The thermophilic Campylobacter jejuni and Campylobacter coli are considered weakly clonal populations where incongruences between genetic markers are assumed to be due to random horizontal transfer of genomic DNA. In order to investigate the population genetics structure we extracted a set of 1180 core gene families (CGF) from 27 sequenced genomes of C. jejuni and C. coli. We adopted a principal component analysis (PCA) on the normalized evolutionary distances in order to reveal any patterns in the evolutionary signals contained within the various CGFs.

Antibody-mediated immunity is highly protective against disease. The majority of current vaccines confer protection through humoral immunity, but there is high variability in responsiveness across populations. Identifying immune mechanisms that mediate low antibody responsiveness may provide potential strategies to boost vaccine efficacy. Here, we report diverse antibody responsiveness to unadjuvanted as well as adjuvanted immunization in substrains of BALB/c mice, resulting in high and low antibody response phenotypes. Furthermore, these antibody phenotypes were not affected by changes in environmental factors such as the gut microbiota composition. Antigen-specific B cells following immunization had a marked difference in capability to class switch, resulting in perturbed IgG isotype antibody production. In vitro, a B-cell intrinsic defect in the regulation of class-switch recombination was identified in mice with low IgG antibody production. Whole genome sequencing identified polymorphisms associated with the magnitude of antibody produced, and we propose candidate genes that may regulate isotype class-switching capability. This study highlights that mice sourced from different vendors can have significantly altered humoral immune response profiles, and provides a resource to interrogate genetic regulators of antibody responsiveness. Together these results further our understanding of immune heterogeneity and suggest additional research on the genetic influences of adjuvanted vaccine strategies is warranted for enhancing vaccine efficacy.

The rumen is known to harbor dense populations of bacteriophages (phages) predicted to be capable of infecting a diverse range of rumen bacteria. While bacterial genome sequencing projects are revealing the presence of phages which can integrate their DNA into the genome of their host to form stable, lysogenic associations, little is known of the genetics of phages which utilize lytic replication. These phages infect and replicate within the host, culminating in host lysis, and the release of progeny phage particles. While lytic phages for rumen bacteria have been previously isolated, their genomes have remained largely uncharacterized. Here we report the first complete genome sequences of lytic phage isolates specifically infecting three genera of rumen bacteria: Bacteroides, Ruminococcus, and Streptococcus. All phages were classified within the viral order Caudovirales and include two phage morphotypes, representative of the Siphoviridae and Podoviridae families. The phage genomes displayed modular organization and conserved viral genes were identified which enabled further classification and determination of closest phage relatives. Co-examination of bacterial host genomes led to the identification of several genes responsible for modulating phage:host interactions, including CRISPR/Cas elements and restriction-modification phage defense systems. These findings provide new genetic information and insights into how lytic phages may interact with bacteria of the rumen microbiome.

The bacterial production of acetate via reductive acetogenesis along the Wood-Ljungdahl metabolic pathway is an important source of this molecule in several environments, ranging from industrial bioreactors to the human gastrointestinal tract. Here, we contributed to the study of reductive acetogens by considering mathematical modelling techniques for the prediction of bacterial growth and acetate production. We found that the incorporation of a hydrogen uptake concentration threshold into the models improves their predictions and we calculated this threshold as 86.2 mM (95% confidence interval 6.1-132.6 mM). Monod kinetics and first-order kinetics models, with the inclusion of two candidate threshold terms or reversible Michaelis-Menten kinetics, were compared to experimental data and the optimal formulation for predicting both growth and metabolism was found. The models were then used to compare the efficacy of two growth media for acetogens. We found that the recently described general acetogen medium was superior to the DSMZ medium in terms of unbiased estimation of acetogen growth and investigated the contribution of yeast extract concentration to acetate production and bacterial growth in culture. The models and their predictions will be useful to those studying both industrially and environmentally relevant reductive acetogenesis and allow for straightforward adaptation to similar cases with different organisms.

