RESULTS:
1 - 14 of 14 for "Alexei Sorokin"
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.
Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the Bacillus cereus Group occasionally associated with food poisoning
An aerobic endospore-forming bacillus (NVH 391-98T) was isolated during a severe food poisoning outbreak in France in 1998 and four other similar strains have since been isolated also mostly from food poisoning cases. Based on 16S rRNA gene sequence similarity these strains were shown to belong to the Bacillus cereus Group (over 97 % similarity with the current Group species) and phylogenetic distance from other validly described species of the genus Bacillus was less than 95 %. Based on 16S rRNA gene sequence similarity and MLST data these novel strains were shown to form a robust and well-separated cluster in the B. cereus Group and constituted the most distant cluster from species of this Group. Major fatty acids (iso-C15 : 0 C16 : 0 iso-C17 : 0 anteiso-C15 : 0 iso-C16 : 0 iso-C13 : 0) supported the affiliation of these strains to the genus Bacillus and more specifically to the B. cereus Group. NVH 391-98T taxon was more specifically characterized by an abundance of iso-C15 : 0 and low amounts of iso-C13 : 0 compared with other members of the B. cereus Group. Genome similarity together with DNA–DNA hybridization values and physiological and biochemical tests made it possible to genotypically and phenotypically differentiate NVH 391-98T taxon from the six current B. cereus Group species. NVH 391-98T therefore represents a novel species for which the name Bacillus cytotoxicus sp. nov. is proposed with the type strain NVH 391-98T ( = DSM 22905T = CIP 110041T).
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 the Bacillus subtilis YxdJ response regulator as the inducer of expression for the cognate ABC transporter YxdLM
The genome of Bacillus subtilis like those of some other AT-rich Gram-positive bacteria has the uncommon feature of containing several copies of arrangements in which the genes encoding two-component and cognate ABC transporter systems are adjacent. As the function of one of these systems the product of the yxd locus is still unknown it was analysed further in order to get some clues on the physiological role of the gene products it encodes. The yxdJ gene was shown to encode a DNA-binding protein that directly controls transcription of the neighbouring operon encoding the ABC transporter YxdLM. Primer extension and DNase protection experiments allowed precise definition of the yxdLM transcription start and controlling region. Two putative direct repeats were identified that are proposed to be the YxdJ response regulator binding sites. Whole-cell transcriptome analyses revealed that the YxdJ regulon is extremely restricted. In addition to the yxdJKLMyxeA operon only a few genes involved in modifications of the bacterial cell wall were shown to be regulated by YxdJ.
Two distinct types of rRNA operons in the Bacillus cereus group
The Bacillus cereus group includes insecticidal bacteria (B. thuringiensis) food-borne pathogens (B. cereus and B. weihenstephanensis) and B. anthracis the causative agent of anthrax. The precise number of rRNA operons in 12 strains of the B. cereus group was determined. Most of the tested strains possess 13 operons and the tested psychrotolerant strains contain 14 operons the highest number ever found in bacteria. The separate clustering of the tested psychrotolerant strains was confirmed by partial sequencing of several genes distributed over the chromosomes. Analysis of regions downstream of the 23S rRNA genes in the type strain B. cereus ATCC 14579 indicates that the rRNA operons can be divided into two classes I and II consisting respectively of eight and five operons. Class II operons exhibit multiple tRNA genes downstream of the 5S rRNA gene and a putative promoter sequence in the 23S–5S intergenic region suggesting that 5S rRNA and the downstream tRNA genes can be transcribed independently of the 16S and 23S genes. Similar observations were made in the recently sequenced genome of B. anthracis strain Ames. The existence of these distinct types of rRNA operons suggests an unknown mechanism for regulation of rRNA and tRNA synthesis potentially related to the pool of amino acids available for protein synthesis.
