BADASS: BActeriocin-Diversity ASsessment Software.

BACKGROUND: Bacteriocins are defined as thermolabile peptides produced by bacteria with biological activity against taxonomically related species. These antimicrobial peptides have a wide application including disease treatment, food conservation, and probiotics. However, even with a large industrial and biotechnological application potential, these peptides are still poorly studied and explored. BADASS is software with a user-friendly graphical interface applied to the search and analysis of bacteriocin diversity in whole-metagenome shotgun sequencing data. RESULTS: The search for bacteriocin sequences is performed with tools such as BLAST or DIAMOND using the BAGEL4 database as a reference. The putative bacteriocin sequences identified are used to determine the abundance and richness of the three classes of bacteriocins. Abundance is calculated by comparing the reads identified as bacteriocins to the reads identified as 16S rRNA gene using SILVA database as a reference. BADASS has a complete pipeline that starts with the quality assessment of the raw data. At the end of the analysis, BADASS generates several plots of richness and abundance automatically as well as tabular files containing information about the main bacteriocins detected. The user is able to change the main parameters of the analysis in the graphical interface. To demonstrate how the software works, we used four datasets from WMS studies using default parameters. Lantibiotics were the most abundant bacteriocins in the four datasets. This class of bacteriocin is commonly produced by Streptomyces sp. CONCLUSIONS: With a user-friendly graphical interface and a complete pipeline, BADASS proved to be a powerful tool for prospecting bacteriocin sequences in Whole-Metagenome Shotgun Sequencing (WMS) data. This tool is publicly available at https://sourceforge.net/projects/badass/ .

Diversity of bacteriocins in the microbiome of the Tucuruí Hydroelectric Power Plant water reservoir and three-dimensional structure prediction of a zoocin.

Bacteriocins are antimicrobial peptides expressed by bacteria through ribosomal activity. In this study, we analyzed the diversity of bacteriocin-like genes in the Tucuruí-HPP using a whole-metagenome shotgun sequencing approach. Three layers of the water column were analyzed (photic, aphotic and sediment). Detection of bacteriocin-like genes was performed with blastx using the BAGEL4 database as subject sequences. In order to calculate the abundance of bacteriocin-like genes we also determined the number of 16S rRNA genes using blastn. Taxonomic analysis was performed using RAST server and the metagenome was assembled using IDBA-UD in order to recover the full sequence of a zoocin which had its three-dimensional structure determined. The photic zone presented the highest number of reads affiliated to bacteriocins. The most abundant bacteriocins were sonorensin, Klebicin D , pyocin and colicin. The zoocin model was composed of eight anti-parallel ß-sheets and two α-helices with a Zn2+ ion in the active site. This model was considerably stable during 10 ns of molecular dynamics simulation. We observed a high diversity of bacteriocins in the Tucuruí-HPP, demonstrating that the environment is an inexhaustible source for prospecting these molecules. Finally, the zoocin model can be used for further studies of substrate binding and molecular mechanisms involving peptidoglycan degradation.

Genomic analysis of Chromobacterium haemolyticum: insights into the species resistome, virulence determinants and genome plasticity.

The increasing number of Chromobacterium haemolyticum human infection reports, especially in tropical regions and connected with environmental sources, resulted in an urge to better describe this species. This study aimed to characterize the C. haemolyticum resistome, virulence determinants and genetic platforms related with genome plasticity. A comparative genomic analysis was conducted between clinical C. haemolyticum genomes publicly available and the genome of an environmental isolate obtained in this study. The pangenome of C. haemolyticum was calculated and a total of 3378 core genes were predicted in its core genome, corresponding to 51.7% of the pangenome. Genetic determinants putatively encoding resistance to beta-lactams, fosfomycin, aminoglycosides and trimethoprim were predicted in all genomes, possibly constituting the intrinsic resistome of this species. In terms of resistance to beta-lactams, 4 genes were predicted encoding beta-lactamases of classes A, C and D. Moreover, the analysis of Chromobacterium genomes and C. haemolyticum environmental isolates reinforced the role of this genus as progenitor of the blaKPC gene. Putative virulence factors (VFs) were predicted in all genomes, related to adherence, toxins production, colonization and cell invasion. Secretion systems, including type III, were detected. A significant number of transposases and genomic islands were predicted in C. haemolyticum, in some cases above the average reported for Gram-negative bacterial genomes. We conclude that C. haemolyticum strains, including those of environmental origin, present a noteworthy collection of antibiotic resistance genes and VFs. Furthermore, sequences related to gene mobility and genome plasticity suggest high adaptability potential and a possible role as disseminator of antibiotic resistance.

