in order to evaluate the role of the enzyme. AcmB does not appear to be involved in cell separation but contributes to cellular autolysis.
A 23 911 bp region of the Bacillus subtilis genome comprising genes located upstream and downstream of the Iev operon
Within the framework of the European project to sequence the whole Bacillus subtilis 168 genome a 23 911 bp long chromosomal DNA fragment located around 233° on the B. subtilis genetic map was cloned and sequenced. From the generated sequencing data and the results of the homology search the primary structure of this region was determined. In addition to the whole Iev operon the region contains putative genes for an amino acid permease two different alcohol dehydrogenases a chitosanase a protein belonging to the LysR family of transcriptional regulators a protein related to the MerR transcriptional regulator up to four proteins related to the product of the spoF gene and genes coding for nine more inferred proteins of unknown function.
Identical amino acid sequence of the aroA(G) gene products of Bacillus subtilis 168 and B. subtilis Marburg strain
A DNA fragment containing the aroA(G) gene of Bacillus subtilis 168 encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase-chorismate mutase was cloned and sequenced. The N-terminus of the protein encoded by aroA(G) showed homology with chorismate mutase encoded by aroH of B. subtilis and with the chorismate mutase parts of proteins encoded by the pheA and tyrA genes of Escherichia coli. The C-terminus of the aroA(G) product has sequence simililarity with 3-deoxy-D-manno-octulosonate 8-phosphate synthase of E. coli. It was shown that the proteins encoded by the aroA(G) gene of B. subtilis 168 and the aroA gene of B. subtilis ATCC 6051 Marburg strain are identical so the observed differences in DAHP synthase activity from these two strains must result from other changes.