Identification and optimization of PrsA in Bacillus subtilis for improved yield of amylase.

BACKGROUND: PrsA is an extracytoplasmic folding catalyst essential in Bacillus subtilis. Overexpression of the native PrsA from B. subtilis has repeatedly lead to increased amylase yields. Nevertheless, little is known about how the overexpression of heterologous PrsAs can affect amylase secretion. RESULTS: In this study, the final yield of five extracellular alpha-amylases was increased by heterologous PrsA co-expression up to 2.5 fold. The effect of the overexpression of heterologous PrsAs on alpha-amylase secretion is specific to the co-expressed alpha-amylase. Co-expression of a heterologous PrsA can significantly reduce the secretion stress response. Engineering of the B. licheniformis PrsA lead to a further increase in amylase secretion and reduced secretion stress. CONCLUSIONS: In this work we show how heterologous PrsA overexpression can give a better result on heterologous amylase secretion than the native PrsA, and that PrsA homologs show a variety of specificity towards different alpha-amylases. We also demonstrate that on top of increasing amylase yield, a good PrsA-amylase pairing can lower the secretion stress response of B. subtilis. Finally, we present a new recombinant PrsA variant with increased performance in both supporting amylase secretion and lowering secretion stress.

Draft genome sequences of two protease-producing strains of arsukibacterium, isolated from two cold and alkaline environments.

Arsukibacterium ikkense GCM72(T) and a close relative, Arsukibacterium sp. MJ3, were isolated from two cold and alkaline environments as producers of extracellular proteolytic enzymes active at high pH and low temperature. This report describes the two draft genome sequences, which may serve as sources of future industrial enzymes.

Draft Genome Sequence of the Psychrophilic and Alkaliphilic Rhodonellum psychrophilum Strain GCM71T.

Rhodonellum psychrophilum GCM71(T), isolated from the cold and alkaline submarine ikaite columns in the Ikka Fjord in Greenland, displays optimal growth at 5 to 10°C and pH 10. Here, we report the draft genome sequence of this strain, which may provide insight into the mechanisms of adaptation to these extreme conditions.

Bayesian prediction of bacterial growth temperature range based on genome sequences.

BACKGROUND: The preferred habitat of a given bacterium can provide a hint of which types of enzymes of potential industrial interest it might produce. These might include enzymes that are stable and active at very high or very low temperatures. Being able to accurately predict this based on a genomic sequence, would thus allow for an efficient and targeted search for production organisms, reducing the need for culturing experiments. RESULTS: This study found a total of 40 protein families useful for distinction between three thermophilicity classes (thermophiles, mesophiles and psychrophiles). The predictive performance of these protein families were compared to those of 87 basic sequence features (relative use of amino acids and codons, genomic and 16S rDNA AT content and genome size). When using naïve Bayesian inference, it was possible to correctly predict the optimal temperature range with a Matthews correlation coefficient of up to 0.68. The best predictive performance was always achieved by including protein families as well as structural features, compared to either of these alone. A dedicated computer program was created to perform these predictions. CONCLUSIONS: This study shows that protein families associated with specific thermophilicity classes can provide effective input data for thermophilicity prediction, and that the naïve Bayesian approach is effective for such a task. The program created for this study is able to efficiently distinguish between thermophilic, mesophilic and psychrophilic adapted bacterial genomes.

Genomic characterization of Campylobacter jejuni strain M1.

Campylobacter jejuni strain M1 (laboratory designation 99/308) is a rarely documented case of direct transmission of C. jejuni from chicken to a person, resulting in enteritis. We have sequenced the genome of C. jejuni strain M1, and compared this to 12 other C. jejuni sequenced genomes currently publicly available. Compared to these, M1 is closest to strain 81116. Based on the 13 genome sequences, we have identified the C. jejuni pan-genome, as well as the core genome, the auxiliary genes, and genes unique between strains M1 and 81116. The pan-genome contains 2,427 gene families, whilst the core genome comprised 1,295 gene families, or about two-thirds of the gene content of the average of the sequenced C. jejuni genomes. Various comparison and visualization tools were applied to the 13 C. jejuni genome sequences, including a species pan- and core genome plot, a BLAST Matrix and a BLAST Atlas. Trees based on 16S rRNA sequences and on the total gene families in each genome are presented. The findings are discussed in the background of the proven virulence potential of M1.

