More Is Not Always Better: Evaluation of 1D and 2D-LC-MS/MS Methods for Metaproteomics.

Metaproteomics, the study of protein expression in microbial communities, is a versatile tool for environmental microbiology. Achieving sufficiently high metaproteome coverage to obtain a comprehensive picture of the activities and interactions in microbial communities is one of the current challenges in metaproteomics. An essential step to maximize the number of identified proteins is peptide separation via liquid chromatography (LC) prior to mass spectrometry (MS). Thorough optimization and comparison of LC methods for metaproteomics are, however, currently lacking. Here, we present an extensive development and test of different 1D and 2D-LC approaches for metaproteomic peptide separations. We used fully characterized mock community samples to evaluate metaproteomic approaches with very long analytical columns (50 and 75 cm) and long gradients (up to 12 h). We assessed a total of over 20 different 1D and 2D-LC approaches in terms of number of protein groups and unique peptides identified, peptide spectrum matches (PSMs) generated, the ability to detect proteins of low-abundance species, the effect of technical replicate runs on protein identifications and method reproducibility. We show here that, while 1D-LC approaches are faster and easier to set up and lead to more identifications per minute of runtime, 2D-LC approaches allow for a higher overall number of identifications with up to >10,000 protein groups identified. We also compared the 1D and 2D-LC approaches to a standard GeLC workflow, in which proteins are pre-fractionated via gel electrophoresis. This method yielded results comparable to the 2D-LC approaches, however with the drawback of a much increased sample preparation time. Based on our results, we provide recommendations on how to choose the best LC approach for metaproteomics experiments, depending on the study aims.

Genome sequence, metabolic properties and cyanobacterial attachment of Porphyrobacter sp. HT-58-2 isolated from a filamentous cyanobacterium-microbial consortium.

Tolyporphins are structurally diverse tetrapyrrole macrocycles produced by the cyanobacterial culture HT-58-2. Although tolyporphins were discovered over 25 years ago, little was known about the microbiology of the culture. The studies reported herein expand the description of the community of predominantly alphaproteobacteria associated with the filamentous HT-58-2 cyanobacterium and isolate a dominant bacterium, Porphyrobacter sp. HT-58-2, for which the complete genome is established and growth properties are examined. Fluorescence in situ hybridization (FISH) analysis of the cyanobacterium-microbial community with a probe targeting the 16S rRNA of Porphyrobacter sp. HT-58-2 showed fluorescence emanating from the cyanobacterial sheath. Although genes for the biosynthesis of bacteriochlorophyll a (BChl a) are present in the Porphyrobacter sp. HT-58-2 genome, the pigment was not detected under the conditions examined, implying the absence of phototrophic growth. Comparative analysis of four Porphyrobacter spp. genomes from worldwide collection sites showed significant collinear gene blocks, with two inversions and three deletion regions. Taken together, the results enrich our understanding of the HT-58-2 cyanobacterium-microbial culture.

Quantitation of Tolyporphins, Diverse Tetrapyrrole Secondary Metabolites with Chlorophyll-Like Absorption, from a Filamentous Cyanobacterium-Microbial Community.

INTRODUCTION: Tolyporphins are unusual tetrapyrrole macrocycles produced by a non-axenic filamentous cyanobacterium (HT-58-2). Tolyporphins A-J, L, and M share a common dioxobacteriochlorin core, differ in peripheral substituents, and exhibit absorption spectra that overlap that of the dominant cyanobacterial pigment, chlorophyll a. Identification and accurate quantitation of the various tolyporphins in these chlorophyll-rich samples presents challenges. OBJECTIVE: To develop methods for the quantitative determination of tolyporphins produced under various growth conditions relative to that of chlorophyll a. METHODOLOGY: Chromatographic fractionation of large-scale (440 L) cultures afforded isolated individual tolyporphins. Lipophilic extraction of small-scale (25 mL) cultures, HPLC separation with an internal standard, and absorption detection enabled quantitation of tolyporphin A and chlorophyll a, and by inference the amounts of tolyporphins A-M. Absorption spectroscopy with multicomponent analysis of lipophilic extracts (2 mL cultures) afforded the ratio of all tolyporphins to chlorophyll a. The reported absorption spectral data for the various tolyporphins required re-evaluation for quantitative purposes. RESULTS AND DISCUSSION: The amount of tolyporphin A after 50 days of illumination ranged from 0.13 nmol/mg dry cells (media containing nitrate) to 1.12 nmol/mg (without nitrate), with maximum 0.23 times that of chlorophyll a. Under soluble-nitrogen deprivation after 35-50 days, tolyporphin A represents 1/3-1/2 of the total tolyporphins, and the total amount of tolyporphins is up to 1.8-fold that of chlorophyll a. CONCLUSIONS: The quantitative methods developed herein should facilitate investigation of the biosynthesis of tolyporphins (and other tetrapyrroles) as well as examination of other strains for production of tolyporphins. Copyright © 2017 John Wiley & Sons, Ltd.

