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The methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 8 Panton-Valentine toxin (PVL)-positive USA300 clone has a worldwide distribution. The USA300 North American (NA) variant, harbouring the arginine catabolic mobile element (ACME), is predominant in the USA while the Latin American (LV) variant is predominant in Northern South America. Both variants have failed to become endemic in Europe. We examined here the epidemiology of the USA300 clone in Belgium from 2006 to 2019. A total of 399 clonal complex 8 PVL-positive MRSA isolates received between 2006 and 2019 by the Belgian National Reference Laboratory for S. aureus were investigated for the presence of ACME. Selected ACME-positive (n=102) and ACME-negative (n=16) isolates were sequenced, characterized for the presence of several resistance and virulence molecular markers and subjected to phylogenetic analysis. A total of 300 isolates were USA300-NA (ACME-positive), while only 99 were ACME-negative. Most USA300-NA interspersed in the phylogeny analysis with isolates from other countries, suggesting multiple introductions. However, two big clades were maintained and spread over a decade, peaking between 2010 and 2017 to finally decrease. Few ACME-negative isolates, mainly related to trips to South America, were identified as USA300-LV. The remaining ACME-negative isolates were ST8 SCCmec IVb or ST923 SCCmec IVa (COL923). Two clades of the USA300-NA clone have successfully spread in Belgium, but seem to currently decrease. Related South American variants have been detected for the first time in Belgium, including the emerging COL923 clone.

In the age of antibiotic resistance and precise microbiome engineering, CRISPR-Cas antimicrobials promise to have a substantial impact on the way we treat diseases in the future. However, the efficacy of these antimicrobials and their mechanisms of resistance remain to be elucidated. We systematically investigated how a target E. coli strain can escape killing by episomally-encoded CRISPR-Cas9 antimicrobials. Using Cas9 from Streptococcus pyogenes (SpCas9) we studied the killing efficiency and resistance mutation rate towards CRISPR-Cas9 antimicrobials and elucidated the underlying genetic alterations. We find that killing efficiency is not correlated with the number of cutting sites or the type of target. While the number of targets did not significantly affect efficiency of killing, it did reduce the emergence of chromosomal mutations conferring resistance. The most frequent target of resistance mutations was the plasmid-encoded SpCas9 that was inactivated by bacterial genome rearrangements involving translocation of mobile genetic elements such as insertion elements. This resistance mechanism can be overcome by re-introduction of an intact copy of SpCas9. The work presented here provides a guide to design strategies that reduce resistance and improve the activity of CRISPR-Cas antimicrobials.

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

The methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 8 Panton-Valentine toxin (PVL)-positive USA300 clone has a worldwide distribution. The USA300 North American (NA) variant, harbouring the arginine catabolic mobile element (ACME), is predominant in the USA while the Latin American (LV) variant is predominant in Northern South America. Both variants have failed to become endemic in Europe. We examined here the epidemiology of the USA300 clone in Belgium from 2006 to 2019. A total of 399 clonal complex 8 PVL-positive MRSA isolates received between 2006 and 2019 by the Belgian National Reference Laboratory for S. aureus were investigated for the presence of ACME. Selected ACME-positive (n=102) and ACME-negative (n=16) isolates were sequenced, characterized for the presence of several resistance and virulence molecular markers and subjected to phylogenetic analysis. A total of 300 isolates were USA300-NA (ACME-positive), while only 99 were ACME-negative. Most USA300-NA interspersed in the phylogeny analysis with isolates from other countries, suggesting multiple introductions. However, two big clades were maintained and spread over a decade, peaking between 2010 and 2017 to finally decrease. Few ACME-negative isolates, mainly related to trips to South America, were identified as USA300-LV. The remaining ACME-negative isolates were ST8 SCCmec IVb or ST923 SCCmec IVa (COL923). Two clades of the USA300-NA clone have successfully spread in Belgium, but seem to currently decrease. Related South American variants have been detected for the first time in Belgium, including the emerging COL923 clone., SCOPUS: ar.j, info:eu-repo/semantics/published

Background: Borrelia bavariensis is one of the agents of Lyme Borreliosis (or Lyme disease) in Eurasia. The genome of the Borrelia burgdorferi sensu lato species complex, that includes B. bavariensis, is known to be very complex and fragmented making the assembly of whole genomes with next-generation sequencing data a challenge. Results: We present a genome reconstruction for 33 B. bavariensis isolates from Eurasia based on long-read (Pacific Bioscience, for three isolates) and short-read (Illumina) data. We show that the combination of both sequencing techniques allows proper genome reconstruction of all plasmids in most cases but use of a very close reference is necessary when only short-read sequencing data is available. B. bavariensis genomes combine a high degree of genetic conservation with high plasticity: all isolates share the main chromosome and five plasmids, but the repertoire of other plasmids is highly variable. In addition to plasmid losses and gains through horizontal transfer, we also observe several fusions between plasmids. Although European isolates of B. bavariensis have little diversity in genome content, there is some geographic structure to this variation. In contrast, each Asian isolate has a unique plasmid repertoire and we observe no geographically based differences between Japanese and Russian isolates. Comparing the genomes of Asian and European populations of B. bavariensis suggests that some genes which are markedly different between the two populations may be good candidates for adaptation to the tick vector, (Ixodes ricinus in Europe and I. persulcatus in Asia). Conclusions: We present the characterization of genomes of a large sample of B. bavariensis isolates and show that their plasmid content is highly variable. This study opens the way for genomic studies seeking to understand host and vector adaptation as well as human pathogenicity in Eurasian Lyme Borreliosis agents. © 2020 The Author(s).

