Soda lakes are saline alkaline lakes characterized by high concentrations of sodium carbonate/bicarbonate which lead to a stable elevated pH (>9), and moderate to extremely high salinity. Despite this combination of extreme conditions, biodiversity in soda lakes is high, and the presence of diverse microbial communities provides a driving force for highly active biogeochemical cycles. The sulfur cycle is one of the most important of these and bacterial sulfur oxidation is dominated by members of the obligately chemolithoautotrophic genus Thioalkalivibrio. Currently, 10 species have been described in this genus, but over one hundred isolates have been obtained from soda lake samples. The genomes of 75 strains were sequenced and annotated previously, and used in this study to provide a comprehensive picture of the diversity and distribution of genes related to dissimilatory sulfur metabolism in Thioalkalivibrio. Initially, all annotated genes in 75 Thioalkalivibrio genomes were placed in ortholog groups and filtered by bi-directional best BLAST analysis. Investigation of the ortholog groups containing genes related to sulfur oxidation showed that flavocytochrome c (fcc), the truncated sox system, and sulfite:quinone oxidoreductase (soe) are present in all strains, whereas dissimilatory sulfite reductase (dsr; which catalyzes the oxidation of elemental sulfur) was found in only six strains. The heterodisulfide reductase system (hdr), which is proposed to oxidize sulfur to sulfite in strains lacking both dsr and soxCD, was detected in 73 genomes. Hierarchical clustering of strains based on sulfur gene repertoire correlated closely with previous phylogenomic analysis. The phylogenetic analysis of several sulfur oxidation genes showed a complex evolutionary history. All in all, this study presents a comprehensive investigation of sulfur metabolism-related genes in cultivated Thioalkalivibrio strains and provides several avenues for future research.

Orally ingested bacteria interact with intestinal mucosa and may impact immunity. However, insights in mechanisms involved are limited. In this randomized placebo-controlled cross-over trial, healthy human subjects were given Lactobacillus plantarum supplementation (strain TIFN101, CIP104448, or WCFS1) or placebo for 7 days. To determine whether L. plantarum can enhance immune response, we compared the effects of three stains on systemic and gut mucosal immunity, by among others assessing memory responses against tetanus toxoid (TT)-antigen, and mucosal gene transcription, in human volunteers during induction of mild immune stressor in the intestine, by giving a commonly used enteropathic drug, indomethacin [non-steroidal anti-inflammatory drug (NSAID)]. Systemic effects of the interventions were studies in peripheral blood samples. NSAID was found to induce a reduction in serum CD4+/Foxp3 regulatory cells, which was prevented by L. plantarum TIFN101. T-cell polarization experiments showed L. plantarum TIFN101 to enhance responses against TT-antigen, which indicates stimulation of memory responses by this strain. Cell extracts of the specific L. plantarum strains provoked responses after WCFS1 and TIFN101 consumption, indicating stimulation of immune responses against the specific bacteria. Mucosal immunomodulatory effects were studied in duodenal biopsies. In small intestinal mucosa, TIFN101 upregulated genes associated with maintenance of T- and B-cell function and antigen presentation. Furthermore, L. plantarum TIFN101 and WCFS1 downregulated immunological pathways involved in antigen presentation and shared downregulation of snoRNAs, which may suggest cellular destabilization, but may also be an indicator of tissue repair. Full sequencing of the L. plantarum strains revealed possible gene clusters that might be responsible for the differential biological effects of the bacteria on host immunity. In conclusion, the impact of oral consumption L. plantarum on host immunity is strain dependent and involves responses against bacterial cell components. Some strains may enhance specific responses against pathogens by enhancing antigen presentation and leukocyte maintenance in mucosa. In future studies and clinical settings, caution should be taken in selecting beneficial bacteria as closely related strains can have different effects. Our data show that specific bacterial strains can prevent immune stress induced by commonly consumed painkillers such as NSAID and can have enhancing beneficial effects on immunity of consumers by stimulating antigen presentation and memory responses.