Impact of Dietary Galacto-Oligosaccharide (GOS) on Chicken's Gut Microbiota, Mucosal Gene Expression, and Salmonella Colonization.

Preventing Salmonella colonization in young birds is key to reducing contamination of poultry products for human consumption (eggs and meat). While several Salmonella vaccines have been developed that are capable of yielding high systemic antibodies, it is not clear how effective these approaches are at controlling or preventing Salmonella colonization of the intestinal tract. Effective alternative control strategies are needed to help supplement the bird's ability to prevent Salmonella colonization, specifically by making the cecum less hospitable to Salmonella. In this study, we investigated the effect of the prebiotic galacto-oligosaccharide (GOS) on the cecal microbiome and ultimately the carriage of Salmonella. Day-old pullet chicks were fed control diets or diets supplemented with GOS (1% w/w) and then challenged with a cocktail of Salmonella Typhimurium and Salmonella Enteritidis. Changes in cecal tonsil gene expression, cecal microbiome, and levels of cecal and extraintestinal Salmonella were assessed at 1, 4, 7, 12, and 27 days post infection. While the Salmonella counts were generally lower in the GOS-treated birds, the differences were not significantly different at the end of the experiment. However, these data demonstrated that treatment with the prebiotic GOS can modify both cecal tonsil gene expression and the cecal microbiome, suggesting that this type of treatment may be useful as a tool for altering the carriage of Salmonella in poultry.

Genome Sequence and Composition of a Tolyporphin-Producing Cyanobacterium-Microbial Community.

The cyanobacterial culture HT-58-2 was originally described as a strain of Tolypothrix nodosa with the ability to produce tolyporphins, which comprise a family of distinct tetrapyrrole macrocycles with reported efflux pump inhibition properties. Upon reviving the culture from what was thought to be a nonextant collection, studies of culture conditions, strain characterization, phylogeny, and genomics have been undertaken. Here, HT-58-2 was shown by 16S rRNA analysis to closely align with Brasilonema strains and not with Tolypothrix isolates. Light, fluorescence, and scanning electron microscopy revealed cyanobacterium filaments that are decorated with attached bacteria and associated with free bacteria. Metagenomic surveys of HT-58-2 cultures revealed a diversity of bacteria dominated by Erythrobacteraceae, 97% of which are Porphyrobacter species. A dimethyl sulfoxide washing procedure was found to yield enriched cyanobacterial DNA (presumably by removing community bacteria) and sequence data sufficient for genome assembly. The finished, closed HT-58-2Cyano genome consists of 7.85 Mbp (42.6% G+C) and contains 6,581 genes. All genes for biosynthesis of tetrapyrroles (e.g., heme, chlorophyll a, and phycocyanobilin) and almost all for cobalamin were identified dispersed throughout the chromosome. Among the 6,177 protein-encoding genes, coding sequences (CDSs) for all but two of the eight enzymes for conversion of glutamic acid to protoporphyrinogen IX also were found within one major gene cluster. The cluster also includes 10 putative genes (and one hypothetical gene) encoding proteins with domains for a glycosyltransferase, two cytochrome P450 enzymes, and a flavin adenine dinucleotide (FAD)-binding protein. The composition of the gene cluster suggests a possible role in tolyporphin biosynthesis.IMPORTANCE A worldwide search more than 25 years ago for cyanobacterial natural products with anticancer activity identified a culture (HT-58-2) from Micronesia that produces tolyporphins. Tolyporphins are tetrapyrroles, like chlorophylls, but have several profound structural differences that reside outside the bounds of known biosynthetic pathways. To begin probing the biosynthetic origin and biological function of tolyporphins, our research has focused on studying the cyanobacterial strain, about which almost nothing has been previously reported. We find that the HT-58-2 culture is composed of the cyanobacterium and a community of associated bacteria, complicating the question of which organisms make tolyporphins. Elucidation of the cyanobacterial genome revealed an intriguing gene cluster that contains tetrapyrrole biosynthesis genes and a collection of unknown genes, suggesting that the cluster may be responsible for tolyporphin production. Knowledge of the genome and the gene cluster sharply focuses research to identify related cyanobacterial producers of tolyporphins and delineate the tolyporphin biosynthetic pathway.

