Pseudomonas boanensis sp. nov., a bacterium isolated from river water used for household purposes in Boane District, Mozambique.

A Gram-negative rod with a single polar flagellum was isolated from a freshwater reservoir used for household purposes in Boane District, near Maputo, Mozambique, and designated as strain DB1T. Growth was observed at 30-42 °C (optimum, 30-37 °C) and with 0.5-1.5 % NaCl. Whole-genome-, rpoD- and 16S rRNA-based phylogenies revealed this isolate to be distant from other Pseudomonas species with Pseudomonas resinovorans, Pseudomonas furukawaii and Pseudomonas lalkuanensis being the closest relatives. Phenotypic analyses of strain DB1T showed marked differences with respect to type strains P. resinovorans CCUG 2473T, P. lalkuanensis CCUG 73691T, P. furukawaii CCUG 75672T and Pseudomonas otiditis CCUG 55592T. Taken together, our results indicate that strain DB1T is a representative of a novel species within the genus Pseudomonas for which the name Pseudomonas boanensis is proposed. The type strain is DB1T (=CCUG 62977T=CECT 30359T).

Metagenomics in bioflocs and their effects on gut microbiome and immune responses in Pacific white shrimp.

Biofloc systems generate and accumulate microbial aggregates known as bioflocs. The presence of bioflocs has been shown to change gut bacterial diversity and stimulate innate immunity in shrimp. The microbial niche of bioflocs may therefore have the potential to drive shifts in the shrimp gut microbiota associated with stimulation of innate immunity. We performed shotgun metagenomic analysis and 16S rRNA-based amplicon sequencing to characterize complex bacterial members in bioflocs and the shrimp digestive tract, respectively. Moreover, we determined whether biofloc-grown shrimp with discrete gut microbiomes had an elevation in local immune-related gene expression and systemic immune activities. Our findings demonstrated that the bacterial community in bioflocs changed dynamically during Pacific white shrimp cultivation. Metagenomic analysis revealed that Vibrio comprised 90% of the biofloc population, while Pseualteromonas, Photobacterium, Shewanella, Alteromonas, Bacillus, Lactobacillus, Acinetobacter, Clostridium, Marinifilum, and Pseudomonas were also detected. In the digestive tract, biofloc-grown shrimp maintained the presence of commensal bacteria including Vibrio, Photobacterium, Shewanella, Granulosicoccus, and Ruegeria similar to control shrimp. However, Vibrio and Photobacterium were significantly enriched and declined, respectively, in biofloc-grown shrimp. The presence of bioflocs upregulated immune-related genes encoding serine proteinase and prophenoloxidase in digestive organs which are routinely exposed to gut microbiota. Biofloc-grown shrimp also demonstrated a significant increase in systemic immune status. As a result, the survival rate of biofloc-grown shrimp was substantially higher than that of the control shrimp. Our findings suggested that the high relative abundance of vibrios in bioflocs enriched the number of vibrios in the digestive tract of biofloc-grown shrimp. This shift in gut microbiota composition may be partially responsible for local upregulation of immune-related gene expression in digestive organs and systemic promotion of immune status in circulating hemolymph.

Strategy for Structural Elucidation of Polysaccharides: Elucidation of a Maize Mucilage that Harbors Diazotrophic Bacteria.

The recruitment of a bacterial consortium by the host is a strategy not limited to animals but is also used in plants. A maize aerial root mucilage has been found that harbors nitrogen fixing bacteria that are attracted to the carbohydrate rich environment. This synbiotic relationship is facilitated by a polysaccharide, whose complicated structure has been previously unknown. In this report, we present the characterization of the maize polysaccharide by employing new analytical strategies combining chemical depolymerization, oligosaccharide sequencing, and monosaccharide and glycosidic linkage quantitation. The mucilage contains a single heterogeneous polysaccharide composed of a highly fucosylated and xylosylated galactose backbone with arabinan and mannoglucuronan branches. This unique polysaccharide structure may select for the diazotrophic community by containing monosaccharides and linkages that correspond to the glycosyl hydrolases associated with the microbial community. The elucidation of this complicated structure illustrates the power of the analytical methods, which may serve as a general platform for polysaccharide analysis in the future.

The protective role of commensal gut microbes and their metabolites against bacterial pathogens.

