Ivan Moszer

CotA of Bacillus subtilis Is a Copper-Dependent Laccase

The spore coat protein CotA of Bacillus subtilis displays similarities with multicopper oxidases, including manganese oxidases and laccases. B. subtilis is able to oxidize manganese, but neither CotA nor other sporulation proteins are involved. We demonstrate that CotA is a laccase. Syringaldazine, a specific substrate of laccases, reacted with wild-type spores but not with DeltacotA spores. CotA may participate in the biosynthesis of the brown spore pigment, which appears to be a melanin-like product and to protect against UV light.

From a consortium sequence to a unified sequence: the Bacillus subtilis 168 reference genome a decade later

Comparative genomics is the cornerstone of identification of gene functions. The immense number of living organisms precludes experimental identification of functions except in a handful of model organisms. The bacterial domain is split into large branches, among which the Firmicutes occupy a considerable space. Bacillus subtilis has been the model of Firmicutes for decades and its genome has been a reference for more than 10 years. Sequencing the genome involved more than 30 laboratories, with different expertises, in a attempt to make the most of the experimental information that could be associated with the sequence. This had the expected drawback that the sequencing expertise was quite varied among the groups involved, especially at a time when sequencing genomes was extremely hard work. The recent development of very efficient, fast and accurate sequencing techniques, in parallel with the development of high-level annotation platforms, motivated the present resequencing work. The updated sequence has been reannotated in agreement with the UniProt protein knowledge base, keeping in perspective the split between the paleome (genes necessary for sustaining and perpetuating life) and the cenome (genes required for occupation of a niche, suggesting here that B. subtilis is an epiphyte). This should permit investigators to make reliable inferences to prepare validation experiments in a variety of domains of bacterial growth and development as well as build up accurate phylogenies.

SubtiList: the reference database for the Bacillus subtilis genome

SubtiList is the reference database dedicated to the genome of Bacillus subtilis 168, the paradigm of Gram-positive endospore-forming bacteria. Developed in the framework of the B.subtilis genome project, SubtiList provides a curated dataset of DNA and protein sequences, combined with the relevant annotations and functional assignments. Information about gene functions and products is continuously updated by linking relevant bibliographic references. Recently, sequence corrections arising from both systematic verifications and submissions by individual scientists were included in the reference genome sequence. SubtiList is based on a generic relational data schema and a World Wide Web interface developed for the handling of bacterial genomes, called GenoList. The World Wide Web interface was designed to allow users to easily browse through genome data and retrieve information according to common biological queries. SubtiList also provides more elaborate tools, such as pattern searching, which are tightly connected to the overall browsing system. SubtiList is accessible at http://genolist.pasteur.fr/SubtiList/. Similar bacterial databases are accessible at http://genolist.pasteur.fr/.

Codon usage and lateral gene transfer in Bacillus subtilis.

Bacillus subtilis possesses three classes of genes, differing by their codon preference. One class corresponds to prophages or prophage-like elements, indicative of the existence of systematic lateral gene transfer in this organism. The nature of the selection pressure that operates on codon bias is beginning to be understood.

Evaluation of High-Throughput Sequencing for Identifying Known and Unknown Viruses in Biological Samples

ABSTRACT High-throughput sequencing furnishes a large number of short sequence reads from uncloned DNA and has rapidly become a major tool for identifying viruses in biological samples, and in particular when the target sequence is undefined. In this study, we assessed the analytical sensitivity of a pipeline for detection of viruses in biological samples based on either the Roche-454 genome sequencer or Illumina genome analyzer platforms. We sequenced biological samples artificially spiked with a wide range of viruses with genomes composed of single or double-stranded DNA or RNA, including linear or circular single-stranded DNA. Viruses were added at a very low concentration most often corresponding to 3 or 0.8 times the validated level of detection of quantitative reverse transcriptase PCRs (RT-PCRs). For the viruses represented, or resembling those represented, in public nucleotide sequence databases, we show that the higher output of Illumina is associated with a much greater sensitivity, approaching that of optimized quantitative (RT-)PCRs. In this blind study, identification of viruses was achieved without incorrect identification. Nevertheless, at these low concentrations, the number of reads generated by the Illumina platform was too small to facilitate assembly of contigs without the use of a reference sequence, thus precluding detection of unknown viruses. When the virus load was sufficiently high, de novo assembly permitted the generation of long contigs corresponding to nearly full-length genomes and thus should facilitate the identification of novel viruses.

The complete genome of Bacillus subtilis: from sequence annotation to data management and analysis

The completion of the entire 4.2‐Mb genome sequence of the Gram‐positive bacterium Bacillus subtilis has been a milestone for biological studies on this model organism. This paper describes bioinformatics work related to this joint European and Japanese project: methods and strategies for gene annotation and detection of sequencing errors, using an integrated cooperative computer environment (Imagene); construction of a specialized database for data management and a WWW server for data retrieval (SubtiList); DNA sequence analysis, yielding striking results on oligonucleotide bias, repeated sequences, and codon usage, all landmarks of evolutionary events shaping the B. subtilis genome.