Parallel genetic and phenotypic evolution of DNA superhelicity in experimental populations of Escherichia coli.

DNA supercoiling is the master function that interconnects chromosome structure and global gene transcription. This function has recently been shown to be under strong selection in Escherichia coli. During the evolution of 12 initially identical populations propagated in a defined environment for 20,000 generations, parallel increases in DNA supercoiling were observed in ten populations. The genetic changes associated with the increased supercoiling were examined in one population, and beneficial mutations in the genes topA (encoding topoisomerase I) and fis (encoding a histone-like protein) were identified. To elucidate the molecular basis and impact of these changes, we quantified the level of genetic, phenotypic, and molecular parallelism linked to DNA supercoiling in all 12 evolving populations. First, sequence determination of DNA topology-related loci revealed strong genetic parallelism, with mutations concentrated in three genes (topA, fis, and dusB), although the populations had different alleles at each locus. Statistical analyses of these polymorphisms implied the action of positive selection and, moreover, suggested that fis and dusB, which belong to the same operon, have related functions. Indeed, we demonstrated that DusB regulates the expression of fis by both experimental and phylogenetic analyses. Second, molecular analyses of five mutations in fis and dusB affecting the transcription, translation, and protein activity of Fis also revealed strong parallelism in the resulting phenotypic effects. Third, artificially increasing DNA supercoiling in one of the two populations that lacked DNA topology changes led to a significant fitness increase. The high levels of molecular and genetic parallelism, targeting a small subset of the many genes involved in DNA supercoiling, indicate that changes in DNA superhelicity have been important in the evolution of these populations. Surprisingly, however, most of the evolved alleles we tested had either no detectable or slightly deleterious effects on fitness, despite these signatures of positive selection.

On the origins of a Vibrio species.

Thirty-two genome sequences of various Vibrionaceae members are compared, with emphasis on what makes V. cholerae unique. As few as 1,000 gene families are conserved across all the Vibrionaceae genomes analysed; this fraction roughly doubles for gene families conserved within the species V. cholerae. Of these, approximately 200 gene families that cluster on various locations of the genome are not found in other sequenced Vibrionaceae; these are possibly unique to the V. cholerae species. By comparing gene family content of the analysed genomes, the relatedness to a particular species is identified for two unspeciated genomes. Conversely, two genomes presumably belonging to the same species have suspiciously dissimilar gene family content. We are able to identify a number of genes that are conserved in, and unique to, V. cholerae. Some of these genes may be crucial to the niche adaptation of this species.

GeneWiz browser: An Interactive Tool for Visualizing Sequenced Chromosomes.

We present an interactive web application for visualizing genomic data of prokaryotic chromosomes. The tool (GeneWiz browser) allows users to carry out various analyses such as mapping alignments of homologous genes to other genomes, mapping of short sequencing reads to a reference chromosome, and calculating DNA properties such as curvature or stacking energy along the chromosome. The GeneWiz browser produces an interactive graphic that enables zooming from a global scale down to single nucleotides, without changing the size of the plot. Its ability to disproportionally zoom provides optimal readability and increased functionality compared to other browsers. The tool allows the user to select the display of various genomic features, color setting and data ranges. Custom numerical data can be added to the plot allowing, for example, visualization of gene expression and regulation data. Further, standard atlases are pre-generated for all prokaryotic genomes available in GenBank, providing a fast overview of all available genomes, including recently deposited genome sequences. The tool is available online from http://www.cbs.dtu.dk/services/gwBrowser. Supplemental material including interactive atlases is available online at http://www.cbs.dtu.dk/services/gwBrowser/suppl/.

The genome BLASTatlas-a GeneWiz extension for visualization of whole-genome homology.

