A probable aculeacin A acylase from the Ralstonia solanacearum GMI1000 is N-acyl-homoserine lactone acylase with quorum-quenching activity

Background

Extraintestinal pathogenic Escherichia coli (ExPEC) strains of serotype O1:K1:H7/NM are frequently implicated in neonatal meningitis, urinary tract infections and septicemia in humans. They are also commonly isolated from colibacillosis in poultry. Studies to determine the similarities of ExPEC from different origins have indicated that avian strains potentially have zoonotic properties.

Results

A total of 59 ExPEC O1:K1:H7/NM isolates (21 from avian colibacillosis, 15 from human meningitis, and 23 from human urinary tract infection and septicemia) originated from four countries were characterized by phylogenetic PCR grouping, Multilocus Sequence Typing (MLST), Pulsed Field Gel Electrophoresis (PFGE) and genotyping based on several genes known for their association with ExPEC or avian pathogenic Escherichia coli (APEC) virulence. APEC and human ExPEC isolates differed significantly in their assignments to phylogenetic groups, being phylogroup B2 more prevalent among APEC than among human ExPEC (95% vs. 53%, P = 0.001), whereas phylogroup D was almost exclusively associated with human ExPEC (47% vs. 5%, P = 0.0000). Seven virulence genes showed significant differences, being fimAv _MT78 and sat genes linked to human isolates, while papGII, tsh, iron, cvaC and iss were significantly associated to APEC. By MLST, 39 of 40 ExPEC belonging to phylogroup B2, and 17 of 19 belonging to phylogroup D exhibited the Sequence Types (STs) ST95 and ST59, respectively. Additionally, two novel STs (ST1013 and ST1006) were established. Considering strains sharing the same ST, phylogenetic group, virulence genotype and PFGE cluster to belong to the same subclone, five subclones were detected; one of those grouped six strains of human and animal origin from two countries.

Conclusion

Present results reveal that the clonal group B2 O1:K1:H7/NM ST95, detected in strains of animal and human origin, recovered from different dates and geographic sources, provides evidence that some APEC isolates may act as potential pathogens for humans and, consequently, poultry as a foodborne source, suggesting no host specificity for this type of isolates. A novel and important finding has been the detection of the clonal group D O1:K1:H7/NM ST59 almost exclusively in humans, carrying pathogenic genes linked to the phylogenetic group D. This finding would suggest D O1:K1:H7/NM ST59 as a host specific pathotype for humans.     

Listeria monocytogenes Surface Proteins: from Genome Predictions to Function

Summary: The genome of the human food-borne pathogen Listeria monocytogenes is predicted to encode a high number of surface proteins. This abundance likely reflects the ability of this bacterium to survive in diverse environments, including soil, food, and the human host. This review focuses on the various mechanisms by which listerial proteins are attached at the bacterial surface and their many functions, including peptidoglycan metabolism, protein processing, adhesion to host cells, and invasion of host tissues. Extensive in silico analysis of the domains or motifs present in these mosaic proteins reveals that diverse structural features allow the surface proteome to interact with diverse bacterial or host components. This diversity offers new clues about the molecular bases of Listeria pathogenesis.     

Isolation of probiotic lactobacilli strains harboring l-asparaginase and arginine deiminase genes from human infant feces for their potential application in cancer prevention

The aim of this work was to isolate novel lactobacilli probiotic strains from human feces and screen them for the presence of two valuable antitumor genes—the arginine deiminase-encoding gene arcA and l-asparaginase-encoding gene ansA—for future potential therapeutic application in cancer prevention. Feces samples were collected from Egyptian infants. Forty-two isolates were determined as Lactobacillus sp. and selected for further characterization. Only 20 isolates exhibited good tolerance to pH 1.5, 0.3 % bile salts and moderate tolerance to pancreatic enzymes in addition to antagonistic action. These isolates were screened by PCR for the presence of the arcA and ansA genes. Three strains were selected and identified to subspecies levels by amplification and sequencing of 16S rRNA genes as Lactobacillus gasseri NM112 containing the ansA gene; Lactobacillus fermentum NM212; and Lactobacillus casei NM312 containing the arcA gene, and confirmed by determining enzyme activity. We conclude that these three strains can be suggested as probiotics with potential therapeutic effect against cancer.     