Exposure to an extremely low-frequency electromagnetic field only slightly modifies the proteome of Chromobacterium violaceumATCC 12472.

Several studies of the physiological responses of different organisms exposed to extremely low-frequency electromagnetic fields (ELF-EMF) have been described. In this work, we report the minimal effects of in situ exposure to ELF-EMF on the global protein expression of Chromobacterium violaceum using a gel-based proteomic approach. The protein expression profile was only slightly altered, with five differentially expressed proteins detected in the exposed cultures; two of these proteins (DNA-binding stress protein, Dps, and alcohol dehydrogenase) were identified by MS/MS. The enhanced expression of Dps possibly helped to prevent physical damage to DNA. Although small, the changes in protein expression observed here were probably beneficial in helping the bacteria to adapt to the stress generated by the electromagnetic field.

Omics profiles used to evaluate the gene expression of Exiguobacterium antarcticum B7 during cold adaptation.

BACKGROUND: Exiguobacterium antarcticum strain B7 is a Gram-positive psychrotrophic bacterial species isolated in Antarctica. Although this bacteria has been poorly studied, its genome has already been sequenced. Therefore, it is an appropriate model for the study of thermal adaptation. In the present study, we analyzed the transcriptomes and proteomes of E. antarcticum B7 grown at 0°C and 37°C by SOLiD RNA-Seq, Ion Torrent RNA-Seq and two-dimensional difference gel electrophoresis tandem mass spectrometry (2D-DIGE-MS/MS). RESULTS: We found expression of 2,058 transcripts in all replicates from both platforms and differential expression of 564 genes (absolute log2FC≥1, P-value<0.001) comparing the two temperatures by RNA-Seq. A total of 73 spots were differentially expressed between the two temperatures on 2D-DIGE, 25 of which were identified by MS/MS. Some proteins exhibited patterns of dispersion in the gel that are characteristic of post-translational modifications. CONCLUSIONS: Our findings suggest that the two sequencing platforms yielded similar results and that different omic approaches may be used to improve the understanding of gene expression. To adapt to low temperatures, E. antarcticum B7 expresses four of the six cold-shock proteins present in its genome. The cold-shock proteins were the most abundant in the bacterial proteome at 0°C. Some of the differentially expressed genes are required to preserve transcription and translation, while others encode proteins that contribute to the maintenance of the intracellular environment and appropriate protein folding. The results denote the complexity intrinsic to the adaptation of psychrotrophic organisms to cold environments and are based on two omic approaches. They also unveil the lifestyle of a bacterial species isolated in Antarctica.

Microbial diversity of an anoxic zone of a hydroelectric power station reservoir in Brazilian Amazonia.