The kdgRKAT operon of Bacillus subtilis:detection of the transcript and regulation by the kdgR and ccpA genes
SUMMARY: Transcription of a new catabolic operon in Bacillus subtilis involved in the late stages of galacturonic acid utilization has been studied. The operon consists of four genes: kdgR encoding the putative regulator protein; kdgK encoding 2-keto-3-deoxyg I uconate kinase; kdgA encoding 2-keto-3-deoxyg luconate-6-phosphate aldolase; and kdg encoding a transporter. These four genes are organized in one transcriptional unit and map at 198" of the B. subtik chromosome. Primer extension experiments and Northern blot analysis show that an active σ-dependent promoter precedes kdgR and transcription is terminated at the putative pindependent terminator downstream of kdgr. The operon is negatively regulated by the kdgR and ccpA gene products which belong t o the Lac1 family of transcription regulators. The expression of the genes in this operon can be induced by galacturonate and strongly repressed when glucose is present in the growth medium. Knockout mutations in genes kdgR and ccpA remove respectively the effects of galacturonate and glucose on the transcription of this operon.
Sequencing and functional annotation of the Bacillus subtilis genes in the 200 kb rrnB-dnaB region
The 200 kb region of the Bacillus subtilis chromosome spanning from 255 to 275° on the genetic map was sequenced. The strategy applied based on use of yeast artificial chromosomes and multiplex Long Accurate PCR proved to be very efficient for sequencing a large bacterial chromosome area. A total of 193 genes of this part of the chromosome was classified by level of knowledge and biological category of their functions. Five levels of gene function understanding are defined. These are: (i) experimental evidence is available of gene product or biological function; (ii) strong homology exists for the putative gene product with proteins from other organisms; (Hi) some indication of the function can be derived from homologies with known proteins; (iv) the gene product can be clustered with hypothetical proteins; (v) no indication on the gene function exists. The percentage of detected genes in each category was: 20 28 20 15 and 17 respectively. In the sequenced region a high percentage of genes are implicated in transport and metabolic linking of glycolysis and the citric acid cycle. A functional connection of several genes from this region and the genes close to 140° in the chromosome was also observed.
Sequence of the Bacillus subtilis genome region in the vicinity of the lev operon reveals two new extracytoplasmic function RNA polymerase sigma factors SigV and SigZ
Summary: Two regions with sizes 18 900 and 25400 bp which join previously known contigs containing levRDEFG aadK and bit genes near 235δ of the Bacillus subtilis chromosome were sequenced. Among others two genes which encode proteins homologous to RNA polymerase σ-factors were identified within this region. The gene products designated SigV and SigZ show the highest homology with σ-factors encoded by the gene carQ of Myxococcus xanthus and sigX (formerly orfX20) of B. subtilis correspondingly. All σ-factors which show statistically significant homology to SigV and SigZ belong to the ECF (extracytoplasmic functions) subfamily. SigV and SigZ do not have N-terminal sequence which prevents such proteins from binding to DNA without RNA polymerase core enzyme.
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.
Mapping of the 150 kb spoIIIC-pheA region of the Bacillus subtilis chromosome using Long Accurate PCR and three yeast artificial chromosomes
We constructed a PCR map of the 150 kb spoIIIC-pheA region of the Bacillus subtilis chromosome. It was established using known sequences of the spoIIIC blt aadK sacC spoVB and pheA loci and eight random sequence tags. The tags were generated using PFGE-purified DNA of yeast artificial chromosome (YAC) 11–17 from the yeast clone which carries the major part of this region. The ends of two other YACs were positioned on the map using total DNA extracted from yeast cells carrying them. The procedure allowed the placement of precisely known and new (putative) genes on the physical chromosome map and the generation of sufficient amounts of DNA for sequencing this region. Apart from allowing correction of the genetic map in this region these results demonstrate how a collection of long segments of bacterial chromosome and Long Accurate PCR can be used for reliable high-resolution physical mapping of an extended chromosome area.