The development of fast and inexpensive methods for sequencing bacterial genomes has led to a wealth of data, often with many genomes being sequenced of the same species or closely related organisms. Thus, there is a need for visualization methods that will allow easy comparison of many sequenced genomes to a defined reference strain. The BLASTatlas is one such tool that is useful for mapping and visualizing whole genome homology of genes and proteins within a reference strain compared to other strains or species of one or more prokaryotic organisms. We provide examples of BLASTatlases, including the Clostridium tetani plasmid p88, where homologues for toxin genes can be easily visualized in other sequenced Clostridium genomes, and for a Clostridium botulinum genome, compared to 14 other Clostridium genomes. DNA structural information is also included in the atlas to visualize the DNA chromosomal context of regions. Additional information can be added to these plots, and as an example we have added circles showing the probability of the DNA helix opening up under superhelical tension. The tool is SOAP compliant and WSDL (web services description language) files are located on our website: (http://www.cbs.dtu.dk/ws/BLASTatlas), where programming examples are available in Perl. By providing an interoperable method to carry out whole genome visualization of homology, this service offers bioinformaticians as well as biologists an easy-to-adopt workflow that can be directly called from the programming language of the user, hence enabling automation of repeated tasks. This tool can be relevant in many pangenomic as well as in metagenomic studies, by giving a quick overview of clusters of insertion sites, genomic islands and overall homology between a reference sequence and a data set.

Global features of the Alcanivorax borkumensis SK2 genome.

The global feature of the completely sequenced Alcanivorax borkumensis SK2 type strain chromosome is its symmetry and homogeneity. The origin and terminus of replication are located opposite to each other in the chromosome and are discerned with high signal to noise ratios by maximal oligonucleotide usage biases on the leading and lagging strand. Genomic DNA structure is rather uniform throughout the chromosome with respect to intrinsic curvature, position preference or base stacking energy. The orthologs and paralogs of A. borkumensis genes with the highest sequence homology were found in most cases among gamma-Proteobacteria, with Acinetobacter and P. aeruginosa as closest relatives. A. borkumensis shares a similar oligonucleotide usage and promoter structure with the Pseudomonadales. A comparatively low number of only 18 genome islands with atypical oligonucleotide usage was detected in the A. borkumensis chromosome. The gene clusters that confer the assimilation of aliphatic hydrocarbons, are localized in two genome islands which were probably acquired from an ancestor of the Yersinia lineage, whereas the alk genes of Pseudomonas putida still exhibit the typical Alcanivorax oligonucleotide signature indicating a complex evolution of this major hydrocarbonoclastic trait.

Characterization of probiotic Escherichia coli isolates with a novel pan-genome microarray.

BACKGROUND: Microarrays have recently emerged as a novel procedure to evaluate the genetic content of bacterial species. So far, microarrays have mostly covered single or few strains from the same species. However, with cheaper high-throughput sequencing techniques emerging, multiple strains of the same species are rapidly becoming available, allowing for the definition and characterization of a whole species as a population of genomes--the 'pan-genome'. RESULTS: Using 32 Escherichia coli and Shigella genome sequences we estimate the pan- and core genome of the species. We designed a high-density microarray in order to provide a tool for characterization of the E. coli pan-genome. Technical performance of this pan-genome microarray based on control strain samples (E. coli K-12 and O157:H7) demonstrated a high sensitivity and relatively low false positive rate. A single-channel analysis approach is robust while allowing the possibility for deriving presence/absence predictions for any gene included on our pan-genome microarray. Moreover, the array was highly sufficient to investigate the gene content of non-pathogenic isolates, despite the strong bias towards pathogenic E. coli strains that have been sequenced so far. CONCLUSION: This high-density microarray provides an excellent tool for characterizing the genetic makeup of unknown E. coli strains and can also deliver insights into phylogenetic relationships. Its design poses a considerably larger challenge and involves different considerations than the design of single strain microarrays. Here, lessons learned and future directions will be discussed in order to optimize design of microarrays targeting entire pan-genomes.

The complete genome sequence and analysis of the epsilonproteobacterium Arcobacter butzleri.