Microbial diversity was evaluated in an anoxic zone of Tucuruí Hydroelectric Power Station reservoir in Brazilian Amazonia using a culture-independent approach by amplifying and sequencing fragments of the 16S rRNA gene using metagenomic DNA as a template. Samples obtained from the photic, aphotic (40 m) and sediment (60 m) layers were used to construct six 16S rDNA libraries containing a total of 1,152 clones. The sediment, aphotic and photic layers presented 64, 33 and 35 unique archaeal operational taxonomic units (OTUs). The estimated richness of these layers was evaluated to be 153, 106 and 79 archaeal OTUs, respectively, using the abundance-based coverage estimator (ACE) and 114, 83 and 77 OTUs using the Chao1 estimator. For bacterial sequences, 114, 69 and 57 OTUs were found in the sediment, aphotic and photic layers, which presented estimated richnesses of 1,414, 522 and 197 OTUs (ACE) and 1,059, 1,014 and 148 OTUs (Chao1), respectively. Phylogenetic analyses of the sequences obtained revealed a high richness of microorganisms which participate in the carbon cycle, namely, methanogenic archaea and methanotrophic proteobacteria. Most sequences obtained belong to non-culturable prokaryotes. The present study offers the first glimpse of the huge microbial diversity of an anoxic area of a man-made lacustrine environment in the tropics.

Occurrence, antibiotic-resistance and virulence of E. coli strains isolated from mangrove oysters (Crassostrea gasar) farmed in estuaries of Amazonia.

Concentration of bacterial species indicative of fecal contamination in the gut of mangrove oysters (Crassostrea gasar) is a major concern for public health and food surveillance. Our work aimed to determine the occurrence, antibiotic-resistance, phylogenetic profile and virulence of Escherichia coli strains isolated from C. gasar farmed in four estuaries of Amazonia. Santo Antônio de Urindeua was the sampling point with the highest number of E. coli cells in oyster samples (104 per 100 g of sample). Twenty-four isolates (52.2%) showed resistance to cephalotin and 18 to amoxicillin (39.1%). Eighteen clonal populations were determined by rep-PCR and were mainly affiliated to the pathogenic and commensal phylo-groups B1 and D. The presence of elt genes suggests that 10 of these clones belong to the Enterotoxigenic Escherichia coli pathotype. Plasmids, mostly of the F incompatibility group, were detected in the majority of the strains. All isolates were susceptible to last-resort antibiotics.

Extended Spectrum Beta-Lactamase-Producing Gram-Negative Bacteria Recovered From an Amazonian Lake Near the City of Belém, Brazil.

Aquatic systems have been described as antibiotic resistance reservoirs, where water may act as a vehicle for the spread of resistant bacteria and resistance genes. We evaluated the occurrence and diversity of third generation cephalosporin-resistant gram-negative bacteria in a lake in the Amazonia region. This water is used for human activities, including consumption after appropriate treatment. Eighteen samples were obtained from six sites in October 2014. Water quality parameters were generally within the legislation limits. Thirty-three bacterial isolates were identified as Escherichia (n = 7 isolates), Acinetobacter, Enterobacter, and Klebsiella (n = 5 each), Pseudomonas (n = 4), Shigella (n = 3), and Chromobacterium, Citrobacter, Leclercia, Phytobacter (1 isolate each). Twenty nine out of 33 isolates (88%) were resistant to most beta-lactams, except carbapenems, and 88% (n = 29) were resistant to antibiotics included in at least three different classes. Among the beta-lactamase genes inspected, the bla CTX-M was the most prevalent (n = 12 positive isolates), followed by bla TEM (n = 5) and bla SHV (n = 4). bla CTX-M-15 (n = 5), bla CTX-M-14 (n = 1) and bla CTX-M-2 (n = 1) variants were detected in conserved genomic contexts: bla CTX-M-15 flanked by ISEcp1 and Orf477; bla CTX-M-14 flanked by ISEcp1 and IS903; and bla CTX-M-2 associated to an ISCR element. For 4 strains the transfer of bla CTX-M was confirmed by conjugation assays. Compared with the recipient, the transconjugants showed more than 500-fold increases in the MICs of cefotaxime and 16 to 32-fold increases in the MICs of ceftazidime. Two isolates (Escherichia coli APC43A and Acinetobacter baumannii APC25) were selected for whole genome analysis. APC43A was predicted as a E. coli pathogen of the high-risk clone ST471 and serotype O154:H18. bla CTX-M-15 as well as determinants related to efflux of antibiotics, were noted in APC43A genome. A. baumannii APC25 was susceptible to carbapenems and antibiotic resistance genes detected in its genome were intrinsic determinants (e.g., bla OXA-208 and bla ADC-like). The strain was not predicted as a human pathogen and belongs to a new sequence type. Operons related to metal resistance were predicted in both genomes as well as pathogenicity and resistance islands. Results suggest a high dissemination of ESBL-producing bacteria in Lake Água Preta which, although not presenting characteristics of a strongly impacted environment, contains multi-drug resistant pathogenic strains.