Multidrug-resistant microorganisms have become a major public health concern around the world. The gut microbiome is a gold mine for bioactive compounds that protect the human body from pathogens. We used a multi-omics approach that integrated whole-genome sequencing (WGS) of 74 commensal gut microbiome isolates with metabolome analysis to discover their metabolic interaction with Salmonella and other antibiotic-resistant pathogens. We evaluated differences in the functional potential of these selected isolates based on WGS annotation profiles. Furthermore, the top altered metabolites in co-culture supernatants of selected commensal gut microbiome isolates were identified including a series of dipeptides and examined for their ability to prevent the growth of various antibiotic-resistant bacteria. Our results provide compelling evidence that the gut microbiome produces metabolites, including the compound class of dipeptides that can potentially be applied for anti-infection medication, especially against antibiotic-resistant pathogens. Our established pipeline for the discovery and validation of bioactive metabolites from the gut microbiome as novel candidates for multidrug-resistant infections represents a new avenue for the discovery of antimicrobial lead structures.

Metagenomic and single-cell RNA-Seq survey of the Helicobacter pylori-infected stomach in asymptomatic individuals.

Helicobacter pylori colonization of the gastric niche can persist for years in asymptomatic individuals. To deeply characterize the host-microbiota environment in H. pylori-infected (HPI) stomachs, we collected human gastric tissues and performed metagenomic sequencing, single-cell RNA-Seq (scRNA-Seq), flow cytometry, and fluorescent microscopy. HPI asymptomatic individuals had dramatic changes in the composition of gastric microbiome and immune cells compared with noninfected individuals. Metagenomic analysis uncovered pathway alterations related to metabolism and immune response. scRNA-Seq and flow cytometry data revealed that, in contrast to murine stomachs, ILC2s are virtually absent in the human gastric mucosa, whereas ILC3s are the dominant population. Specifically, proportion of NKp44+ ILC3s out of total ILCs were highly increased in the gastric mucosa of asymptomatic HPI individuals, and correlated with the abundance of selected microbial taxa. In addition, CD11c+ myeloid cells and activated CD4+ T cells and B cells were expanded in HPI individuals. B cells of HPI individuals acquired an activated phenotype and progressed into a highly proliferating germinal-center stage and plasmablast maturation, which correlated with the presence of tertiary lymphoid structures within the gastric lamina propria. Our study provides a comprehensive atlas of the gastric mucosa-associated microbiome and immune cell landscape when comparing asymptomatic HPI and uninfected individuals.

Comparative Genomics of Cyclic di-GMP Metabolism and Chemosensory Pathways in Shewanella algae Strains: Novel Bacterial Sensory Domains and Functional Insights into Lifestyle Regulation.

Shewanella spp. play important ecological and biogeochemical roles, due in part to their versatile metabolism and swift integration of stimuli. While Shewanella spp. are primarily considered environmental microbes, Shewanella algae is increasingly recognized as an occasional human pathogen. S. algae shares the broad metabolic and respiratory repertoire of Shewanella spp. and thrives in similar ecological niches. In S. algae, nitrate and dimethyl sulfoxide (DMSO) respiration promote biofilm formation strain specifically, with potential implication of taxis and cyclic diguanosine monophosphate (c-di-GMP) signaling. Signal transduction systems in S. algae have not been investigated. To fill these knowledge gaps, we provide here an inventory of the c-di-GMP turnover proteome and chemosensory networks of the type strain S. algae CECT 5071 and compare them with those of 41 whole-genome-sequenced clinical and environmental S. algae isolates. Besides comparative analysis of genetic content and identification of laterally transferred genes, the occurrence and topology of c-di-GMP turnover proteins and chemoreceptors were analyzed. We found S. algae strains to encode 61 to 67 c-di-GMP turnover proteins and 28 to 31 chemoreceptors, placing S. algae near the top in terms of these signaling capacities per Mbp of genome. Most c-di-GMP turnover proteins were predicted to be catalytically active; we describe in them six novel N-terminal sensory domains that appear to control their catalytic activity. Overall, our work defines the c-di-GMP and chemosensory signal transduction pathways in S. algae, contributing to a better understanding of its ecophysiology and establishing S. algae as an auspicious model for the analysis of metabolic and signaling pathways within the genus Shewanella. IMPORTANCE Shewanella spp. are widespread aquatic bacteria that include the well-studied freshwater model strain Shewanella oneidensis MR-1. In contrast, the physiology of the marine and occasionally pathogenic species Shewanella algae is poorly understood. Chemosensory and c-di-GMP signal transduction systems integrate environmental stimuli to modulate gene expression, including the switch from a planktonic to sessile lifestyle and pathogenicity. Here, we systematically dissect the c-di-GMP proteome and chemosensory pathways of the type strain S. algae CECT 5071 and 41 additional S. algae isolates. We provide insights into the activity and function of these proteins, including a description of six novel sensory domains. Our work will enable future analyses of the complex, intertwined c-di-GMP metabolism and chemotaxis networks of S. algae and their ecophysiological role.