Marine microorganisms inhabiting nutrient-depleted waters play critical roles in global biogeochemical cycles due to their abundance and broad distribution. Many of these microbes share similar genomic features including small genome size, low % G + C content, short intergenic regions, and low nitrogen content in encoded amino acid residue side chains (N-ARSC), but the evolutionary drivers of these characteristics are unclear. Here, we compared the strength of purifying selection across the Marinimicrobia, a candidate phylum which encompasses a broad range of phylogenetic groups with disparate genomic features, by estimating the ratio of nonsynonymous and synonymous substitutions (dN/dS) in conserved marker genes. Our analysis reveals that epipelagic Marinimicrobia that exhibit features consistent with genome streamlining have significantly lower dN/dS values when compared with their mesopelagic counterparts. We also found a significant positive correlation between median dN/dS values and % G + C content, N-ARSC, and intergenic region length. We did not identify a significant correlation between dN/dS ratios and estimated genome size, suggesting the strength of selection is not a primary factor shaping genome size in this group. Our findings are generally consistent with genome streamlining theory, which postulates that many genomic features of abundant epipelagic bacteria are the result of adaptation to oligotrophic nutrient conditions. Our results are also in agreement with previous findings that genome streamlining is common in epipelagic waters, suggesting that microbes inhabiting this region of the ocean have been shaped by strong selection together with prevalent nutritional constraints characteristic of this environment.

Synthetic genome recoding is a new means of generating designed organisms with altered phenotypes. Synonymous mutations introduced into the protein coding region tolerate modifications in DNA or mRNA without modifying the encoded proteins. Synonymous genome-wide recoding has allowed the synthetic generation of different small-genome viruses with modified phenotypes and biological properties. Recently, a decreased cost of chemically synthesizing DNA and improved methods for assembling DNA fragments (e.g. lambda red recombination and CRISPR-based editing) have enabled the construction of an Escherichia coli variant with a 4-Mb synthetic synonymously recoded genome with a reduced number of sense codons (n = 59) encoding the 20 canonical amino acids. Synonymous genome recoding is increasing our knowledge of microbial interactions with innate immune responses, identifying functional genome structures, and strategically ameliorating cis-inhibitory signaling sequences related to splicing, replication (in eukaryotes), and complex microbe functions, unraveling the relevance of codon usage for the temporal regulation of gene expression and the microbe mutant spectrum and adaptability. New biotechnological and therapeutic applications of this methodology can easily be envisaged. In this review, we discuss how synonymous genome recoding may impact our knowledge of microbial biology and the development of new and better therapeutic methodologies.

Synthetic genome recoding is a new means of generating designed organisms with altered phenotypes. Synonymous mutations introduced into the protein coding region tolerate modifications in DNA or mRNA without modifying the encoded proteins. Synonymous genome-wide recoding has allowed the synthetic generation of different small-genome viruses with modified phenotypes and biological properties. Recently, a decreased cost of chemically synthesizing DNA and improved methods for assembling DNA fragments (e.g. lambda red recombination and CRISPR-based editing) have enabled the construction of an Escherichia coli variant with a 4-Mb synthetic synonymously recoded genome with a reduced number of sense codons (n = 59) encoding the 20 canonical amino acids. Synonymous genome recoding is increasing our knowledge of microbial interactions with innate immune responses, identifying functional genome structures, and strategically ameliorating cis-inhibitory signaling sequences related to splicing, replication (in eukaryotes), and complex microbe functions, unraveling the relevance of codon usage for the temporal regulation of gene expression and the microbe mutant spectrum and adaptability. New biotechnological and therapeutic applications of this methodology can easily be envisaged. In this review, we discuss how synonymous genome recoding may impact our knowledge of microbial biology and the development of new and better therapeutic methodologies.

BACKGROUND: Next-generation sequencing (NGS) is increasingly being translated into routine public health practice, affecting the surveillance and control of many pathogens. The purpose of this scoping review is to identify and characterize the recent literature concerning the application of bacterial pathogen genomics for public health practice and to assess the added value, challenges, and needs related to its implementation from an epidemiologist's perspective. METHODS: In this scoping review, a systematic PubMed search with forward and backward snowballing was performed to identify manuscripts in English published between January 2015 and September 2018. Included studies had to describe the application of NGS on bacterial isolates within a public health setting. The studied pathogen, year of publication, country, number of isolates, sampling fraction, setting, public health application, study aim, level of implementation, time orientation of the NGS analyses, and key findings were extracted from each study. Due to a large heterogeneity of settings, applications, pathogens, and study measurements, a descriptive narrative synthesis of the eligible studies was performed. RESULTS: Out of the 275 included articles, 164 were outbreak investigations, 70 focused on strategy-oriented surveillance, and 41 on control-oriented surveillance. Main applications included the use of whole-genome sequencing (WGS) data for (1) source tracing, (2) early outbreak detection, (3) unraveling transmission dynamics, (4) monitoring drug resistance, (5) detecting cross-border transmission events, (6) identifying the emergence of strains with enhanced virulence or zoonotic potential, and (7) assessing the impact of prevention and control programs. The superior resolution over conventional typing methods to infer transmission routes was reported as an added value, as well as the ability to simultaneously characterize the resistome and virulome of the studied pathogen. However, the full potent

The past few years observed a breakthrough of genome sequences of bacteria of Rhodococcus genus with significant biodegradation abilities. Invaluable knowledge from genome data and their functional analysis can be applied to develop and design strategies for attenuating damages caused by hydrocarbon contamination. With the advent of high-throughput -omic technologies, it is currently possible to utilize the functional properties of diverse catabolic genes, analyze an entire system at the level of molecule (DNA, RNA, protein, and metabolite), simultaneously predict and construct catabolic degradation pathways. In this review, the genes involved in the biodegradation of hydrocarbons and several emerging plasticizer compounds in Rhodococcus strains are described in detail (aliphatic, aromatics, PAH, phthalate, polyethylene, and polyisoprene). The metabolic biodegradation networks predicted from omics-derived data along with the catabolic enzymes exploited in diverse biotechnological and bioremediation applications are characterized.