Comparison of Widely Used Listeria monocytogenes Strains EGD, 10403S, and EGD-e Highlights Genomic Differences Underlying Variations in Pathogenicity

ABSTRACT For nearly 3 decades, listeriologists and immunologists have used mainly three strains of the same serovar (1/2a) to analyze the virulence of the bacterial pathogen Listeria monocytogenes. The genomes of two of these strains, EGD-e and 10403S, were released in 2001 and 2008, respectively. Here we report the genome sequence of the third reference strain, EGD, and extensive genomic and phenotypic comparisons of the three strains. Strikingly, EGD-e is genetically highly distinct from EGD (29,016 single nucleotide polymorphisms [SNPs]) and 10403S (30,296 SNPs), and is more related to serovar 1/2c than 1/2a strains. We also found that while EGD and 10403S strains are genetically very close (317 SNPs), EGD has a point mutation in the transcriptional regulator PrfA (PrfA*), leading to constitutive expression of several major virulence genes. We generated an EGD-e PrfA* mutant and showed that EGD behaves like this strain in vitro, with slower growth in broth and higher invasiveness in human cells than those of EGD-e and 10403S. In contrast, bacterial counts in blood, liver, and spleen during infection in mice revealed that EGD and 10403S are less virulent than EGD-e, which is itself less virulent than EGD-e PrfA*. Thus, constitutive expression of PrfA-regulated virulence genes does not appear to provide a significant advantage to the EGD strain during infection in vivo, highlighting the fact that in vitro invasion assays are not sufficient for evaluating the pathogenic potential of L. monocytogenes strains. Together, our results pave the way for deciphering unexplained differences or discrepancies in experiments using different L. monocytogenes strains. IMPORTANCE Over the past 3 decades, Listeria has become a model organism for host-pathogen interactions, leading to critical discoveries in a broad range of fields, including bacterial gene regulation, cell biology, and bacterial pathophysiology. Scientists studying Listeria use primarily three pathogenic strains: EGD, EGD-e, and 10403S. Despite many studies on EGD, it is the only one of the three strains whose genome has not been sequenced. Here we report the sequence of its genome and a series of important genomic and phenotypic differences between the three strains, in particular, a critical mutation in EGD’s PrfA, the main regulator of Listeria virulence. Our results show that the three strains display differences which may play an important role in the virulence differences observed between the strains. Our findings will be of critical relevance to listeriologists and immunologists who have used or may use Listeria as a tool to study the pathophysiology of listeriosis and immune responses. Over the past 3 decades, Listeria has become a model organism for host-pathogen interactions, leading to critical discoveries in a broad range of fields, including bacterial gene regulation, cell biology, and bacterial pathophysiology. Scientists studying Listeria use primarily three pathogenic strains: EGD, EGD-e, and 10403S. Despite many studies on EGD, it is the only one of the three strains whose genome has not been sequenced. Here we report the sequence of its genome and a series of important genomic and phenotypic differences between the three strains, in particular, a critical mutation in EGD’s PrfA, the main regulator of Listeria virulence. Our results show that the three strains display differences which may play an important role in the virulence differences observed between the strains. Our findings will be of critical relevance to listeriologists and immunologists who have used or may use Listeria as a tool to study the pathophysiology of listeriosis and immune responses.

Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline

Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates.

ANAEROBIC TRANSCRIPTION ACTIVATION IN BACILLUS SUBTILIS : IDENTIFICATION OF DISTINCT FNR-DEPENDENT AND -INDEPENDENT REGULATORY MECHANISMS

Bacillus subtilis is able to grow anaerobically using alternative electron acceptors, including nitrate or fumarate. We characterized an operon encoding the dissimilatory nitrate reductase subunits homologous to the Escherichia coli narGHJI operon and the narK gene encoding a protein with nitrite extrusion activity. Downstream from narK and co‐transcribed with it a gene (fnr) encoding a protein homologous to E.coli FNR was found. Disruption of fnr abolished both nitrate and fumarate utilization as electron acceptors and anaerobic induction of narK. Four putative FNR binding sites were found in B.subtilis sequences. The consensus sequence, centred at position −41.5, is identical to the consensus for the DNA site for E.coli CAP. Bs‐FNR contained a four cysteine residue cluster at its C‐terminal end. This is in contrast to Ec‐FNR, where a similar cluster is present at the N‐terminal end. It is possible that oxygen modulates the activity of both activators by a similar mechanism involving iron. Unlike in E.coli, where fnr expression is weakly repressed by anaerobiosis, fnr gene expression in B.subtilis is strongly activated by anaerobiosis. We have identified in the narK‐fnr intergenic region a promotor activated by anaerobiosis independently of FNR. Thus induction of genes involved in anaerobic respiration requires in B.subtilis at least two levels of regulation: activation of fnr transcription and activation of FNR to induce transcription of FNR‐dependent promoters.

Reannotation of the genome sequence of Clostridium difficile strain 630

A regular update of genome annotations is a prerequisite step to help maintain the accuracy and relevance of the information they contain. Five years after the first publication of the complete genome sequence of Clostridium difficile strain 630, we manually reannotated each of the coding sequences (CDSs), using a high-level annotation platform. The functions of more than 500 genes annotated previously with putative functions were reannotated based on updated sequence similarities to proteins whose functions have been recently identified by experimental data from the literature. We also modified 222 CDS starts, detected 127 new CDSs and added the enzyme commission numbers, which were not supplied in the original annotation. In addition, an intensive project was undertaken to standardize the names of genes and gene products and thus harmonize as much as possible with the HAMAP project. The reannotation is stored in a relational database that will be available on the MicroScope web-based platform (https://www.genoscope.cns.fr/agc/microscope/mage/viewer.php?S_id=752&wwwpkgdb=a78e3466ad5db29aa8fe49e8812de8a7). The original submission stored in the (International Nucleotide Sequence Database Collaboration) INSDC nucleotide sequence databases was also updated.