Plant polysaccharide breakdown by microbes in the rumen is fundamental to digestion in ruminant livestock. Bacterial species belonging to the rumen genera Butyrivibrio and Pseudobutyrivibrio are important degraders and utilizers of lignocellulosic plant material. These bacteria degrade polysaccharides and ferment the released monosaccharides to yield short-chain fatty acids that are used by the ruminant for growth and the production of meat, milk, and fiber products. Although rumen Butyrivibrio and Pseudobutyrivibrio species are regarded as common rumen inhabitants, their polysaccharide-degrading and carbohydrate-utilizing enzymes are not well understood. In this study, we analyzed the genomes of 40 Butyrivibrio and 6 Pseudobutyrivibrio strains isolated from the plant-adherent fraction of New Zealand dairy cows to explore the polysaccharide-degrading potential of these important rumen bacteria. Comparative genome analyses combined with phylogenetic analysis of their 16S rRNA genes and short-chain fatty acid production patterns provide insight into the genomic diversity and physiology of these bacteria and divide Butyrivibrio into 3 species clusters. Rumen Butyrivibrio bacteria were found to encode a large and diverse spectrum of degradative carbohydrate-active enzymes (CAZymes) and binding proteins. In total, 4,421 glycoside hydrolases (GHs), 1,283 carbohydrate esterases (CEs), 110 polysaccharide lyases (PLs), 3,605 glycosyltransferases (GTs), and 1,706 carbohydrate-binding protein modules (CBM) with predicted activities involved in the depolymerization and transport of the insoluble plant polysaccharides were identified. Butyrivibrio genomes had similar patterns of CAZyme families but varied greatly in the number of genes within each category in the Carbohydrate-Active Enzymes database (CAZy), suggesting some level of functional redundancy. These results suggest that rumen Butyrivibrio species occupy similar niches but apply different degradation strategies to be able to coexist in the rumen.IMPORTANCE Feeding a global population of 8 billion people and climate change are the primary challenges facing agriculture today. Ruminant livestock are important food-producing animals, and maximizing their productivity requires an understanding of their digestive systems and the roles played by rumen microbes in plant polysaccharide degradation. Members of the genera Butyrivibrio and Pseudobutyrivibrio are a phylogenetically diverse group of bacteria and are commonly found in the rumen, where they are a substantial source of polysaccharide-degrading enzymes for the depolymerization of lignocellulosic material. Our findings have highlighted the immense enzymatic machinery of Butyrivibrio and Pseudobutyrivibrio species for the degradation of plant fiber, suggesting that these bacteria occupy similar niches but apply different degradation strategies in order to coexist in the competitive rumen environment.

Hydrogen cross-feeding microbes form a functionally important subset of the human colonic microbiota. The three major hydrogenotrophic functional groups of the colon: sulphate-reducing bacteria (SRB), methanogens and reductive acetogens, have been linked to wide ranging impacts on host physiology, health and wellbeing.

Eight Faecalibacterium sp. strains were isolated from feces of healthy human volunteers. Here, we describe their genome sequences. The genome sizes ranged from 2.78 Mbp to 3.23 Mbp, with an average GC content of 56.6% and encoding 2,795 protein-coding genes on average.

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes that produce a wide range of bioactive peptides, such as siderophores, toxins, and antibacterial and insecticidal agents. NRPSs are dynamic proteins characterized by extensive interdomain communications as a consequence of their assembly-line mode of synthesis. Hence, crystal structures of multidomain fragments of NRPSs have aided in elucidating crucial interdomain interactions that occur during different steps of the NRPS catalytic cycle. One crucial yet unexplored interaction is that between the reductase (R) domain and the peptide carrier protein (PCP) domain. R domains are members of the short-chain dehydrogenase/reductase family and function as termination domains that catalyze the reductive release of the final peptide product from the terminal PCP domain of the NRPS. Here, we report the crystal structure of an archaeal NRPS PCP-R didomain construct. This is the first NRPS R domain structure to be determined together with the upstream PCP domain and is also the first structure of an archaeal NRPS to be reported. The structure reveals that a novel helix-turn-helix motif, found in NRPS R domains but not in other short-chain dehydrogenase/reductase family members, plays a major role in the interface between the PCP and R domains. The information derived from the described PCP-R interface will aid in gaining further mechanistic insights into the peptide termination reaction catalyzed by the R domain and may have implications in engineering NRPSs to synthesize novel peptide products.

Lactococcus lactis subsp. cremoris HP(T) has been widely used in studies of the metabolism of lactococcal dairy starter cultures. A comparison of the draft HP(T) genome with those from other strains of L. lactis subsp. cremoris will aid our understanding of the domestication and evolution of these important industrial cultures.

Lactobacillus fermentum is commonly found in food products, and some strains are known to have beneficial effects on human health. However, our previous research indicated that L. fermentum AGR1487 decreases in vitro intestinal barrier integrity. The hypothesis was that cell surface structures of AGR1487 are responsible for the observed in vitro effect. AGR1487 was compared to another human oral L. fermentum strain, AGR1485, which does not cause the same effect. The examination of phenotypic traits associated with the composition of cell surface structures showed that compared to AGR1485, AGR1487 had a smaller genome, utilized different sugars, and had greater tolerance to acid and bile. The effect of the two strains on intestinal barrier integrity was determined using two independent measures of paracellular permeability of the intestinal epithelial Caco-2 cell line. The transepithelial electrical resistance (TEER) assay specifically measures ion permeability, whereas the mannitol flux assay measures the passage of uncharged molecules. Both live and UV-inactivated AGR1487 decreased TEER across Caco-2 cells implicating the cell surfaces structures in the effect. However, only live AGR1487, and not UV-inactivated AGR1487, increased the rate of passage of mannitol, implying that a secreted component(s) is responsible for this effect. These differences in barrier integrity results are likely due to the TEER and mannitol flux assays measuring different characteristics of the epithelial barrier, and therefore imply that there are multiple mechanisms involved in the effect of AGR1487 on barrier integrity.