Group A Streptococcus (GAS; Streptococcus pyogenes) is a bacterial pathogen for which a commercial vaccine for humans is not available. Employing the advantages of high-throughput DNA sequencing technology to vaccine design, we have analyzed 2,083 globally sampled GAS genomes. The global GAS population structure reveals extensive genomic heterogeneity driven by homologous recombination and overlaid with high levels of accessory gene plasticity. We identified the existence of more than 290 clinically associated genomic phylogroups across 22 countries, highlighting challenges in designing vaccines of global utility. To determine vaccine candidate coverage, we investigated all of the previously described GAS candidate antigens for gene carriage and gene sequence heterogeneity. Only 15 of 28 vaccine antigen candidates were found to have both low naturally occurring sequence variation and high (>99%) coverage across this diverse GAS population. This technological platform for vaccine coverage determination is equally applicable to prospective GAS vaccine antigens identified in future studies., info:eu-repo/semantics/published

Marine microorganisms inhabiting nutrient-depleted waters play critical roles in global biogeochemical cycles due to their abundance and broad distribution. Many of these microbes share similar genomic features including small genome size, low % G + C content, short intergenic regions, and low nitrogen content in encoded amino acid residue side chains (N-ARSC), but the evolutionary drivers of these characteristics are unclear. Here, we compared the strength of purifying selection across the Marinimicrobia, a candidate phylum which encompasses a broad range of phylogenetic groups with disparate genomic features, by estimating the ratio of nonsynonymous and synonymous substitutions (dN/dS) in conserved marker genes. Our analysis reveals that epipelagic Marinimicrobia that exhibit features consistent with genome streamlining have significantly lower dN/dS values when compared with their mesopelagic counterparts. We also found a significant positive correlation between median dN/dS values and % G + C content, N-ARSC, and intergenic region length. We did not identify a significant correlation between dN/dS ratios and estimated genome size, suggesting the strength of selection is not a primary factor shaping genome size in this group. Our findings are generally consistent with genome streamlining theory, which postulates that many genomic features of abundant epipelagic bacteria are the result of adaptation to oligotrophic nutrient conditions. Our results are also in agreement with previous findings that genome streamlining is common in epipelagic waters, suggesting that microbes inhabiting this region of the ocean have been shaped by strong selection together with prevalent nutritional constraints characteristic of this environment.

Plants intimately associate with diverse bacteria. Plant-associated bacteria have ostensibly evolved genes that enable them to adapt to plant environments. However, the identities of such genes are mostly unknown, and their functions are poorly characterized. We sequenced 484 genomes of bacterial isolates from roots of Brassicaceae, poplar, and maize. We then compared 3,837 bacterial genomes to identify thousands of plant-associated gene clusters. Genomes of plant-associated bacteria encode more carbohydrate metabolism functions and fewer mobile elements than related non-plant-associated genomes do. We experimentally validated candidates from two sets of plant-associated genes: one involved in plant colonization, and the other serving in microbe-microbe competition between plant-associated bacteria. We also identified 64 plant-associated protein domains that potentially mimic plant domains; some are shared with plant-associated fungi and oomycetes. This work expands the genome-based understanding of plant-microbe interactions and provides potential leads for efficient and sustainable agriculture through microbiome engineering.

Determining bacterial fitness represents a major challenge and no single parameter can accurately predict the ability of a certain pathogen to succeed. The M strain of Mycobacterium tuberculosis managed to spread and establish in the community and caused the largest multidrug-resistant tuberculosis outbreak in Latin America. We have previously shown that the M strain can manipulate the host immune response, but we still have no direct evidence, other than epidemiology, that can account for the enhanced fitness of the M strain. Our objective was to further characterize the performance of the outbreak strain M in different fitness assays. Two main aspects were evaluated: (1) molecular characterization of selected isolates from the M outbreak and related strains and (2) comparative fitness and in vivo performance of representative M strain isolates vs. the non-prosperous M strain variant 410. Our approach confirmed the multifaceted nature of fitness. Altogether, we conclude that the epidemiologically abortive strain 410 was vulnerable to drug-driven pressure, a weak competitor, and a stronger inductor of protective response in vivo. Conversely, the isolate 6548, representative of the M outbreak peak, had a growth disadvantage but performed very well in competition and induced lung damage at advanced stages in spite of reaching relatively low CFU counts. Integration of these observations supports the idea that the M strain managed to find a unique path to success.

Determining bacterial fitness represents a major challenge and no single parameter can accurately predict the ability of a certain pathogen to succeed. The M strain of Mycobacterium tuberculosis managed to spread and establish in the community and caused the largest multidrug-resistant tuberculosis outbreak in Latin America. We have previously shown that the M strain can manipulate the host immune response, but we still have no direct evidence, other than epidemiology, that can account for the enhanced fitness of the M strain. Our objective was to further characterize the performance of the outbreak strain M in different fitness assays. Two main aspects were evaluated: (1) molecular characterization of selected isolates from the M outbreak and related strains and (2) comparative fitness and in vivo performance of representative M strain isolates vs. the non-prosperous M strain variant 410. Our approach confirmed the multifaceted nature of fitness. Altogether, we conclude that the epidemiologically abortive strain 410 was vulnerable to drug-driven pressure, a weak competitor, and a stronger inductor of protective response in vivo. Conversely, the isolate 6548, representative of the M outbreak peak, had a growth disadvantage but performed very well in competition and induced lung damage at advanced stages in spite of reaching relatively low CFU counts. Integration of these observations supports the idea that the M strain managed to find a unique path to success.

