Application of phenotypic microarrays to environmental microbiology

Environmental organisms are extremely diverse and only a small fraction has been successfully cultured in the laboratory. Culture in micro wells provides a method for rapid screening of a wide variety of growth conditions and commercially available plates contain a large number of substrates, nutrient sources, and inhibitors, which can provide an assessment of the phenotype of an organism. This review describes applications of phenotype arrays to anaerobic and thermophilic microorganisms, use of the plates in stress response studies, in development of culture media for newly discovered strains, and for assessment of phenotype of environmental communities. Also discussed are considerations and challenges in data interpretation and visualization, including data normalization, statistics, and curve fitting

Complete genome sequence of Sanguibacter keddieii type strain (ST-74T)

Sanguibacter keddieii is the type species of the genus Sanguibacter, the only genus within the family of Sanguibacteraceae. Phylogenetically, this family is located in the neighborhood of the genus Oerskovia and the family Cellulomonadaceae within the actinobacterial suborder Micrococcineae. The strain described in this report was isolated from blood of apparently healthy cows. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the family Sanguibacteraceae, and the 4,253,413 bp long single replicon genome with its 3735 protein-coding and 70 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project

Glycoside Hydrolases from a targeted Compost Metagenome, activity-screening and functional characterization

<p>Abstract</p> <p>Background</p> <p>Metagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes. Discovery of new glycoside hydrolases with improved biocatalytic properties for the efficient conversion of lignocellulosic material to biofuels is a critical challenge in the development of economically viable routes from biomass to fuels and chemicals.</p> <p>Results</p> <p>Twenty-two putative ORFs (open reading frames) were identified from a switchgrass-adapted compost community based on sequence homology to related gene families. These ORFs were expressed in <it>E. coli</it> and assayed for predicted activities. Seven of the ORFs were demonstrated to encode active enzymes, encompassing five classes of hemicellulases. Four enzymes were over expressed <it>in vivo</it>, purified to homogeneity and subjected to detailed biochemical characterization. Their pH optima ranged between 5.5 - 7.5 and they exhibit moderate thermostability up to ~60-70°C.</p> <p>Conclusions</p> <p>Seven active enzymes were identified from this set of ORFs comprising five different hemicellulose activities. These enzymes have been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering towards the goal of developing efficient enzyme cocktails for biomass degradation under diverse process conditions.</p

The Polymorphic Membrane Protein G Has a Neutral Effect and the Plasmid Glycoprotein 3 an Antagonistic Effect on the Ability of the Major Outer Membrane Protein to Elicit Protective Immune Responses against a Chlamydia muridarum Respiratory Challenge

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen. The number of chlamydial infections continuous to increase and there is an urgent need for a safe and efficacious vaccine. To assess the ability of the Chlamydia muridarum polymorphic membrane protein G (PmpG) and the plasmid glycoprotein 3 (Pgp3) as single antigens, and in combination with the major outer-membrane protein (MOMP) to induce protection, BALB/c mice were immunized utilizing CpG-1826 and Montanide ISA 720 VG as adjuvants. Following vaccination with MOMP, significant humoral and cell-mediated immune responses were observed, while immunization with PmpG, or Pgp3, elicited weaker immune responses. Weaker immune responses were induced with MOMP+Pgp3 compared with MOMP alone. Following the intranasal challenge with C. muridarum, mice vaccinated with MOMP showed robust protection against body-weight loss, inflammatory responses in the lungs and number of Chlamydia recovered from the lungs. PmpG and Pgp3 elicited weaker protective responses. Mice immunized with MOMP+PmpG, were no better protected than animals vaccinated with MOMP only, while Pgp3 antagonized the protection elicited by MOMP. In conclusion, PmpG and Pgp3 elicited limited protective immune responses in mice against a respiratory challenge with C. muridarum and failed to enhance the protection induced by MOMP alone. The virulence of Pgp3 may result from its antagonistic effect on the immune protection induced by MOMP

Metagenomic analysis of intertidal hypersaline microbial mats from Elkhorn Slough, California, grown with and without molybdate