Mycobacterium tuberculosis is characterised by limited genomic diversity, which makes the application of whole genome sequencing particularly attractive for clinical and epidemiological investigation. However, in order to confidently infer transmission events, an accurate knowledge of the rate of change in the genome over relevant timescales is required.

A key complication in comparative genomics for reliable gene function prediction is the existence of duplicated genes. To study the effect of gene duplication on function prediction, we analyze orthologs between pairs of genomes where in one genome the orthologous gene has duplicated after the speciation of the two genomes (i.e. inparalogs). For these duplicated genes we investigate whether the gene that is most similar on the sequence level is also the gene that has retained the ancestral gene-neighborhood. Although the majority of investigated cases show a consistent pattern between sequence similarity and gene-neighborhood conservation, a substantial fraction, 29-38%, is inconsistent. The observation of inconsistency is not the result of a chance outcome owing to a lack of divergence time between inparalogs, but rather it seems to be the result of a chance outcome caused by very similar rates of sequence evolution of both inparalogs relative to their ortholog. If one-to-one orthologous relationships are required, it is advisable to combine contextual information (i.e. gene-neighborhood in prokaryotes and co-expression in eukaryotes) with protein sequence information to predict the most probable functional equivalent ortholog in the presence of inparalogs.

A pangenome is the total of genes present in strains of the same species. Pangenome microarrays allow determining the genomic content of bacterial strains more accurately than conventional comparative genome hybridization microarrays. PanCGHweb is the first tool that effectively calls genotype based on pangenome microarray data.

Thiocyanate is a C1 compound containing carbon, nitrogen, and sulfur. It is a (by)product in a number of natural and industrial processes. Because thiocyanate is toxic to many organisms, including humans, its removal from industrial waste streams is an important problem. Although a number of bacteria can use thiocyanate as a nitrogen source, only a few can use it as an electron donor. There are two distinct pathways to use thiocyanate: (i) the "carbonyl sulfide pathway," which has been extensively studied, and (ii) the "cyanate pathway," whose key enzyme, thiocyanate dehydrogenase, was recently purified and studied. Three species of Thioalkalivibrio, a group of haloalkaliphilic sulfur-oxidizing bacteria isolated from soda lakes, have been described as thiocyanate oxidizers: (i) Thioalkalivibrio paradoxus ("cyanate pathway"), (ii) Thioalkalivibrio thiocyanoxidans ("cyanate pathway") and (iii) Thioalkalivibrio thiocyanodenitrificans ("carbonyl sulfide pathway"). In this study we provide a comparative genome analysis of these described thiocyanate oxidizers, with genomes ranging in size from 2.5 to 3.8 million base pairs. While focusing on thiocyanate degradation, we also analyzed the differences in sulfur, carbon, and nitrogen metabolism. We found that the thiocyanate dehydrogenase gene is present in 10 different Thioalkalivibrio strains, in two distinct genomic contexts/genotypes. The first genotype is defined by having genes for flavocytochrome c sulfide dehydrogenase upstream from the thiocyanate dehydrogenase operon (present in two strains including the type strain of Tv. paradoxus), whereas in the second genotype these genes are located downstream, together with two additional genes of unknown function (present in eight strains, including the type strains of Tv. thiocyanoxidans). Additionally, we found differences in the presence/absence of genes for various sulfur oxidation pathways, such as sulfide:quinone oxidoreductase, dissimilatory sulfite reductase, and sulfite dehydrogenase. One strain (Tv. thiocyanodenitrificans) lacks genes encoding a carbon concentrating mechanism and none of the investigated genomes were shown to contain known bicarbonate transporters. This study gives insight into the genomic variation of thiocyanate oxidizing bacteria and may lead to improvements in the application of these organisms in the bioremediation of industrial waste streams.