Plants intimately associate with diverse bacteria. Plant-associated bacteria have ostensibly evolved genes that enable them to adapt to plant environments. However, the identities of such genes are mostly unknown, and their functions are poorly characterized. We sequenced 484 genomes of bacterial isolates from roots of Brassicaceae, poplar, and maize. We then compared 3,837 bacterial genomes to identify thousands of plant-associated gene clusters. Genomes of plant-associated bacteria encode more carbohydrate metabolism functions and fewer mobile elements than related non-plant-associated genomes do. We experimentally validated candidates from two sets of plant-associated genes: one involved in plant colonization, and the other serving in microbe-microbe competition between plant-associated bacteria. We also identified 64 plant-associated protein domains that potentially mimic plant domains; some are shared with plant-associated fungi and oomycetes. This work expands the genome-based understanding of plant-microbe interactions and provides potential leads for efficient and sustainable agriculture through microbiome engineering.

The moose (Alces alces) is a ruminant that harvests energy from fiber-rich lignocellulose material through carbohydrate-active enzymes (CAZymes) produced by its rumen microbes. We applied shotgun metagenomics to rumen contents from six moose to obtain insights into this microbiome. Following binning, 99 metagenome-assembled genomes (MAGs) belonging to 11 prokaryotic phyla were reconstructed and characterized based on phylogeny and CAZyme profile. The taxonomy of these MAGs reflected the overall composition of the metagenome, with dominance of the phyla Bacteroidetes and Firmicutes. Unlike in other ruminants, Spirochaetes constituted a significant proportion of the community and our analyses indicate that the corresponding strains are primarily pectin digesters. Pectin-degrading genes were also common in MAGs of Ruminococcus, Fibrobacteres and Bacteroidetes and were overall overrepresented in the moose microbiome compared with other ruminants. Phylogenomic analyses revealed several clades within the Bacteriodetes without previously characterized genomes. Several of these MAGs encoded a large numbers of dockerins, a module usually associated with cellulosomes. The Bacteroidetes dockerins were often linked to CAZymes and sometimes encoded inside polysaccharide utilization loci, which has never been reported before. The almost 100 CAZyme-annotated genomes reconstructed in this study provide an in-depth view of an efficient lignocellulose-degrading microbiome and prospects for developing enzyme technology for biorefineries.

The aim of this study is to obtain a systems-level understanding of the interactions between and corrinoid-supplying microorganisms by analyzing community structures and functional compositions, activities, and dynamics in trichloroethene (TCE)-dechlorinating enrichments. Metagenomes and metatranscriptomes of the dechlorinating enrichments with and without exogenous cobalamin were compared. Seven putative draft genomes were binned from the metagenomes. At an early stage (2 days), more transcripts of genes in the bin-genome were detected in the metatranscriptome of the enrichment without exogenous cobalamin than in the one with the addition of cobalamin. Among these genes, sporulation-related genes exhibited the highest differential expression when cobalamin was not added, suggesting a possible release route of corrinoids from corrinoid producers. Other differentially expressed genes include those involved in energy conservation and nutrient transport (including cobalt transport). The most highly expressed corrinoid biosynthesis pathway was also assigned to the bin-genome. Targeted quantitative PCR (qPCR) analyses confirmed higher transcript abundances of those corrinoid biosynthesis genes in the enrichment without exogenous cobalamin than in the enrichment with cobalamin. Furthermore, the corrinoid salvaging and modification pathway of was upregulated in response to the cobalamin stress. This study provides important insights into the microbial interactions and roles played by members of dechlorinating communities under cobalamin-limited conditions. The key chloroethene-dechlorinating bacterium is a cobalamin auxotroph, thus acquiring corrinoids from other community members. Therefore, it is important to investigate the microbe-microbe interactions between and the corrinoid-providing microorganisms in a community. This study provides systems-level information, i.e., taxonomic and functional compositions and dynamics of the supportive microorganisms in dechlorinating

The moose (Alces alces) is a ruminant that harvests energy from fiber-rich lignocellulose material through carbohydrate-active enzymes (CAZymes) produced by its rumen microbes. We applied shotgun metagenomics to rumen contents from six moose to obtain insights into this microbiome. Following binning, 99 metagenome-assembled genomes (MAGs) belonging to 11 prokaryotic phyla were reconstructed and characterized based on phylogeny and CAZyme profile. The taxonomy of these MAGs reflected the overall composition of the metagenome, with dominance of the phyla Bacteroidetes and Firmicutes. Unlike in other ruminants, Spirochaetes constituted a significant proportion of the community and our analyses indicate that the corresponding strains are primarily pectin digesters. Pectin-degrading genes were also common in MAGs of Ruminococcus, Fibrobacteres and Bacteroidetes and were overall overrepresented in the moose microbiome compared with other ruminants. Phylogenomic analyses revealed several clades within the Bacteriodetes without previously characterized genomes. Several of these MAGs encoded a large numbers of dockerins, a module usually associated with cellulosomes. The Bacteroidetes dockerins were often linked to CAZymes and sometimes encoded inside polysaccharide utilization loci, which has never been reported before. The almost 100 CAZyme-annotated genomes reconstructed in this study provide an in-depth view of an efficient lignocellulose-degrading microbiome and prospects for developing enzyme technology for biorefineries.

The aim of this study is to obtain a systems-level understanding of the interactions between and corrinoid-supplying microorganisms by analyzing community structures and functional compositions, activities, and dynamics in trichloroethene (TCE)-dechlorinating enrichments. Metagenomes and metatranscriptomes of the dechlorinating enrichments with and without exogenous cobalamin were compared. Seven putative draft genomes were binned from the metagenomes. At an early stage (2 days), more transcripts of genes in the bin-genome were detected in the metatranscriptome of the enrichment without exogenous cobalamin than in the one with the addition of cobalamin. Among these genes, sporulation-related genes exhibited the highest differential expression when cobalamin was not added, suggesting a possible release route of corrinoids from corrinoid producers. Other differentially expressed genes include those involved in energy conservation and nutrient transport (including cobalt transport). The most highly expressed corrinoid biosynthesis pathway was also assigned to the bin-genome. Targeted quantitative PCR (qPCR) analyses confirmed higher transcript abundances of those corrinoid biosynthesis genes in the enrichment without exogenous cobalamin than in the enrichment with cobalamin. Furthermore, the corrinoid salvaging and modification pathway of was upregulated in response to the cobalamin stress. This study provides important insights into the microbial interactions and roles played by members of dechlorinating communities under cobalamin-limited conditions. The key chloroethene-dechlorinating bacterium is a cobalamin auxotroph, thus acquiring corrinoids from other community members. Therefore, it is important to investigate the microbe-microbe interactions between and the corrinoid-providing microorganisms in a community. This study provides systems-level information, i.e., taxonomic and functional compositions and dynamics of the supportive microorganisms in dechlorinating