The microbiome of a polluted urban lake harbors pathogens with diverse antimicrobial resistance and virulence genes.

Bacterial resistance to antibiotics is one of the greatest threats to the modern human population. Paradoxically, urban settlements are often culpable in generating such resistance by influencing the adaptation of bacterial communities via pollution of natural ecosystems. Urban lakes are well-known examples of this problem, as they often receive discharges of both domestic and industrial wastewater. In this study, we used shotgun metagenome sequencing to examine the microbial diversity of water and sediment samples of Lake Alalay, a polluted urban lake near Cochabamba, Bolivia. We found that Proteobacteria dominated the relative abundance of both water and sediment samples at levels over 25% and that a significant proportion of the microbial diversity could not be classified (about 9% in water and 22% in sediment). Further metagenomic investigation of antimicrobial resistance (AR) genes identified 277 and 150 AR genes in water and sediment samples, respectively. These included genes with functional annotations for resistance to fluoroquinolones, tetracyclines, phenicols, macrolides, beta-lactams, and rifamycin. A high number of genes involved in bacterial virulence also occurred in both water and sediment samples (169 and 283, respectively), where the virulence gene pscP normally found in the Pseudomonas aeruginosa type III secretion system had the highest relative abundance. Isolated and identified bacteria from water samples also revealed the presence of pathogenic bacteria among the microbiota of Lake Alalay. Seeing as most AR and virulence genes detected in this study are commonly described in nosocomial infections, we provide evidence suggesting that the microbial ecosystem of Lake Alalay presents a severe health risk to the surrounding population.

Identification of Nitrogen Fixation Genes in Lactococcus Isolated from Maize Using Population Genomics and Machine Learning.

Sierra Mixe maize is a landrace variety from Oaxaca, Mexico, that utilizes nitrogen derived from the atmosphere via an undefined nitrogen fixation mechanism. The diazotrophic microbiota associated with the plant's mucilaginous aerial root exudate composed of complex carbohydrates was previously identified and characterized by our group where we found 23 lactococci capable of biological nitrogen fixation (BNF) without containing any of the proposed essential genes for this trait (nifHDKENB). To determine the genes in Lactococcus associated with this phenotype, we selected 70 lactococci from the dairy industry that are not known to be diazotrophic to conduct a comparative population genomic analysis. This showed that the diazotrophic lactococcal genomes were distinctly different from the dairy isolates. Examining the pangenome followed by genome-wide association study and machine learning identified genes with the functions needed for BNF in the maize isolates that were absent from the dairy isolates. Many of the putative genes received an 'unknown' annotation, which led to the domain analysis of the 135 homologs. This revealed genes with molecular functions needed for BNF, including mucilage carbohydrate catabolism, glycan-mediated host adhesion, iron/siderophore utilization, and oxidation/reduction control. This is the first report of this pathway in this organism to underpin BNF. Consequently, we proposed a model needed for BNF in lactococci that plausibly accounts for BNF in the absence of the nif operon in this organism.

Diazotrophic bacteria from maize exhibit multifaceted plant growth promotion traits in multiple hosts.

Sierra Mixe maize is a geographically remote landrace variety grown on nitrogen-deficient fields in Oaxaca, Mexico that meets its nutritional requirements without synthetic fertilizer by associating with free-living diazotrophs comprising the microbiota of its aerial root mucilage. We selected nearly 500 diazotrophic (N2-fixing) bacteria isolated from Sierra Mixe maize mucilage and sequenced their genomes. Comparative genomic analysis demonstrated that isolates represented diverse genera and composed three major diazotrophic groups based on nitrogen fixation gene content. In addition to nitrogen fixation, we examined deamination of 1-amino-1-cyclopropanecarboxylic acid, biosynthesis of indole-3-acetic acid, and phosphate solubilization as alternative mechanisms of direct plant growth promotion (PGP). Genome mining showed that isolates of all diazotrophic groups possessed marker genes for multiple mechanisms of direct plant growth promotion (PGP). Implementing in vitro assays corroborated isolate genotypes by measuring each isolate's potential to confer the targeted PGP traits and revealed phenotypic variation among isolates based on diazotrophic group assignment. Investigating the ability of mucilage diazotrophs to confer PGP by direct inoculation of clonally propagated potato plants in planta led to the identification of 16 bio-stimulant candidates. Conducting nitrogen-stress greenhouse experiments demonstrated that potato inoculation with a synthetic community of bio-stimulant candidates, as well as with its individual components, resulted in PGP phenotypes. We further demonstrated that one diazotrophic isolate conferred PGP to a conventional maize variety under nitrogen-stress in the greenhouse. These results indicate that, while many diazotrophic isolates from Sierra Mixe maize possessed genotypes and in vitro phenotypes for targeted PGP traits, a subset of these organisms promoted the growth of potato and conventional maize, potentially through the use of multiple promotion mechanisms.