Genomic Architecture of the Two Cold-Adapted Genera Exiguobacterium and Psychrobacter: Evidence of Functional Reduction in the Exiguobacterium antarcticum B7 Genome.

Exiguobacterium and Psychrobacter are bacterial genera with several cold-adapted species. These extremophiles are commonly isolated from the same habitats in Earth's cryosphere and have great ecological and biotechnological relevance. Thus, through comparative genomic analyses, it was possible to understand the functional diversity of these psychrotrophic and psychrophilic species and present new insights into the microbial adaptation to cold. The nucleotide identity between Exiguobacterium genomes was >90%. Three genomic islands were identified in the E. antarcticum B7 genome. These islands contained genes involved in flagella biosynthesis and chemotaxis, as well as enzymes for carotenoid biosynthesis. Clustering of cold shock proteins by Ka/Ks ratio suggests the occurrence of a positive selection over these genes. Neighbor-joining clustering of complete genomes showed that the E. sibiricum was the most closely related to E. antarcticum. A total of 92 genes were shared between Exiguobacterium and Psychrobacter. A reduction in the genomic content of E. antarcticum B7 was observed. It presented the smallest genome size of its genus and a lower number of genes because of the loss of many gene families compared with the other genomes. In our study, eight genomes of Exiguobacterium and Psychrobacter were compared and analysed. Psychrobacter showed higher genomic plasticity and E. antarcticum B7 presented a large decrease in genomic content without changing its ability to grow in cold environments.

A Proteomic Perspective on the Bacterial Adaptation to Cold: Integrating OMICs Data of the Psychrotrophic Bacterium Exiguobacterium antarcticum B7.

Since the publication of one of the first studies using 2D gel electrophoresis by Patrick H. O'Farrell in 1975, several other studies have used that method to evaluate cellular responses to different physicochemical variations. In environmental microbiology, bacterial adaptation to cold environments is a "hot topic" because of its application in biotechnological processes. As in other fields, gel-based and gel-free proteomic methods have been used to determine the molecular mechanisms of adaptation to cold of several psychrotrophic and psychrophilic bacterial species. In this review, we aim to describe and discuss these main molecular mechanisms of cold adaptation, referencing proteomic studies that have made significant contributions to our current knowledge in the area. Furthermore, we use Exiguobacterium antarcticum B7 as a model organism to present the importance of integrating genomic, transcriptomic, and proteomic data. This species has been isolated in Antarctica and previously studied at all three omic levels. The integration of these data permitted more robust conclusions about the mechanisms of bacterial adaptation to cold.

Genomic analysis of four strains of Corynebacterium pseudotuberculosis bv. Equi isolated from horses showing distinct signs of infection.