The moose (Alces alces) is a ruminant that harvests energy from fiber-rich lignocellulose material through carbohydrate-active enzymes (CAZymes) produced by its rumen microbes. We applied shotgun metagenomics to rumen contents from six moose to obtain insights into this microbiome. Following binning, 99 metagenome-assembled genomes (MAGs) belonging to 11 prokaryotic phyla were reconstructed and characterized based on phylogeny and CAZyme profile. The taxonomy of these MAGs reflected the overall composition of the metagenome, with dominance of the phyla Bacteroidetes and Firmicutes. Unlike in other ruminants, Spirochaetes constituted a significant proportion of the community and our analyses indicate that the corresponding strains are primarily pectin digesters. Pectin-degrading genes were also common in MAGs of Ruminococcus, Fibrobacteres and Bacteroidetes and were overall overrepresented in the moose microbiome compared with other ruminants. Phylogenomic analyses revealed several clades within the Bacteriodetes without previously characterized genomes. Several of these MAGs encoded a large numbers of dockerins, a module usually associated with cellulosomes. The Bacteroidetes dockerins were often linked to CAZymes and sometimes encoded inside polysaccharide utilization loci, which has never been reported before. The almost 100 CAZyme-annotated genomes reconstructed in this study provide an in-depth view of an efficient lignocellulose-degrading microbiome and prospects for developing enzyme technology for biorefineries.

We describe 2 fatal cases of methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 septicemia in persons who had no contact with livestock. Whole-genome sequencing of the isolated MRSA strains strongly suggest that both were of animal origin and that the patients had been infected through 2 independent person-to-person transmission chains.

Snakin-1, a peptide produced by higher plants, has broad-spectrum antibiotic activity, inhibiting organisms ranging from Bacteria to Eukaryotes. However, the mode of action against target organisms is poorly understood. As a first step to elucidate the mechanism, we screened a mutation library of Pseudomonas fluorescens Pf-5 in LB and agar medium supplemented with alfalfa snakin-1 (MsSN1). We identified three biofilm formation-related Pseudomonas mutants that showed increased resistance to MsSN1. Genetic, physiological and bioinformatics analysis validated the results of the mutant screens, indicating that bacterial adhesion protein lapA is probably the target of MsSN1. Collectively, these findings suggest that snakin-1 acts on microbial adhesion properties.

Typing of meticillin resistant Staphylococcus aureus (MRSA) by whole genome sequencing (WGS) is performed routinely in Copenhagen since January 2013. We describe the relatedness, based on WGS data and epidemiological data, of 341 MRSA isolates. These comprised all MRSA (n = 300) identified in Copenhagen in the first five months of 2013. Moreover, because MRSA of staphylococcal protein A (spa)-type 304 (t304), sequence type (ST) 6 had been associated with a continuous neonatal ward outbreak in Copenhagen starting in 2011, 41 t304 isolates collected in the city between 2010 and 2012 were also included. Isolates from 2013 found to be of t304, ST6 (n=14) were compared to the 41 earlier isolates. In the study, isolates of clonal complex (CC) 22 were examined in detail, as this CC has been shown to include the hospitalacquired epidemic MRSA (EMRSA-15) clone. Finally, all MRSA ST80 were also further analysed, as representatives of an important community-acquired MRSA in Europe. Overall the analysis identified 85 spa-types and 35 STs from 17 CCs. WGS confirmed the relatedness of epidemiologically linked t304 neonatal outbreak isolates. Several non-outbreak related patients had isolates closely related to the neonatal isolates suggesting unrecognised community chains of transmission and insufficient epidemiological data. Only four CC22 isolates were related to EMRSA-15. No community spread was observed among the 13 ST80 isolates. WGS successfully replaced conventional typing and added information to epidemiological surveillance. Creation of a MRSA database allows clustering of isolates based on single nucleotide polymorphism (SNP) calling and has improved our understanding of MRSA transmission., Typing of meticillin resistant Staphylococcus aureus (MRSA) by whole genome sequencing (WGS) is performed routinely in Copenhagen since January 2013. We describe the relatedness, based on WGS data and epidemiological data, of 341 MRSA isolates. These comprised all MRSA (n = 300) identified in Copenhagen in the first five months of 2013. Moreover, because MRSA of staphylococcal protein A (spa)-type 304 (t304), sequence type (ST) 6 had been associated with a continuous neonatal ward outbreak in Copenhagen starting in 2011, 41 t304 isolates collected in the city between 2010 and 2012 were also included. Isolates from 2013 found to be of t304, ST6 (n=14) were compared to the 41 earlier isolates. In the study, isolates of clonal complex (CC) 22 were examined in detail, as this CC has been shown to include the hospital-acquired epidemic MRSA (EMRSA-15) clone. Finally, all MRSA ST80 were also further analysed, as representatives of an important community-acquired MRSA in Europe. Overall the analysis identified 85 spa-types and 35 STs from 17 CCs. WGS confirmed the relatedness of epidemiologically linked t304 neonatal outbreak isolates. Several non-outbreak related patients had isolates closely related to the neonatal isolates suggesting unrecognised community chains of transmission and insufficient epidemiological data. Only four CC22 isolates were related to EMRSA-15. No community spread was observed among the 13 ST80 isolates. WGS successfully replaced conventional typing and added information to epidemiological surveillance. Creation of a MRSA database allows clustering of isolates based on single nucleotide polymorphism (SNP) calling and has improved our understanding of MRSA transmission.