Organization of the Bacillus subtilis 168 chromosome between kdg and the attachment site of the SPβ prophage: use of Long Accurate PCR and yeast artificial chromosomes for sequencing
Within the Bacillus subtilis genome sequencing project the region between lysA and ilvA was assigned to our laboratory. In this report we present the sequence of the last 36 kb of this region between the kdg operon and the attachment site of the SPβ prophage. A two-step strategy was used for the sequencing. In the first step total chromosomal DNA was cloned in phage M13-based vectors and the clones carrying inserts from the target region were identified by hybridization with a cognate yeast artificial chromosome (YAC) from our collection. Sequencing of the clones allowed us to establish a number of contigs. In the second step the contigs were mapped by Long Accurate (LA) PCR and the remaining gaps closed by sequencing of the PCR products. The level of sequence inaccuracy due to LA PCR errors appeared to be about 1 in 10000 which does not affect significantly the final sequence quality. This two-step strategy is efficient and we suggest that it can be applied to sequencing of longer chromosomal regions. The 36 kb sequence contains 38 coding sequences (CDSs) 19 of which encode unknown proteins. Seven genetic loci already mapped in this region xpt metB ilvA ilvD thyB dfrA and degR were identified. Eleven CDSs were found to display significant similarities to known proteins from the data banks suggesting possible functions for some of the novel genes: cspD may encode a cold shock protein; bcsA the first bacterial homologue of chalcone synthase; exol a 5′ to 3′ exonuclease similar to that of DNA polymerase I of Escherichia coli; and bsaA a stress-response-associated protein. The protein encoded by ypIP has homology with the transcriptional NifA-like regulators. The arrangement of the genes relative to possible promoters and terminators suggests 19 potential transcription units.
Sequence analysis of the Bacillus subtilis chromosome region between the serA and kdg loci cloned in a yeast artificial chromosome
The standard strategies of genome sequencing based on λ-vector or cosmid libraries are only partially applicable to AT-rich Gram-positive bacteria because of the problem of instability of their chromosomal DNA in heterologous hosts like Escherichia coli . One complete collection of ordered clones known for such bacteria is that of Bacillus subtilis established by using yeast artificial chromosomes (YACs). This paper reports the results of the direct use of one of the YAC clones from the above collection for the sequencing of the region cloned in it. The strategy applied consisted of the following: (i) construction of M13 banks of the partially purified YAC DNA and sequencing of 800 M13 clones chosen at random; (ii) directed selection of M13 clones to sequence by using marginal contig fragments as hybridization probes; (iii) direct sequencing of joining PCR fragments obtained by combinations of primers corresponding to the ends of representative contigs. The complete 104 109 bp insert sequence of this YAC clone was thus established. The strategy used allowed us to avoid resequencing the two largest previously sequenced contigs (13695 and 20303 bp) of the YAC insert. We propose that the strategy used can be applied to the sequencing of the whole bacterial genome without intermediate cloning as well as for larger inserts of eukaryotic origin cloned ir YACs. Sequencing of the insert of the YAC clone 15-6B allowed us to establish the contiguous sequence of 127 kb from spollA to kdg. The organization of the newly determined region is presented. Of the 138 ORFs identified in the spollA-kdg region 57 have no clear putative function from their homology to proteins in the databases.
Nucleotide sequence of the Bacillus subtilis dnaD gene
The dnaD gene of Bacillus subtilis was identified within a 104 kb DNA segment cloned into a yeast artificial chromosome. The nucleotide sequence of the wild type and dnaD23 mutant genes were determined. dnaD is predicted to encode a protein of 232 amino acids with no similarity to proteins in the data banks.
The Bacillus subtilis chromosome region encoding homologues of the Escherichia coli mssA and rpsA gene products
SUMMARY: A gene was found in Bacillus subtilis which encodes a protein highly homologous to the Escherichia coli rpsA gene product the S1 ribosomal protein. The B. subtilis protein contains the domain responsible for binding to ribosomes and two S1 motifs instead of four as found in the E. coli protein. The B. subtilis protein is similar in this way to the equivalent protein of plant chloroplast ribosomes supposed to be the counterpart of E. coli S1. The gene is expressed during vegetative growth in B. subtilis at the transcriptional and translational levels as judged by Northern hybridization and expression in a translational fusion with a reporter gene. In contrast to the E. coli situation it can be inactivated without dramatic effects on cell viability. Southern hybridization of the B. subtilis DNA fragment encoding this gene revealed specific homologous fragments in all other Gram-positive bacteria tested. The hybridization pattern with B. stearothermophilus suggests the presence of at least two homologous genes in this bacterium. We show that in B. subtilis the ORF preceding the rpsA homologue encodes a protein which is highly similar to the product of the E. coli mssA gene which is located upstream of rpsA. Again in contrast to the E. coli situation where these genes are co-transcribed in B. subtilis they are separated by a transcription terminator and the mssA homologue is transcribed during sporulation. We suggest that during the evolution very similar structures and genetic organization of these two genes were conserved but acquired different functions in Gram-negative and Gram-positive bacteria.