Equally important and challenging as genome annotation, is the subsequent classification of predicted genes into their respective pathways. The Kyoto Encyclopedia of Genes and Genomes (KEGG) represents a database consisting of known genes and their respective biochemical functionalities. Although accessible online, analyses of multiple genes are time consuming and are not suitable for analyzing data sets that are proprietary.

Lateral gene transfer (LGT) appears to promote genotypic and phenotypic variation in microbial communities in a range of environments, including the mammalian intestine. However, the extent and mechanisms of LGT in intestinal microbial communities of non-mammalian hosts remains poorly understood.

Ruminant animals contribute significantly to the global value of agriculture and rely on a complex microbial community for efficient digestion. However, little is known of how this microbial-host relationship develops and is maintained. To begin to address this, we have determined the ability of three Bifidobacterium species isolated from the faeces of newborn calves to grow on carbohydrates typical of a newborn ruminant diet. Genome sequences have been determined for these bacteria with analysis of the genomes providing insights into the host association and identification of several genes that may mediate interactions with the ruminant gastrointestinal tract. The present study provides a starting point from which we can define the role of potential beneficial microbes in the nutrition of young ruminants and begin to influence the interactions between the microbiota and the host. The differences observed in genomic content hint at niche partitioning among the bifidobacterial species analysed and the different strategies they employ to successfully adapt to this habitat.

Methane is a potent greenhouse gas, 25 times more efficient at trapping heat than carbon dioxide. Ruminant methane emissions contribute almost 30% to anthropogenic sources of global atmospheric methane levels and a reduction in methane emissions would significantly contribute to slowing global temperature rises. Here we demonstrate the use of a lytic enyzme, PeiR, from a methanogen virus that infects Methanobrevibacter ruminantium M1 as an effective agent inhibiting a range of rumen methanogen strains in pure culture. We determined the substrate specificity of soluble PeiR and demonstrated that the enzyme is capable of hydrolysing the pseudomurein cell walls of methanogens. Subsequently, peiR was fused to the polyhydroxyalkanoate (PHA) synthase gene phaC and displayed on the surface of PHA bionanoparticles (BNPs) expressed in Eschericia coli via one-step biosynthesis. These tailored BNPs were capable of lysing not only the original methanogen host strain, but a wide range of other rumen methanogen strains in vitro. Methane production was reduced by up to 97% for 5 days post-inoculation in the in vitro assay. We propose that tailored BNPs carrying anti-methanogen enzymes represent a new class of methane inhibitors. Tailored BNPs can be rapidly developed and may be able to modulate the methanogen community in vivo with the aim to lower ruminant methane emissions without impacting animal productivity.

Lactobacillus animalis 381-IL-28 is an integral component of a multistrain commercial culture with food biopreservative and pathogen biocontrol functionality. A draft sequence of the L. animalis 381-IL-28 genome is described in this paper.

Methane (CH(4)) is a potent greenhouse gas (GHG), having a global warming potential 21 times that of carbon dioxide (CO(2)). Methane emissions from agriculture represent around 40% of the emissions produced by human-related activities, the single largest source being enteric fermentation, mainly in ruminant livestock. Technologies to reduce these emissions are lacking. Ruminant methane is formed by the action of methanogenic archaea typified by Methanobrevibacter ruminantium, which is present in ruminants fed a wide variety of diets worldwide. To gain more insight into the lifestyle of a rumen methanogen, and to identify genes and proteins that can be targeted to reduce methane production, we have sequenced the 2.93 Mb genome of M. ruminantium M1, the first rumen methanogen genome to be completed.

Streptococcus gallolyticus LL009 produces gallocin D, a narrow spectrum two component bacteriocin with potent activity against vancomycin-resistant enterococci. Gallocin D is distinct from gallocin A, a separate two component bacteriocin produced by S. gallolyticus. Although the gene clusters encoding gallocin A and gallocin D have a high degree of gene synteny, the structural genes are highly variable and appear to have undergone gene shuffling with other streptococcal species. Gallocin D was analysed in laboratory-based experiments. The mature peptides are 3,343 ± 1 Da and 3,019 ± 1 Da and could be readily synthesized and display activity against a vancomycin resistant Enterococcus strain EC300 with a MIC value of 1.56 µM. Importantly, these bacteriocins could contribute to the ability of S. gallolyticus to colonize the colon where they have been associated with colorectal cancer.