Here we report the draft genomes and annotation of four N-acyl homoserine lactone (AHL)-producing members from the family Sphingomonadaceae. Comparative genomic analyses of 62 Sphingomonadaceae genomes were performed to gain insights into the distribution of the canonical luxI/R-type quorum sensing (QS) network within this family. Forty genomes contained at least one luxR homolog while the genome of Sphingobium yanoikuyae B1 contained seven Open Reading Frames (ORFs) that have significant homology to that of luxR. Thirty-three genomes contained at least one luxI homolog while the genomes of Sphingobium sp. SYK6, Sphingobium japonicum, and Sphingobium lactosutens contained four luxI. Using phylogenetic analysis, the sphingomonad LuxR homologs formed five distinct clades with two minor clades located near the plant associated bacteria (PAB) LuxR solo clade. This work for the first time shows that 13 Sphingobium and one Sphingomonas genome(s) contain three convergently oriented genes composed of two tandem luxR genes proximal to one luxI (luxR-luxR-luxI). Interestingly, luxI solos were identified in two Sphingobium species and may represent species that contribute to AHL-based QS system by contributing AHL molecules but are unable to perceive AHLs as signals. This work provides the most comprehensive description of the luxI/R circuitry and genome-based taxonomical description of the available sphingomonad genomes to date indicating that the presence of luxR solos and luxI solos are not an uncommon feature in members of the Sphingomonadaceae family.

In this study, we report the screening of four Arcobacter species for the presence of small and large plasmids. Plasmids were present in 9.9% of the 273 examined strains. One Arcobacter cryaerophilus and four Arcobacter butzleri plasmids were selected for further sequencing. The size of three small plasmids isolated from A. butzleri and the one from A. cryaerophilus strains ranged between 4.8 and 5.1 kb, and the size of the large plasmid, isolated from A. butzleri, was 27.4 kbp. The G+C content of all plasmids ranged between 25.4% and 26.2%. A total of 95% of the large plasmid sequence represents coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried 35 coding sequences, including seven genes in a contiguous region of 11.6 kbp that encodes an orthologous type IV secretion system found in the Wolinella succinogenes genome, Helicobacter pylori and Campylobacter jejuni plasmids, which makes this plasmid interesting for further exploration., info:eu-repo/semantics/published

Species of Anoxybacillus are widespread in geothermal springs, manure, and milk-processing plants. The genus is composed of 22 species and two subspecies, but the relationship between its lifestyle and genome is little understood. In this study, two high-quality draft genomes were generated from Anoxybacillus spp. SK3-4 and DT3-1, isolated from Malaysian hot springs. De novo assembly and annotation were performed, followed by comparative genome analysis with the complete genome of Anoxybacillus flavithermus WK1 and two additional draft genomes, of A. flavithermus TNO-09.006 and A. kamchatkensis G10. The genomes of Anoxybacillus spp. are among the smaller of the family Bacillaceae. Despite having smaller genomes, their essential genes related to lifestyle adaptations at elevated temperature, extreme pH, and protection against ultraviolet are complete. Due to the presence of various competence proteins, Anoxybacillus spp. SK3-4 and DT3-1 are able to take up foreign DNA fragments, and some of these transferred genes are important for the survival of the cells. The analysis of intact putative prophage genomes shows that they are highly diversified. Based on the genome analysis using SEED, many of the annotated sequences are involved in carbohydrate metabolism. The presence of glycosyl hydrolases among the Anoxybacillus spp. was compared, and the potential applications of these unexplored enzymes are suggested here. This is the first study that compares Anoxybacillus genomes from the aspect of lifestyle adaptations, the capacity for horizontal gene transfer, and carbohydrate metabolism.

BACKGROUND: Typhoid fever is an infectious disease of global importance that is caused by Salmonella enterica subsp. enterica serovar Typhi (S. Typhi). This disease causes an estimated 200,000 deaths per year and remains a serious global health threat. S. Typhi is strictly a human pathogen, and some recovered individuals become long-term carriers who continue to shed the bacteria in their faeces, thus becoming main reservoirs of infection. RESULTS: A comparative genomics analysis combined with a phylogenomic analysis revealed that the strains from the outbreak and carrier were closely related with microvariations and possibly derived from a common ancestor. Additionally, the comparative genomics analysis with all of the other completely sequenced S. Typhi genomes revealed that strains BL196 and CR0044 exhibit unusual genomic variations despite S. Typhi being generally regarded as highly clonal. The two genomes shared distinct chromosomal architectures and uncommon genome features; notably, the presence of a ~10 kb novel genomic island containing uncharacterised virulence-related genes, and zot in particular. Variations were also detected in the T6SS system and genes that were related to SPI-10, insertion sequences, CRISPRs and nsSNPs among the studied genomes. Interestingly, the carrier strain CR0044 harboured far more genetic polymorphisms (83% mutant nsSNPs) compared with the closely related BL196 outbreak strain. Notably, the two highly related virulence-determinant genes, rpoS and tviE, were mutated in strains BL196 and CR0044, respectively, which revealed that the mutation in rpoS is stabilising, while that in tviE is destabilising. These microvariations provide novel insight into the optimisation of genes by the pathogens. However, the sporadic strain was found to be far more conserved compared with the others. CONCLUSIONS: The uncommon genomic variations in the two closely related BL196 and CR0044 strains suggests that S. Typhi is more