We characterised carbohydrate utilisation of 20 newly sequenced Bacillus cereus strains isolated from food products and food processing environments and two laboratory strains, B. cereus ATCC 10987 and B. cereus ATCC 14579. Subsequently, genome sequences of these strains were analysed together with 11 additional B. cereus reference genomes to provide an overview of the different types of carbohydrate transporters and utilization systems found in B. cereus strains. The combined application of API tests, defined growth media experiments and comparative genomics enabled us to link the carbohydrate utilisation capacity of 22 B. cereus strains with their genome content and in some cases to the panC phylogenetic grouping. A core set of carbohydrates including glucose, fructose, maltose, trehalose, N-acetyl-glucosamine, and ribose could be used by all strains, whereas utilisation of other carbohydrates like xylose, galactose, and lactose, and typical host-derived carbohydrates such as fucose, mannose, N-acetyl-galactosamine and inositol is limited to a subset of strains. Finally, the roles of selected carbohydrate transporters and utilisation systems in specific niches such as soil, foods and the human host are discussed.

There is a growing interest in the Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) of microbes, fungi and plants because they can produce bioactive peptides such as antibiotics. The ability to identify the substrate specificity of the enzyme's adenylation (A) and acyl-transferase (AT) domains is essential to rationally deduce or engineer new products. We here report on a Hidden Markov Model (HMM)-based ensemble method to predict the substrate specificity at high quality. We collected a new reference set of experimentally validated sequences. An initial classification based on alignment and Neighbor Joining was performed in line with most of the previously published prediction methods. We then created and tested single substrate specific HMMs and found that their use improved the correct identification significantly for A as well as for AT domains. A major advantage of the use of HMMs is that it abolishes the dependency on multiple sequence alignment and residue selection that is hampering the alignment-based clustering methods. Using our models we obtained a high prediction quality for the substrate specificity of the A domains similar to two recently published tools that make use of HMMs or Support Vector Machines (NRPSsp and NRPS predictor2, respectively). Moreover, replacement of the single substrate specific HMMs by ensembles of models caused a clear increase in prediction quality. We argue that the superiority of the ensemble over the single model is caused by the way substrate specificity evolves for the studied systems. It is likely that this also holds true for other protein domains. The ensemble predictor has been implemented in a simple web-based tool that is available at http://www.cmbi.ru.nl/NRPS-PKS-substrate-predictor/.

No abstract available

No abstract available

No abstract available

No abstract available

Sigma-54 is a central regulator in many pathogenic bacteria and has been linked to a multitude of cellular processes like nitrogen assimilation and important functional traits such as motility, virulence, and biofilm formation. Until now it has remained obscure whether these phenomena and the control by Sigma-54 share an underlying theme.

The identification of translation initiation sites (TISs) constitutes an important aspect of sequence-based genome analysis. An erroneous TIS annotation can impair the identification of regulatory elements and N-terminal signal peptides, and also may flaw the determination of descent, for any particular gene. We have formulated a reference-free method to score the TIS annotation quality. The method is based on a comparison of the observed and expected distribution of all TISs in a particular genome given prior gene-calling. We have assessed the TIS annotations for all available NCBI RefSeq microbial genomes and found that approximately 87% is of appropriate quality, whereas 13% needs substantial improvement. We have analyzed a number of factors that could affect TIS annotation quality such as GC-content, taxonomy, the fraction of genes with a Shine-Dalgarno sequence and the year of publication. The analysis showed that only the first factor has a clear effect. We have then formulated a straightforward Principle Component Analysis-based TIS identification strategy to self-organize and score potential TISs. The strategy is independent of reference data and a priori calculations. A representative set of 277 genomes was subjected to the analysis and we found a clear increase in TIS annotation quality for the genomes with a low quality score. The PCA-based annotation was also compared with annotation with the current tool of reference, Prodigal. The comparison for the model genome of Escherichia coli K12 showed that both methods supplement each other and that prediction agreement can be used as an indicator of a correct TIS annotation. Importantly, the data suggest that the addition of a PCA-based strategy to a Prodigal prediction can be used to 'flag' TIS annotations for re-evaluation and in addition can be used to evaluate a given annotation in case a Prodigal annotation is lacking.