The genomes of four strains (MB11, MB14, MB30, and MB66) of the species Corynebacterium pseudotuberculosis biovar equi were sequenced on the Ion Torrent PGM platform, completely assembled, and their gene content and structure were analyzed. The strains were isolated from horses with distinct signs of infection, including ulcerative lymphangitis, external abscesses on the chest, or internal abscesses on the liver, kidneys, and lungs. The average size of the genomes was 2.3 Mbp, with 2169 (Strain MB11) to 2235 (Strain MB14) predicted coding sequences (CDSs). An optical map of the MB11 strain generated using the KpnI restriction enzyme showed that the approach used to assemble the genome was satisfactory, producing good alignment between the sequence observed in vitro and that obtained in silico. In the resulting Neighbor-Joining dendrogram, the C. pseudotuberculosis strains sequenced in this study were clustered into a single clade supported by a high bootstrap value. The structural analysis showed that the genomes of the MB11 and MB14 strains were very similar, while the MB30 and MB66 strains had several inverted regions. The observed genomic characteristics were similar to those described for other strains of the same species, despite the number of inversions found. These genomes will serve as a basis for determining the relationship between the genotype of the pathogen and the type of infection that it causes.

Assessing the Genotypic Differences between Strains of Corynebacterium pseudotuberculosis biovar equi through Comparative Genomics.

Seven genomes of Corynebacterium pseudotuberculosis biovar equi were sequenced on the Ion Torrent PGM platform, generating high-quality scaffolds over 2.35 Mbp. This bacterium is the causative agent of disease known as "pigeon fever" which commonly affects horses worldwide. The pangenome of biovar equi was calculated and two phylogenomic approaches were used to identify clustering patterns within Corynebacterium genus. Furthermore, other comparative analyses were performed including the prediction of genomic islands and prophages, and SNP-based phylogeny. In the phylogenomic tree, C. pseudotuberculosis was divided into two distinct clades, one formed by nitrate non-reducing species (biovar ovis) and another formed by nitrate-reducing species (biovar equi). In the latter group, the strains isolated from California were more related to each other, while the strains CIP 52.97 and 1/06-A formed the outermost clade of the biovar equi. A total of 1,355 core genes were identified, corresponding to 42.5% of the pangenome. This pangenome has one of the smallest core genomes described in the literature, suggesting a high genetic variability of biovar equi of C. pseudotuberculosis. The analysis of the similarity between the resistance islands identified a higher proximity between the strains that caused more severe infectious conditions (infection in the internal organs). Pathogenicity islands were largely conserved between strains. Several genes that modulate the pathogenicity of C. pseudotuberculosis were described including peptidases, recombination enzymes, micoside synthesis enzymes, bacteriocins with antimicrobial activity and several others. Finally, no genotypic differences were observed between the strains that caused the three different types of infection (external abscess formation, infection with abscess formation in the internal organs, and ulcerative lymphangitis). Instead, it was noted that there is a higher phenetic correlation between strains isolated at California compared to the other strains. Additionally, high variability of resistance islands suggests gene acquisition through several events of horizontal gene transfer.

Reconstruction of the Fatty Acid Biosynthetic Pathway of Exiguobacterium antarcticum B7 Based on Genomic and Bibliomic Data.

Exiguobacterium antarcticum B7 is extremophile Gram-positive bacteria able to survive in cold environments. A key factor to understanding cold adaptation processes is related to the modification of fatty acids composing the cell membranes of psychrotrophic bacteria. In our study we show the in silico reconstruction of the fatty acid biosynthesis pathway of E. antarcticum B7. To build the stoichiometric model, a semiautomatic procedure was applied, which integrates genome information using KEGG and RAST/SEED. Constraint-based methods, namely, Flux Balance Analysis (FBA) and elementary modes (EM), were applied. FBA was implemented in the sense of hexadecenoic acid production maximization. To evaluate the influence of the gene expression in the fluxome analysis, FBA was also calculated using the log2⁡FC values obtained in the transcriptome analysis at 0°C and 37°C. The fatty acid biosynthesis pathway showed a total of 13 elementary flux modes, four of which showed routes for the production of hexadecenoic acid. The reconstructed pathway demonstrated the capacity of E. antarcticum B7 to de novo produce fatty acid molecules. Under the influence of the transcriptome, the fluxome was altered, promoting the production of short-chain fatty acids. The calculated models contribute to better understanding of the bacterial adaptation at cold environments.