Genomic analyses of microbial populations in their natural environment remain limited by the difficulty to assemble full genomes of individual species. Consequently, the chromosome organization of microorganisms has been investigated in a few model species, but the extent to which the features described can be generalized to other taxa remains unknown. Using controlled mixes of bacterial and yeast species, we developed meta3C, a metagenomic chromosome conformation capture approach that allows characterizing individual genomes and their average organization within a mix of organisms. Not only can meta3C be applied to species already sequenced, but a single meta3C library can be used for assembling, scaffolding and characterizing the tridimensional organization of unknown genomes. By applying meta3C to a semi-complex environmental sample, we confirmed its promising potential. Overall, this first meta3C study highlights the remarkable diversity of microorganisms chromosome organization, while providing an elegant and integrated approach to metagenomic analysis., info:eu-repo/semantics/published

Microbial synthesis of functional polymers has become increasingly important for industrial biotechnology. For the first time, it became possible to synthesize controllable composition of poly(3-hydroxyalkanoate) (P3HA) consisting of 3-hydroxydodecanoate (3HDD) and phenyl group on the side-chain when chromosome of Pseudomonas entomophila was edited to weaken its β-oxidation. Cultured in the presence of 5-phenylvaleric acid (PVA), the edited P. entomophila produced only homopolymer poly(3-hydroxy-5-phenylvalerate) or P(3HPhV). While copolyesters P(3HPhV-co-3HDD) of 3-hydroxy-5-phenylvalerate (3HPhV) and 3-hydroxydodecanoate (3HDD) were synthesized when the strain was grown on mixtures of PVA and dodecanoic acid (DDA). Compositions of 3HPhV in P(3HPhV-co-3HDD) were controllable ranging from 3% to 32% depending on DDDA/PVA ratios. Nuclear magnetic resonance (NMR) spectra clearly indicated that the polymers were homopolymer of P(3HPhV) and random copolymers of 3HPhV and 3HDD. Their mechanical and thermal properties varied dramatically depending on the monomer ratios. Our results demonstrated the possibility to produce tailor-made, novel functional PHA using the chromosome edited P. entomophila.

In this study, we report the screening of four Arcobacter species for the presence of small and large plasmids. Plasmids were present in 9.9% of the 273 examined strains. One Arcobacter cryaerophilus and four Arcobacter butzleri plasmids were selected for further sequencing. The size of three small plasmids isolated from A. butzleri and the one from A. cryaerophilus strains ranged between 4.8 and 5.1 kb, and the size of the large plasmid, isolated from A. butzleri, was 27.4 kbp. The G+C content of all plasmids ranged between 25.4% and 26.2%. A total of 95% of the large plasmid sequence represents coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried 35 coding sequences, including seven genes in a contiguous region of 11.6 kbp that encodes an orthologous type IV secretion system found in the Wolinella succinogenes genome, Helicobacter pylori and Campylobacter jejuni plasmids, which makes this plasmid interesting for further exploration., info:eu-repo/semantics/published

The genome-scale model (GEM) of metabolism in the bacterium Escherichia coli K-12 has been in development for over a decade and is now in wide use. GEM-enabled studies of E. coli have been primarily focused on six applications: (1) metabolic engineering, (2) model-driven discovery, (3) prediction of cellular phenotypes, (4) analysis of biological network properties, (5) studies of evolutionary processes, and (6) models of interspecies interactions. In this review, we provide an overview of these applications along with a critical assessment of their successes and limitations, and a perspective on likely future developments in the field. Taken together, the studies performed over the past decade have established a genome-scale mechanistic understanding of genotype-phenotype relationships in E. coli metabolism that forms the basis for similar efforts for other microbial species. Future challenges include the expansion of GEMs by integrating additional cellular processes beyond metabolism, the identification of key constraints based on emerging data types, and the development of computational methods able to handle such large-scale network models with sufficient accuracy.

Adaptation of cells to environmental changes requires dynamic interactions between metabolic and regulatory networks, but studies typically address only one or a few layers of regulation. For nutritional shifts between two preferred carbon sources of Bacillus subtilis, we combined statistical and model-based data analyses of dynamic transcript, protein, and metabolite abundances and promoter activities. Adaptation to malate was rapid and primarily controlled posttranscriptionally compared with the slow, mainly transcriptionally controlled adaptation to glucose that entailed nearly half of the known transcription regulation network. Interactions across multiple levels of regulation were involved in adaptive changes that could also be achieved by controlling single genes. Our analysis suggests that global trade-offs and evolutionary constraints provide incentives to favor complex control programs.

Accurate strain identification is essential for anyone working with bacteria. For many species, multilocus sequence typing (MLST) is considered the "gold standard" of typing, but it is traditionally performed in an expensive and time-consuming manner. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available to scientists and routine diagnostic laboratories. Currently, the cost is below that of traditional MLST. The new challenges will be how to extract the relevant information from the large amount of data so as to allow for comparison over time and between laboratories. Ideally, this information should also allow for comparison to historical data. We developed a Web-based method for MLST of 66 bacterial species based on WGS data. As input, the method uses short sequence reads from four sequencing platforms or preassembled genomes. Updates from the MLST databases are downloaded monthly, and the best-matching MLST alleles of the specified MLST scheme are found using a BLAST-based ranking method. The sequence type is then determined by the combination of alleles identified. The method was tested on preassembled genomes from 336 isolates covering 56 MLST schemes, on short sequence reads from 387 isolates covering 10 schemes, and on a small test set of short sequence reads from 29 isolates for which the sequence type had been determined by traditional methods. The method presented here enables investigators to determine the sequence types of their isolates on the basis of WGS data. This method is publicly available at www.cbs.dtu.dk/services/MLST.