Global Profiling of Carbohydrate Active Enzymes in Human Gut Microbiome

Motivation

Carbohydrate Active enzyme (CAZyme) families, encoded by human gut microflora, play a crucial role in breakdown of complex dietary carbohydrates into components that can be absorbed by our intestinal epithelium. Since nutritional wellbeing of an individual is dependent on the nutrient harvesting capability of the gut microbiome, it is important to understand how CAZyme repertoire in the gut is influenced by factors like age, geography and food habits.

Results

This study reports a comprehensive in-silico analysis of CAZyme profiles in the gut microbiomes of 448 individuals belonging to different geographies, using similarity searches of the corresponding gut metagenomic contigs against the carbohydrate active enzymes database. The study identifies a core group of 89 CAZyme families that are present across 85% of the gut microbiomes. The study detects several geography/age-specific trends in gut CAZyme repertoires of the individuals. Notably, a group of CAZymes having a positive correlation with BMI has been identified. Further this group of BMI-associated CAZymes is observed to be specifically abundant in the Firmicutes phyla. One of the major findings from this study is identification of three distinct groups of individuals, referred to as ''CAZotypes'', having similar CAZyme profiles. Distinct taxonomic drivers for these CAZotypes as well as the probable dietary basis for such trends have also been elucidated. The results of this study provide a global view of CAZyme profiles across individuals of various geographies and age-groups. These results re-iterate the need of a more precise understanding of the role of carbohydrate active enzymes in human nutrition.     

Functional Environmental Screening of a Metagenomic Library Identifies stlA; A Unique Salt Tolerance Locus from the Human Gut Microbiome

Functional environmental screening of metagenomic libraries is a powerful means to identify and assign function to novel genes and their encoded proteins without any prior sequence knowledge. In the current study we describe the identification and subsequent analysis of a salt-tolerant clone from a human gut metagenomic library. Following transposon mutagenesis we identified an unknown gene (stlA, for “salt tolerance locus A”) with no current known homologues in the databases. Subsequent cloning and expression in Escherichia coli MKH13 revealed that stlA confers a salt tolerance phenotype in its surrogate host. Furthermore, a detailed in silico analysis was also conducted to gain additional information on the properties of the encoded StlA protein. The stlA gene is rare when searched against human metagenome datasets such as MetaHit and the Human Microbiome Project and represents a novel and unique salt tolerance determinant which appears to be found exclusively in the human gut environment.     

Bartonella choladocola sp. nov. and Bartonella apihabitans sp. nov., two novel species isolated from honey bee gut

Bartonella is one of the noncore bacterial genera in the honey bee (Apis mellifera) gut. So far, only one species, Bartonella apis, has been described from the honey bee gut. Previous analyses based on the genomic information of isolates and metagenome-assembled genomes suggested the existence of multiple Bartonella species in the bee guts. Here, 10 strains were isolated and characterized from the gut of A. mellifera from Jilin Province, China. New isolates shared ＞95% 16S rRNA gene sequence similarity with other species of the genus Bartonella. Phylogenetic analysis revealed that new isolates clustered with other type strains of Bartonella, and the bee gut Bartonella could be classified into three clades. The in silico DDH and average nucleotide identity values between strains of different clusters from the honey bee gut are 29.1–32.5% and 87.6–89.3%, all below the recommended 70.0% and 95% cutoff points. Cells are Gram-staining-negative rods and can grow on the surface of Brain Heart Infusion agar plates supplemented with defibrinated sheep blood in an aerobic environment with 5% CO₂ at 35–37 °C. Strains from different species varied in both phenotypic and chemotaxonomic characterizations. Comparative genomic analysis indicated that B. choladocola had unique sets of genes encoding invasin, representing the potential for this species to both live as a gut symbiont and also as an erythrocytic pathogen. Thus, we propose two novel species Bartonella choladocola sp. nov. whose type strain is W8125^T(=JCM 35030^T = ACCC 62057^T), and Bartonella apihabitans sp. nov. whose type strain is W8097^T(=JCM 35029^T = ACCC 62056^T).     

Comparative Analysis of Carbohydrate Active Enzymes in Clostridium termitidis CT1112 Reveals Complex Carbohydrate Degradation Ability

Clostridium termitidis strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, Nasutitermes lujae. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes), sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in C. termitidis, the recently sequenced strain CT1112 of C. termitidis was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in C. termitidis, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199) and carbohydrate binding modules (95) were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.     