Understanding the molecular circuitry of cell lineage specification in the early mouse embryo.

Pluripotent stem cells hold great promise for cell-based therapies in regenerative medicine. However, critical to understanding and exploiting mechanisms of cell lineage specification, epigenetic reprogramming, and the optimal environment for maintaining and differentiating pluripotent stem cells is a fundamental knowledge of how these events occur in normal embryogenesis. The early mouse embryo has provided an excellent model to interrogate events crucial in cell lineage commitment and plasticity, as well as for embryo-derived lineage-specific stem cells and induced pluripotent stem (iPS) cells. Here we provide an overview of cell lineage specification in the early (preimplantation) mouse embryo focusing on the transcriptional circuitry and epigenetic marks necessary for successive differentiation events leading to the formation of the blastocyst.

Exposure of Campylobacter jejuni to 6 degrees C: effects on heat resistance and electron transport activity.

Human infection with Campylobacter jejuni is frequently associated with the consumption of foods, especially chicken meat, which have been exposed to a range of temperatures during processing, storage, and cooking. Despite the public health importance of C. jejuni, little is known about the effects of cold exposure (refrigeration) on the subsequent ability of this pathogen to survive heat challenge. This work examined the effect of rapid exposure to 6 degrees C for 24 h on the heat resistance at 52 degrees C of 19 C. jejuni strains originally isolated from various sources. The resulting death curves were analyzed with the Weibull model. Unlike cold-exposed cells of Escherichia coli and Salmonella, which have been reported to show significant increased sensitivity to heat, such exposure had only a marginal effect on heat resistance of the C. jejuni strains in this study. A possible explanation for this effect is that rapid chilling renders C. jejuni cells unable to adapt to reduced temperatures in an active manner. This hypothesis is supported by the observation that exposure to 6 degrees C for 24 h resulted in a significant and marked reduction in electron transport system activity when compared with controls at 37 degrees C.

The response of Campylobacter jejuni to low temperature differs from that of Escherichia coli.

Human infection with Campylobacter jejuni is often associated with the consumption of foods that have been exposed to both chilling and high temperatures. Despite the public health importance of this pathogen, little is known about the effects of cold exposure on its ability to survive a subsequent heat challenge. This work examined the effect of rapid exposure to chilling, as would occur in poultry processing, on the heat resistance at 56 degrees C of two C. jejuni strains, 11168 and 2097e48, and of Escherichia coli K-12. Unlike E. coli K-12, whose cold-exposed cells showed increased sensitivity to 56 degrees C, such exposure had only a marginal effect on subsequent heat resistance in C. jejuni. This may be explained by the finding that during rapid chilling, unlike E. coli cells, C. jejuni cells are unable to alter their fatty acid composition and do not adapt to cold exposure. However, their unaltered fatty acid composition is more suited to survival when cells are exposed to high temperatures. This hypothesis is supported by the fact that in C. jejuni, the ratio of unsaturated to saturated fatty acids was not significantly different after cold exposure, but it was in E. coli. The low-temperature response of C. jejuni is very different from that of other food-borne pathogens, and this may contribute to its tolerance to further heat stresses.