Abstract Cyanobacterial mats are laminated microbial ecosystems which occur in highly diverse environments and which may provide a possible model for early life on Earth. Their ability to produce hydrogen also makes them of interest from a biotechnological and bioenergy perspective. Samples of an intertidal microbial mat from the Elkhorn Slough estuary in Monterey Bay, California, were transplanted to a greenhouse at NASA Ames Research Center to study a 24-h diel cycle, in the presence or absence of molybdate (which inhibits biohydrogen consumption by sulfate reducers). Here, we present metagenomic analyses of four samples that will be used as references for future metatranscriptomic analyses of this diel time series

Complete genome sequence of “Enterobacter lignolyticus” SCF1

In an effort to discover anaerobic bacteria capable of lignin degradation, we isolated "Enterobacter lignolyticus" SCF1 on minimal media with alkali lignin as the sole source of carbon. This organism was isolated anaerobically from tropical forest soils collected from the Short Cloud Forest site in the El Yunque National Forest in Puerto Rico, USA, part of the Luquillo Long-Term Ecological Research Station. At this site, the soils experience strong fluctuations in redox potential and are net methane producers. Because of its ability to grow on lignin anaerobically, we sequenced the genome. The genome of "E. lignolyticus" SCF1 is 4.81 Mbp with no detected plasmids, and includes a relatively small arsenal of lignocellulolytic carbohydrate active enzymes. Lignin degradation was observed in culture, and the genome revealed two putative laccases, a putative peroxidase, and a complete 4-hydroxyphenylacetate degradation pathway encoded in a single gene cluster

Complete genome sequence of “Enterobacter lignolyticus” SCF1

In an effort to discover anaerobic bacteria capable of lignin degradation, we isolated "Enterobacter lignolyticus" SCF1 on minimal media with alkali lignin as the sole source of carbon. This organism was isolated anaerobically from tropical forest soils collected from the Short Cloud Forest site in the El Yunque National Forest in Puerto Rico, USA, part of the Luquillo Long-Term Ecological Research Station. At this site, the soils experience strong fluctuations in redox potential and are net methane producers. Because of its ability to grow on lignin anaerobically, we sequenced the genome. The genome of "E. lignolyticus" SCF1 is 4.81 Mbp with no detected plasmids, and includes a relatively small arsenal of lignocellulolytic carbohydrate active enzymes. Lignin degradation was observed in culture, and the genome revealed two putative laccases, a putative peroxidase, and a complete 4-hydroxyphenylacetate degradation pathway encoded in a single gene cluster

Scatterplots of contig properties for select genus bins in thermophilic and mesophilic communities.

<p>Plotted contigs correspond to (A) <i>Micromonospora</i> (<i>Actinobacteria</i>) in thermophilic community, (B) <i>Mycobacterium</i> (<i>Actinobacteria</i>) in thermophilic community, (C) <i>Pseudoxanthomonas</i> (<i>Proteobacteria</i>) in thermophilic community, (D) <i>Pseudoxanthomonas</i> (<i>Proteobacteria</i>) in mesophilic community, (E) <i>Chryseobacterium</i> (<i>Bacteroidetes</i>) in mesophilic community, (F) <i>Niabella</i> (<i>Bacteroidetes</i>) in thermophilic community, (G) <i>Niastella</i> (<i>Bacteroidetes</i>) in mesophilic community, and (H) <i>Chelativorans</i> (<i>Proteobacteria</i>) in thermophilic community. Genera presented in A–E account for >50% of total dissimilarity between thermophilic and mesophilic communities. Notable clusters with high abundance or large contigs are labeled for reference in subsequent analyses.</p

Rarefaction curves from pyrotag data for enriched mesophilic and thermophilic microbial communities.

<p>Dashed lines indicate ±1 standard error.</p

Pylum composition of microbial communities from mesophilic and thermophilic enrichments on rice straw.

<p>Pylum composition of microbial communities from mesophilic and thermophilic enrichments on rice straw.</p