Prevalence and Abundance of Uncultivated Megasphaera-Like Bacteria in the Human Vaginal Environment

Cultivation-independent analysis of 16S rRNA gene sequences in vaginal samples revealed two previously unrecognized, uncultivated Megasphaera-like phylotypes. Phylogenetic analysis and environmental distribution suggest that these Megasphaera types may be unique to the vaginal environment. Quantitative PCR suggests that both phylotypes are present in higher concentrations in women with bacterial vaginosis.     

Discovery of a Conjugative Megaplasmid in Bifidobacterium breve

Bifidobacterium breve is a common and sometimes very abundant inhabitant of the human gut. Genome sequencing of B. breve JCM 7017 revealed the presence of an extrachromosomal element, designated pMP7017 consisting of >190 kb, thus representing the first reported bifidobacterial megaplasmid. In silico characterization of this element revealed several genomic features supporting a stable establishment of the megaplasmid in its host, illustrated by predicted CRISPR-Cas functions that are known to protect the host against intrusion of foreign DNA. Interestingly, pMP7017 is also predicted to encode a conjugative DNA transfer apparatus and, consistent with this notion, we demonstrate here the conjugal transfer of pMP7017 to representative strains of B. breve and B. longum subsp. longum. We also demonstrate the presence of a megaplasmid with homology to pMP7017 in three B. longum subsp. longum strains.     

A CAZyme-Rich Genome of a Taxonomically Novel Rhodophyte-Associated Carrageenolytic Marine Bacterium

Carbohydrate-active enzymes (CAZymes) have significant biotechnological potential as agents for degradation or modification of polysaccharides/glycans. As marine macroalgae are known to be rich in various types of polysaccharides, seaweed-associated bacteria are likely to be a good source of these CAZymes. A genomics approach can be used to explore CAZyme abundance and diversity, but it can also provide deep insights into the biology of CAZyme producers and, in particular, into molecular mechanisms that mediate their interaction with their hosts. In this study, a Gram-negative, aerobic, rod-shaped, carrageenolytic, and culturable marine bacterium designated as AOL6 was isolated from a diseased thallus of a carrageenan-producing farmed rhodophyte, Kappaphycus alvarezii (Gigartinales, Rhodophyta). The whole genome of this bacterium was sequenced and characterized. Sequence reads were assembled producing a high-quality genome assembly. The estimated genome size of the bacterium is 4.4 Mb and a G+C content of 52%. Molecular phylogenetic analysis based on a complete sequence of 16S rRNA, rpoB, and a set of 38 single-copy genes suggests that the bacterium is an unknown species and represents a novel genus in the family Cellvibrionaceae that is most closely related to the genera Teredinibacter and Saccharophagus. Genome comparison with T. turnerae T7901 and S. degradans 2-40 reveals several features shared by the three species, including a large number of CAZymes that comprised >?5% of the total number of protein-coding genes. The high proportion of CAZymes found in the AOL6 genome exceeds that of other known carbohydrate degraders, suggesting a significant capacity to degrade a range of polysaccharides including κ-carrageenan; 34% of these CAZymes have signal peptide sequences for secretion. Three putative κ-carrageenase-encoding genes were identified from the genome of the bacterium via in silico analysis, consistent with the results of the zymography assay (with κ-carrageenan as substrate). Genome analysis also indicated that AOL6 relies exclusively on type 2 secretion system (T2SS) for secreting proteins (possibly including glycoside hydrolases). In relation to T2SS, the product of the pilZ gene was predicted to be highly expressed, suggesting specialization for cell adhesion and secretion of virulence factors. The assignment of proteins to clusters of orthologous groups (COGs) revealed a pattern characteristic of r-strategists. Majority of two-component system proteins identified in the AOL6 genome were also predicted to be involved in chemotaxis and surface colonization. These genomic features suggest that AOL6 is an opportunistic pathogen, adapted to colonizing polysaccharide-rich hosts, including carrageenophytes.     

Identification and Characterization of Potential Therapeutic Candidates in Emerging Human Pathogen Mycobacterium abscessus: A Novel Hierarchical In Silico Approach