Lactic acid bacteria (LAB) are utilized widely for the fermentation of foods. In the current post-genomic era, tools have been developed that explore genetic diversity among LAB strains aiming to link these variations to differential phenotypes observed in the strains investigated. However, these genotype-phenotype matching approaches fail to assess the role of conserved genes in the determination of physiological characteristics of cultures by environmental conditions. This manuscript describes a complementary approach in which Lactobacillus plantarum WCFS1 was fermented under a variety of conditions that differ in temperature, pH, as well as NaCl, amino acid, and O(2) levels. Samples derived from these fermentations were analyzed by full-genome transcriptomics, paralleled by the assessment of physiological characteristics, e.g., maximum growth rate, yield, and organic acid profiles. A data-storage and -mining suite designated FermDB was constructed and exploited to identify correlations between fermentation conditions and industrially relevant physiological characteristics of L. plantarum, as well as the associated transcriptome signatures. Finally, integration of the specific fermentation variables with the transcriptomes enabled the reconstruction of the gene-regulatory networks involved. The fermentation-genomics platform presented here is a valuable complementary approach to earlier described genotype-phenotype matching strategies which allows the identification of transcriptome signatures underlying physiological variations imposed by different fermentation conditions.

In the past decades, various protein subcellular-location (SCL) predictors have been developed. Most of these predictors, like TMHMM 2.0, SignalP 3.0, PrediSi and Phobius, aim at the identification of one or a few SCLs, whereas others such as CELLO and Psortb.v.2.0 aim at a broader classification. Although these tools and pipelines can achieve a high precision in the accurate prediction of signal peptides and transmembrane helices, they have a much lower accuracy when other sequence characteristics are concerned. For instance, it proved notoriously difficult to identify the fate of proteins carrying a putative type I signal peptidase (SPIase) cleavage site, as many of those proteins are retained in the cell membrane as N-terminally anchored membrane proteins. Moreover, most of the SCL classifiers are based on the classification of the Swiss-Prot database and consequently inherited the inconsistency of that SCL classification. As accurate and detailed SCL prediction on a genome scale is highly desired by experimental researchers, we decided to construct a new SCL prediction pipeline: LocateP.

Thioalkalivibrio is a genus of obligate chemolithoautotrophic haloalkaliphilic sulfur-oxidizing bacteria. Their habitat are soda lakes which are dual extreme environments with a pH range from 9.5 to 11 and salt concentrations up to saturation. More than 100 strains of this genus have been isolated from various soda lakes all over the world, but only ten species have been effectively described yet. Therefore, the assignment of the remaining strains to either existing or novel species is important and will further elucidate their genomic diversity as well as give a better general understanding of this genus. Recently, the genomes of 76 Thioalkalivibrio strains were sequenced. On these, we applied different methods including (i) 16S rRNA gene sequence analysis, (ii) Multilocus Sequence Analysis (MLSA) based on eight housekeeping genes, (iii) Average Nucleotide Identity based on BLAST (ANIb) and MUMmer (ANIm), (iv) Tetranucleotide frequency correlation coefficients (TETRA), (v) digital DNA:DNA hybridization (dDDH) as well as (vi) nucleotide- and amino acid-based Genome BLAST Distance Phylogeny (GBDP) analyses. We detected a high genomic diversity by revealing 15 new "genomic" species and 16 new "genomic" subspecies in addition to the ten already described species. Phylogenetic and phylogenomic analyses showed that the genus is not monophyletic, because four strains were clearly separated from the other Thioalkalivibrio by type strains from other genera. Therefore, it is recommended to classify the latter group as a novel genus. The biogeographic distribution of Thioalkalivibrio suggested that the different "genomic" species can be classified as candidate disjunct or candidate endemic species. This study is a detailed genome-based classification and identification of members within the genus Thioalkalivibrio. However, future phenotypical and chemotaxonomical studies will be needed for a full species description of this genus.