BACKGROUND: The advances and decreasing economical cost of whole genome sequencing (WGS), will soon make this technology available for routine infectious disease epidemiology. In epidemiological studies, outbreak isolates have very little diversity and require extensive genomic analysis to differentiate and classify isolates. One of the successfully and broadly used methods is analysis of single nucletide polymorphisms (SNPs). Currently, there are different tools and methods to identify SNPs including various options and cut-off values. Furthermore, all current methods require bioinformatic skills. Thus, we lack a standard and simple automatic tool to determine SNPs and construct phylogenetic tree from WGS data.RESULTS: Here we introduce snpTree, a server for online-automatic SNPs analysis. This tool is composed of different SNPs analysis suites, perl and python scripts. snpTree can identify SNPs and construct phylogenetic trees from WGS as well as from assembled genomes or contigs. WGS data in fastq format are aligned to reference genomes by BWA while contigs in fasta format are processed by Nucmer. SNPs are concatenated based on position on reference genome and a tree is constructed from concatenated SNPs using FastTree and a perl script. The online server was implemented by HTML, Java and python script.The server was evaluated using four published bacterial WGS data sets (V. cholerae, S. aureus CC398, S. Typhimurium and M. tuberculosis). The evaluation results for the first three cases was consistent and concordant for both raw reads and assembled genomes. In the latter case the original publication involved extensive filtering of SNPs, which could not be repeated using snpTree.CONCLUSIONS: The snpTree server is an easy to use option for rapid standardised and automatic SNP analysis in epidemiological studies also for users with limited bioinformatic experience. The web server is freely accessible at http://www.cbs.dtu.dk/services/snpTree-1.0/.

Accurate strain identification is essential for anyone working with bacteria. For many species, multilocus sequence typing (MLST) is considered the "gold standard" of typing, but it is traditionally performed in an expensive and time-consuming manner. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available to scientists and routine diagnostic laboratories. Currently, the cost is below that of traditional MLST. The new challenges will be how to extract the relevant information from the large amount of data so as to allow for comparison over time and between laboratories. Ideally, this information should also allow for comparison to historical data. We developed a Web-based method for MLST of 66 bacterial species based on WGS data. As input, the method uses short sequence reads from four sequencing platforms or preassembled genomes. Updates from the MLST databases are downloaded monthly, and the best-matching MLST alleles of the specified MLST scheme are found using a BLAST-based ranking method. The sequence type is then determined by the combination of alleles identified. The method was tested on preassembled genomes from 336 isolates covering 56 MLST schemes, on short sequence reads from 387 isolates covering 10 schemes, and on a small test set of short sequence reads from 29 isolates for which the sequence type had been determined by traditional methods. The method presented here enables investigators to determine the sequence types of their isolates on the basis of WGS data. This method is publicly available at www.cbs.dtu.dk/services/MLST.

BACKGROUND: The advances and decreasing economical cost of whole genome sequencing (WGS), will soon make this technology available for routine infectious disease epidemiology. In epidemiological studies, outbreak isolates have very little diversity and require extensive genomic analysis to differentiate and classify isolates. One of the successfully and broadly used methods is analysis of single nucletide polymorphisms (SNPs). Currently, there are different tools and methods to identify SNPs including various options and cut-off values. Furthermore, all current methods require bioinformatic skills. Thus, we lack a standard and simple automatic tool to determine SNPs and construct phylogenetic tree from WGS data.RESULTS: Here we introduce snpTree, a server for online-automatic SNPs analysis. This tool is composed of different SNPs analysis suites, perl and python scripts. snpTree can identify SNPs and construct phylogenetic trees from WGS as well as from assembled genomes or contigs. WGS data in fastq format are aligned to reference genomes by BWA while contigs in fasta format are processed by Nucmer. SNPs are concatenated based on position on reference genome and a tree is constructed from concatenated SNPs using FastTree and a perl script. The online server was implemented by HTML, Java and python script.The server was evaluated using four published bacterial WGS data sets (V. cholerae, S. aureus CC398, S. Typhimurium and M. tuberculosis). The evaluation results for the first three cases was consistent and concordant for both raw reads and assembled genomes. In the latter case the original publication involved extensive filtering of SNPs, which could not be repeated using snpTree.CONCLUSIONS: The snpTree server is an easy to use option for rapid standardised and automatic SNP analysis in epidemiological studies also for users with limited bioinformatic experience. The web server is freely accessible at http://www.cbs.dtu.dk/services/snpTree-1.0/.

Adaptation of cells to environmental changes requires dynamic interactions between metabolic and regulatory networks, but studies typically address only one or a few layers of regulation. For nutritional shifts between two preferred carbon sources of Bacillus subtilis, we combined statistical and model-based data analyses of dynamic transcript, protein, and metabolite abundances and promoter activities. Adaptation to malate was rapid and primarily controlled posttranscriptionally compared with the slow, mainly transcriptionally controlled adaptation to glucose that entailed nearly half of the known transcription regulation network. Interactions across multiple levels of regulation were involved in adaptive changes that could also be achieved by controlling single genes. Our analysis suggests that global trade-offs and evolutionary constraints provide incentives to favor complex control programs.

Due to the complexity of the protocols and a limited knowledge of the nature of microbial communities, simulating metagenomic sequences plays an important role in testing the performance of existing tools and data analysis methods with metagenomic data. We developed metagenomic read simulators with platform-specific (Sanger, pyrosequencing, Illumina) base-error models, and simulated metagenomes of differing community complexities. We first evaluated the effect of rigorous quality control on Illumina data. Although quality filtering removed a large proportion of the data, it greatly improved the accuracy and contig lengths of resulting assemblies. We then compared the quality-trimmed Illumina assemblies to those from Sanger and pyrosequencing. For the simple community (10 genomes) all sequencing technologies assembled a similar amount and accurately represented the expected functional composition. For the more complex community (100 genomes) Illumina produced the best assemblies and more correctly resembled the expected functional composition. For the most complex community (400 genomes) there was very little assembly of reads from any sequencing technology. However, due to the longer read length the Sanger reads still represented the overall functional composition reasonably well. We further examined the effect of scaffolding of contigs using paired-end Illumina reads. It dramatically increased contig lengths of the simple community and yielded minor improvements to the more complex communities. Although the increase in contig length was accompanied by increased chimericity, it resulted in more complete genes and a better characterization of the functional repertoire. The metagenomic simulators developed for this research are freely available.