Genomic characterization of a diazotrophic microbiota associated with maize aerial root mucilage.

A geographically isolated maize landrace cultivated on nitrogen-depleted fields without synthetic fertilizer in the Sierra Mixe region of Oaxaca, Mexico utilizes nitrogen derived from the atmosphere and develops an extensive network of mucilage-secreting aerial roots that harbors a diazotrophic (N2-fixing) microbiota. Targeting these diazotrophs, we selected nearly 600 microbes of a collection obtained from mucilage and confirmed their ability to incorporate heavy nitrogen (15N2) metabolites in vitro. Sequencing their genomes and conducting comparative bioinformatic analyses showed that these genomes had substantial phylogenetic diversity. We examined each diazotroph genome for the presence of nif genes essential to nitrogen fixation (nifHDKENB) and carbohydrate utilization genes relevant to the mucilage polysaccharide digestion. These analyses identified diazotrophs that possessed the canonical nif gene operons, as well as many other operon configurations with concomitant fixation and release of >700 different 15N labeled metabolites. We further demonstrated that many diazotrophs possessed alternative nif gene operons and confirmed their genomic potential to derive chemical energy from mucilage polysaccharide to fuel nitrogen fixation. These results confirm that some diazotrophic bacteria associated with Sierra Mixe maize were capable of incorporating atmospheric nitrogen into their small molecule extracellular metabolites through multiple nif gene configurations while others were able to fix nitrogen without the canonical (nifHDKENB) genes.

Characterization of novel glycosyl hydrolases discovered by cell wall glycan directed monoclonal antibody screening and metagenome analysis of maize aerial root mucilage.

An indigenous maize landrace from the Sierra Mixe region of Oaxaca, Mexico exhibits extensive formation of aerial roots which exude large volumes of a polysaccharide-rich gel matrix or "mucilage" that harbors diazotrophic microbiota. We hypothesize that the mucilage associated microbial community carries out multiple functions, including disassembly of the mucilage polysaccharide. In situ, hydrolytic assay of the mucilage revealed endogenous arabinofuranosidase, galactosidase, fucosidase, mannosidase and xylanase activities. Screening the mucilage against plant cell wall glycan-specific monoclonal antibodies recognized the presence of carbohydrate epitopes of hemicellulosic polysaccharides like xyloglucan (both non-fucosylated and fucosylated), xylan (both substituted and unsubstituted xylan domains) and pectic-arabinogalactans, all of which are potential carbon sources for mucilage microbial residents. Mucilage metagenome annotation using MG-RAST identified the members forming the microbial community, and gene fragments with predicted functions associated with carbohydrate disassembly. Data from the in situ hydrolytic activity and monoclonal antibody screening assays were used to guide the selection of five full length genes with predicted glycosyl hydrolase function from the GenBank database that were similar to gene fragments of high relative abundance in the mucilage metagenomes. These five genes were then synthesized for recombinant production in Escherichia coli. Here we report the characterization of an α-N-arabinofuranosidase (GH51) and an oligosaccharide reducing-end xylanase (GH8) from Flavobacterium johnsoniae; an α-L-fucosidase (GH29) and a xylan ß-1,4 xylosidase (GH39) from Spirosoma linguale, and a ß-mannosidase (GH2) from Agrobacterium fabrum. Biochemical characterization of these enzymes revealed a ß-Mannosidase that also exhibits a secondary activity towards the cleavage of galactosyl residues. We also describe two xylanases (GH8 and GH39) from underexplored glycosyl hydrolase families, one thermostable α-L-Fucosidase (GH29) and a thermostable α-N-Arabinofuranosidase (GH51).