The Beijing strain is one of the most successful genotypes of Mycobacterium tuberculosis worldwide and appears to be highly homogenous according to existing genotyping methods. To type Beijing strains reliably we developed a robust typing scheme using single nucleotide polymorphisms (SNPs) and regions of difference (RDs) derived from whole-genome sequencing data of eight Beijing strains. SNP/RD typing of 259 M. tuberculosis isolates originating from 45 countries worldwide discriminated 27 clonal complexes within the Beijing genotype family. A total of 16 Beijing clonal complexes contained more than one isolate of known origin, of which two clonal complexes were strongly associated with South African origin. The remaining 14 clonal complexes encompassed isolates from different countries. Even highly resolved clonal complexes comprised isolates from distinct geographical sites. Our results suggest that Beijing strains spread globally on multiple occasions and that the tuberculosis epidemic caused by the Beijing genotype is at least partially driven by modern migration patterns. The SNPs and RDs presented in this study will facilitate future molecular epidemiological and phylogenetic studies on Beijing strains.

The vaginal microbiota, in particular Lactobacillus species, play an important role in female health through modulation of immunity, countering pathogens and maintaining a pH below 4.7. We report the isolation and genome sequence of Lactobacillus pentosus strain KCA1 (formally known as L. plantarum) from the vagina of a healthy Nigerian woman. The genome was sequenced using Illumina GA II technology. The resulting 16,920,226 paired-end reads were assembled with the Velvet tool. Contigs were annotated using the RAST server, and manually curated. A comparative analysis with the available genomes of L. pentosus IG1 and L. plantarum WCFS1 showed that over 15% of the predicted functional activities are found only in this strain. The strain has a chromosome sequence of 3,418,159 bp with a G+C content of 46.4%, and is devoid of plasmids. Novel gene clusters or variants of known genes relative to the reference genomes were found. In particular, the strain has loci encoding additional putative mannose phosphotransferase systems. Clusters of genes include those for utilization of hydantoin, isopropylmalate, malonate, rhamnosides, and genes for assimilation of polyglycans, suggesting the metabolic versatility of L. pentosus KCA1. Loci encoding putative phage defense systems were also found including clustered regularly interspaced short palindromic repeats (CRISPRs), abortive infection (Abi) systems and toxin-antitoxin systems (TA). A putative cluster of genes for biosynthesis of a cyclic bacteriocin precursor, here designated as pentocin KCA1 (penA) were identified. These findings add crucial information for understanding the genomic and geographic diversity of vaginal lactobacilli.

Lactobacillus paracasei is a member of the normal human and animal gut microbiota and is used extensively in the food industry in starter cultures for dairy products or as probiotics. With the development of low-cost, high-throughput sequencing techniques it has become feasible to sequence many different strains of one species and to determine its "pan-genome". We have sequenced the genomes of 34 different L. paracasei strains, and performed a comparative genomics analysis. We analysed genome synteny and content, focussing on the pan-genome, core genome and variable genome. Each genome was shown to contain around 2800-3100 protein-coding genes, and comparative analysis identified over 4200 ortholog groups that comprise the pan-genome of this species, of which about 1800 ortholog groups make up the conserved core. Several factors previously associated with host-microbe interactions such as pili, cell-envelope proteinase, hydrolases p40 and p75 or the capacity to produce short branched-chain fatty acids (bkd operon) are part of the L. paracasei core genome present in all analysed strains. The variome consists mainly of hypothetical proteins, phages, plasmids, transposon/conjugative elements, and known functions such as sugar metabolism, cell-surface proteins, transporters, CRISPR-associated proteins, and EPS biosynthesis proteins. An enormous variety and variability of sugar utilization gene cassettes were identified, with each strain harbouring between 25-53 cassettes, reflecting the high adaptability of L. paracasei to different niches. A phylogenomic tree was constructed based on total genome contents, and together with an analysis of horizontal gene transfer events we conclude that evolution of these L. paracasei strains is complex and not always related to niche adaptation. The results of this genome content comparison was used, together with high-throughput growth experiments on various carbohydrates, to perform gene-trait matching analysis, in order to link the distribution pattern of a specific phenotype to the presence/absence of specific sets of genes.