De novo synthesis of fatty acids is regulated by FapR protein in Exiguobacterium antarcticum B7, a psychrotrophic bacterium isolated from Antarctica.

BACKGROUND: FapR protein from the psychrotrophic species Exiguobacterium antarcticum B7 was expressed and purified, and subsequently evaluated for its capacity to bind to the promoter regions of the fabH1-fabF and fapR-plsX-fabD-fabG operons, using electrophoretic mobility shift assay. The genes that compose these operons encode for enzymes involved in the de novo synthesis of fatty acids molecules. In Bacillus subtilis, FapR regulates the expression of these operons, and consequently has influence in the synthesis of long or short-chain fatty acids. To analyze the bacterial cold adaptation, this is an important metabolic pathway because psychrotrophic microrganisms tend to synthesize short and branched-chain unsaturated fatty acids at cold to maintain cell membrane fluidity. RESULTS: In this work, it was observed that recombinant protein was able to bind to the promoter of the fully amplified fabH1-fabF and fapR-plsX-fabD-fabG operons. However, FapR was unable to bind to the promoter of fapR-plsX-fabD-fabG operon when synthesized only up to the protein-binding palindrome 5'-TTAGTACCAGATACTAA-3', thus showing the importance of the entire promoter sequence for the correct protein-DNA interaction. CONCLUSIONS: Through this observation, we demonstrate that the FapR protein possibly regulates the same operons as described for other species, which emphasizes its importance to cold adaptation process of E. antarcticum B7, a psychrotrophic bacterium isolated at Antarctica.

Whole-Genome Sequence of Corynebacterium pseudotuberculosis Strain 226, Isolated from the Abscess of a Goat in California.

Corynebacterium pseudotuberculosis is the etiological agent of a caseous lymphadenitis disease. Herein, we present the first complete genome sequencing of C. pseudotuberculosis strain 226, isolated from an abscess of the sub-iliac lymph node of a goat from California (USA). The genome contains 2,138 coding sequences (CDSs), 12 rRNAs, 49 tRNAs, and 72 pseudogenes.

Genome Sequence of Corynebacterium pseudotuberculosis MB20 bv. equi Isolated from a Pectoral Abscess of an Oldenburg Horse in California.

The genome of Corynebacterium pseudotuberculosis MB20 bv. equi was sequenced using the Ion Personal Genome Machine (PGM) platform, and showed a size of 2,363,089 bp, with 2,365 coding sequences and a GC content of 52.1%. These results will serve as a basis for further studies on the pathogenicity of C. pseudotuberculosis bv. equi.

Proteomics Analysis of the Effects of Cyanate on Chromobacterium violaceum Metabolism.

Chromobacterium violaceum is a gram-negative betaproteobacterium that has been isolated from various Brazilian ecosystems. Its genome contains the cyn operon, which gives it the ability to metabolize highly toxic cyanate into ammonium and carbon dioxide. We used a proteomics approach to investigate the effects of cyanate on the metabolism of this bacterium. The proteome of cells grown with and without cyanate was compared on 2-D gels. Differential spots were digested and identified by mass spectrometry. The bacterium was able to grow at concentrations of up to 1 mM cyanate. Eighteen spots were differentially expressed in the presence of cyanate, of which 16 were downregulated and only two were upregulated. An additional 12 spots were detected only in extracts of cells unexposed to cyanate, and one was expressed only by the exposed cells. Fourteen spots were identified, corresponding to 13 different proteins. We conclude that cyanate promotes expression of enzymes that combat oxidative stress and represses enzymes of the citric acid cycle, strongly affecting the energetic metabolism of the cell. Other proteins that were under-expressed in bacteria exposed to cyanate are involved in amino-acid metabolism or are hypothetical proteins, demonstrating that cyanate also affects expression of genes that are not part of the cyn operon.