The Mycobacteria are a genus of Actinobacteria that include human pathogens such as Mycobacterium tuberculosis (TB). Active TB disease can spread by airborne transmission to healthcare workers and to their community. The HHMI SEA-PHAGES program has contributed to discovering bacteriophages that are able to infect M. smegmatis MC2 155, a close relative of M. tuberculosis. This collection of diverse Mycobacteriophages is an excellent resource for trialling bacteriophage-sourced enzymes in novel applications. Herein we measured the ability Mycobacteriophage endolysins to lyse their host strain when functionally fused to biodegradable polyhydroxyalkanoate (PHA) nanobeads. PHA nanobeads facilitate both the expression and the application of enzymes to surfaces and have been demonstrated to stabilize a wide array of proteins for practical applications whilst eliminating the challenges of traditional protein purification. We selected two Lysin A and six Lysin B homologs to be functionally fused to the polyhydroxyalkanoate synthase C (PhaC). Expression of these constructs resulted in functional lysins displayed on the surface of PHA nanobeads. The lysins thus directionally displayed on nanobeads lysed up to 79% of the M. smegmatis MC2 155 population using 80 mg/mL of nanobeads in pure culture. In order to determine whether the nanobeads would be effective as a protective layer in PPE we adapted a fabric-based test and observed a maximum of 1 log loss of the cell population after 5 h of exposure on a textile (91% cell lysis). Lysin B enzymes performed better than the Lysin A enzymes as a protective barrier on textiles surface assays. These results suggest that bacterial endolysins are efficient in their action when displayed on PHA nanobeads and can cause significant population mortality in as little as 45 min. Our results provide the proof-of-principle that Mycobacteriophage endolysins can be used on functionalized nanobeads where they can protect surfaces such as personal protective equipment (PPE) that routinely come into contact with aerosolised bacteria.

Salmonella enterica serovar Typhimurium is a foodborne pathogen causing occasional outbreaks of enteric infections in humans. Salmonella has one of the largest pools of temperate phages in its genome that possess evolutionary significance for pathogen. In this study, we characterized a novel temperate phage Salmonella phage BIS20 (BIS20) with unique tail fiber genes. It belongs to the subfamily Peduovirinae genus Eganvirus and infects Salmonella Typhimurium strain (SE-BS17; Acc. NO MZ503545) of poultry origin. Phage BIS20 was viable only at biological pH and temperature ranges (pH7 and 37 °C). Despite being temperate BIS20 significantly slowed down the growth of host strain for 24 h as compared to control (P < 0.009). Phage BIS20 features 29,477-base pair (bp) linear DNA genome with 53% GC content and encodes for 37 putative ORFs. These ORFs have mosaic arrangement as indicated by its ORF similarity to various phages and prophages in NCBI. Genome analysis indicates its similarity to Salmonella enterica serovar Senftenberg prophage (SEStP) sequence (Nucleotide similarity 87.7%) and Escherichia virus 186 (~ 82.4% nucleotide similarity). Capsid genes were conserved however those associated with tail fiber formation and assembly were unique to all members of genus Eganvirus. We found strong evidence of recombination hotspot in tail fiber gene. Our study identifies BIS20 as a new species of genus Eganvirus temperate phages as its maximum nucleotide similarity is 82.4% with any phage in NCBI. Our findings may contribute to understanding of origin of new temperate phages.

Here we report the draft genome of Yersinia entomophaga type strain MH96T. The genome shows 93.8% nucleotide sequence identity to that of Yersinia nurmii type strain APN3a-cT, and comprises a single chromosome of approximately 4,275,531 bp. In silico analysis identified that, in addition to the previously documented Y. entomophaga Yen-TC gene cluster, the genome encodes a diverse array of toxins, including two type III secretion systems, and five rhs-associated gene clusters. As well as these multicomponent systems, several orthologs of known insect toxins, such as VIP2 toxin and the binary toxin PirAB, and distant orthologs of some mammalian toxins, including repeats-in-toxin, a cytolethal distending toxin, hemolysin-like genes and an adenylate cyclase were identified. The genome also contains a large number of hypothetical proteins and orthologs of known effector proteins, such as LopT, as well as genes encoding a wide range of proteolytic determinants, including metalloproteases and pathogen fitness determinants, such as genes involved in iron metabolism. The bioinformatic data derived from the current in silico analysis, along with previous information on the pathobiology of Y. entomophaga against its insect hosts, suggests that a number of these virulence systems are required for survival in the hemocoel and incapacitation of the insect host.