Mycobacterium abscessus, a non-tuberculous rapidly growing mycobacterium, is recognized as an emerging human pathogen causing a variety of infections ranging from skin and soft tissue infections to severe pulmonary infections. Lack of an optimal treatment regimen and emergence of multi-drug resistance in clinical isolates necessitate the development of better/new drugs against this pathogen. The present study aims at identification and qualitative characterization of promising drug targets in M. abscessus using a novel hierarchical in silico approach, encompassing three phases of analyses. In phase I, five sets of proteins were mined through chokepoint, plasmid, pathway, virulence factors, and resistance genes and protein network analysis. These were filtered in phase II, in order to find out promising drug target candidates through subtractive channel of analysis. The analysis resulted in 40 therapeutic candidates which are likely to be essential for the survival of the pathogen and non-homologous to host, human anti-targets, and gut flora. Many of the identified targets were found to be involved in different metabolisms (viz., amino acid, energy, carbohydrate, fatty acid, and nucleotide), xenobiotics degradation, and bacterial pathogenicity. Finally, in phase III, the candidate targets were qualitatively characterized through cellular localization, broad spectrum, interactome, functionality, and druggability analysis. The study explained their subcellular location identifying drug/vaccine targets, possibility of being broad spectrum target candidate, functional association with metabolically interacting proteins, cellular function (if hypothetical), and finally, druggable property. Outcome of the present study could facilitate the identification of novel antibacterial agents for better treatment of M. abscesses infections.     

Cellulose-degrading bacteria associated with the invasive woodwasp Sirex noctilio

Sirex noctilio is an invasive wood-feeding wasp that threatens the world''s commercial and natural pine forests. Successful tree colonization by this insect is contingent on the decline of host defenses and the ability to utilize the woody substrate as a source of energy. We explored its potential association with bacterial symbionts that may assist in nutrient acquisition via plant biomass deconstruction using growth assays, culture-dependent and -independent analysis of bacterial frequency of association and whole-genome analysis. We identified Streptomyces and γ-Proteobacteria that were each associated with 94% and 88% of wasps, respectively. Streptomyces isolates grew on all three cellulose substrates tested and across a range of pH 5.6 to 9. On the basis of whole-genome sequencing, three Streptomyces isolates have some of the highest proportions of genes predicted to encode for carbohydrate-active enzymes (CAZyme) of sequenced Actinobacteria. γ-Proteobacteria isolates grew on a cellulose derivative and a structurally diverse substrate, ammonia fiber explosion-treated corn stover, but not on microcrystalline cellulose. Analysis of the genome of a Pantoea isolate detected genes putatively encoding for CAZymes, the majority predicted to be active on hemicellulose and more simple sugars. We propose that a consortium of microorganisms, including the described bacteria and the fungal symbiont Amylostereum areolatum, has complementary functions for degrading woody substrates and that such degradation may assist in nutrient acquisition by S. noctilio, thus contributing to its ability to be established in forested habitats worldwide.     

Butyrate-Producing Bacteria,Including Mucin Degraders,from the Swine Intestinal Tract

To identify bacteria with potential for influencing gut health, 980 anaerobes were cultured from the swine intestinal tract and analyzed for butyrate production. Fifteen isolates in the order Clostridiales produced butyrate and had butyryl coenzyme A (CoA):acetate CoA transferase activity. Three of the isolates grew on mucin, suggesting an intimate association with host intestinal mucosa.     

A Longitudinal Study of the Feline Faecal Microbiome Identifies Changes into Early Adulthood Irrespective of Sexual Development

Companion animals provide an excellent model for studies of the gut microbiome because potential confounders such as diet and environment can be more readily controlled for than in humans. Additionally, domestic cats and dogs are typically neutered early in life, enabling an investigation into the potential effect of sex hormones on the microbiome. In a longitudinal study to investigate the potential effects of neutering, neutering age and gender on the gut microbiome during growth, the faeces of kittens (16 male, 14 female) were sampled at 18, 30 and 42 weeks of age. DNA was shotgun sequenced on the Illumina platform and sequence reads were annotated for taxonomy and function by comparison to a database of protein coding genes. In a statistical analysis of diversity, taxonomy and functional potential of the microbiomes, age was identified as the only factor with significant associations. No significant effects were detected for gender, neutering, or age when neutered (19 or 31 weeks). At 18 weeks of age the microbiome was dominated by the genera Lactobacillus and Bifidobacterium (35% and 20% average abundance). Structural and functional diversity was significantly increased by week 30 but there was no further significant increase. At 42 weeks of age the most abundant genera were Bacteroides (16%), Prevotella (14%) and Megasphaera (8%). Significant differences in functional potential included an enrichment for genes in energy metabolism (carbon metabolism and oxidative phosphorylation) and depletion in cell motility (flagella and chemotaxis). We conclude that the feline faecal microbiome is predominantly determined by age when diet and environment are controlled for. We suggest this finding may also be informative for studies of the human microbiome, where control over such factors is usually limited.