BACKGROUND: Arcobacter butzleri is a member of the epsilon subdivision of the Proteobacteria and a close taxonomic relative of established pathogens, such as Campylobacter jejuni and Helicobacter pylori. Here we present the complete genome sequence of the human clinical isolate, A. butzleri strain RM4018. METHODOLOGY/PRINCIPAL FINDINGS: Arcobacter butzleri is a member of the Campylobacteraceae, but the majority of its proteome is most similar to those of Sulfuromonas denitrificans and Wolinella succinogenes, both members of the Helicobacteraceae, and those of the deep-sea vent Epsilonproteobacteria Sulfurovum and Nitratiruptor. In addition, many of the genes and pathways described here, e.g. those involved in signal transduction and sulfur metabolism, have been identified previously within the epsilon subdivision only in S. denitrificans, W. succinogenes, Sulfurovum, and/or Nitratiruptor, or are unique to the subdivision. In addition, the analyses indicated also that a substantial proportion of the A. butzleri genome is devoted to growth and survival under diverse environmental conditions, with a large number of respiration-associated proteins, signal transduction and chemotaxis proteins and proteins involved in DNA repair and adaptation. To investigate the genomic diversity of A. butzleri strains, we constructed an A. butzleri DNA microarray comprising 2238 genes from strain RM4018. Comparative genomic indexing analysis of 12 additional A. butzleri strains identified both the core genes of A. butzleri and intraspecies hypervariable regions, where <70% of the genes were present in at least two strains. CONCLUSION/SIGNIFICANCE: The presence of pathways and loci associated often with non-host-associated organisms, as well as genes associated with virulence, suggests that A. butzleri is a free-living, water-borne organism that might be classified rightfully as an emerging pathogen. The genome sequence and analyses presented in this study are an important first step in understanding the physiology and genetics of this organism, which constitutes a bridge between the environment and mammalian hosts.

RNAmmer: consistent and rapid annotation of ribosomal RNA genes.

The publication of a complete genome sequence is usually accompanied by annotations of its genes. In contrast to protein coding genes, genes for ribosomal RNA (rRNA) are often poorly or inconsistently annotated. This makes comparative studies based on rRNA genes difficult. We have therefore created computational predictors for the major rRNA species from all kingdoms of life and compiled them into a program called RNAmmer. The program uses hidden Markov models trained on data from the 5S ribosomal RNA database and the European ribosomal RNA database project. A pre-screening step makes the method fast with little loss of sensitivity, enabling the analysis of a complete bacterial genome in less than a minute. Results from running RNAmmer on a large set of genomes indicate that the location of rRNAs can be predicted with a very high level of accuracy. Novel, unannotated rRNAs are also predicted in many genomes. The software as well as the genome analysis results are available at the CBS web server.

Ten years of bacterial genome sequencing: comparative-genomics-based discoveries.

It has been more than 10 years since the first bacterial genome sequence was published. Hundreds of bacterial genome sequences are now available for comparative genomics, and searching a given protein against more than a thousand genomes will soon be possible. The subject of this review will address a relatively straightforward question: "What have we learned from this vast amount of new genomic data?" Perhaps one of the most important lessons has been that genetic diversity, at the level of large-scale variation amongst even genomes of the same species, is far greater than was thought. The classical textbook view of evolution relying on the relatively slow accumulation of mutational events at the level of individual bases scattered throughout the genome has changed. One of the most obvious conclusions from examining the sequences from several hundred bacterial genomes is the enormous amount of diversity--even in different genomes from the same bacterial species. This diversity is generated by a variety of mechanisms, including mobile genetic elements and bacteriophages. An examination of the 20 Escherichia coli genomes sequenced so far dramatically illustrates this, with the genome size ranging from 4.6 to 5.5 Mbp; much of the variation appears to be of phage origin. This review also addresses mobile genetic elements, including pathogenicity islands and the structure of transposable elements. There are at least 20 different methods available to compare bacterial genomes. Metagenomics offers the chance to study genomic sequences found in ecosystems, including genomes of species that are difficult to culture. It has become clear that a genome sequence represents more than just a collection of gene sequences for an organism and that information concerning the environment and growth conditions for the organism are important for interpretation of the genomic data. The newly proposed Minimal Information about a Genome Sequence standard has been developed to obtain this information.

Origin of replication in circular prokaryotic chromosomes.

To predict origins of replication in prokaryotic chromosomes, we analyse the leading and lagging strands of 200 chromosomes for differences in oligomer composition and show that these correlate strongly with taxonomic grouping, lifestyle and molecular details of the replication process. While all bacteria have a preference for Gs over Cs on the leading strand, we discover that the direction of the A/T skew is determined by the polymerase-alpha subunit that replicates the leading strand. The strength of the strand bias varies greatly between both phyla and environments and appears to correlate with growth rate. Finally we observe much greater diversity of skew among archaea than among bacteria. We have developed a program that accurately locates the origins of replication by measuring the differences between leading and lagging strand of all oligonucleotides up to 8 bp in length. The program and results for all publicly available genomes are available from http://www.cbs.dtu.dk/services/GenomeAtlas/suppl/origin.