The web server probeCheck, freely accessible at http://www.microbial-ecology.net/probecheck, provides a pivotal forum for rapid specificity and coverage evaluations of probes and primers against ...selected databases of phylogenetic and functional marker genes. Currently, 24 widely used sequence collections including the Ribosomal Database Project (RDP) II, Greengenes, SILVA and the Functional Gene Pipeline/Repository can be queried. For this purpose, probeCheck integrates a new online version of the popular ARB probe match tool with free energy (ΔG) calculations for each perfectly matched and mismatched probe-target hybrid, allowing assessment of the theoretical binding stabilities of oligo-target and non-target hybrids. For each output sequence, the accession number, the GenBank taxonomy and a link to the respective entry at GenBank, EMBL and, if applicable, the query database are displayed. Filtering options allow customizing results on the output page. In addition, probeCheck is linked with probe match tools of RDP II and Greengenes, NCBI blast, the Oligonucleotide Properties Calculator, the two-state folding tool of the DINAMelt server and the rRNA-targeted probe database probeBase. Taken together, these features provide a multifunctional platform with maximal flexibility for the user in the choice of databases and options for the evaluation of published and newly developed probes and primers.
HuR is an RNA-binding protein implicated in immune homeostasis and various cancers, including colorectal cancer. HuR binding to AU-rich elements within the 3' untranslated region of mRNAs encoding ...oncogenes, growth factors, and various cytokines leads message stability and translation. In this study, we evaluated HuR as a small-molecule target for preventing colorectal cancer in high-risk groups such as those with familial adenomatosis polyposis (FAP) or inflammatory bowel disease (IBD). In human specimens, levels of cytoplasmic HuR were increased in colonic epithelial cells from patients with IBD, IBD-cancer, FAP-adenoma, and colorectal cancer, but not in patients with IBD-dysplasia. Intraperitoneal injection of the HuR small-molecule inhibitor MS-444 in AOM/DSS mice, a model of IBD and inflammatory colon cancer, augmented DSS-induced weight loss and increased tumor multiplicity, size, and invasiveness. MS-444 treatment also abrogated tumor cell apoptosis and depleted tumor-associated eosinophils, accompanied by a decrease in IL18 and eotaxin-1. In contrast, HuR inhibition in APC
mice, a model of FAP and colon cancer, diminished the number of small intestinal tumors generated. In this setting, fecal microbiota, evaluated by 16S rRNA gene amplicon sequencing, shifted to a state of reduced bacterial diversity, with an increased representation of
, and
Taken together, our results indicate that HuR activation is an early event in FAP-adenoma but is not present in IBD-dysplasia. Furthermore, our results offer a preclinical proof of concept for HuR inhibition as an effective means of FAP chemoprevention, with caution advised in the setting of IBD.
.
Summary
Genes encoding dissimilatory sulfite reductase (DsrAB) are commonly used as diagnostic markers in ecological studies of sulfite‐ and sulfate‐reducing microorganisms. Here, we developed new ...high‐coverage primer sets for generation of reductive bacterial‐type dsrA and dsrB polymerase chain reaction (PCR) products for highly parallel amplicon sequencing and a bioinformatics workflow for processing and taxonomic classification of short dsrA and dsrB reads. We employed two diverse mock communities that consisted of 45 or 90 known dsrAB sequences derived from environmental clones to precisely evaluate the performance of individual steps of our amplicon sequencing approach on the Illumina MiSeq platform. Although PCR cycle number, gene‐specific primer mismatches and stringent filtering for high‐quality sequences had notable effects on the observed dsrA and dsrB community structures, recovery of most mock community sequences was generally proportional to their relative input abundances. Successful dsrA and dsrB diversity analysis in selected environmental samples further proved that the multiplex amplicon sequencing approach is adequate for monitoring spatial distribution and temporal abundance dynamics of dsrAB‐containing microorganisms. Although tested for reductive bacterial‐type dsrAB, this method is readily applicable for oxidative‐type dsrAB of sulfur‐oxidizing bacteria and also provides guidance for processing short amplicon reads of other functional genes.
Distinct lineages of Gammaproteobacteria clade Woeseiales are globally distributed in marine sediments, based on metagenomic and 16S rRNA gene analysis. Yet little is known about why they are ...dominant or their ecological role in Arctic fjord sediments, where glacial retreat is rapidly imposing change. This study combined 16S rRNA gene analysis, metagenome-assembled genomes (MAGs), and genome-resolved metatranscriptomics uncovered the in situ abundance and transcriptional activity of Woeseiales with burial in four shallow sediment sites of Kongsfjorden and Van Keulenfjorden of Svalbard (79°N). We present five novel Woeseiales MAGs and show transcriptional evidence for metabolic plasticity during burial, including sulfur oxidation with reverse dissimilatory sulfite reductase (dsrAB) down to 4 cm depth and nitrite reduction down to 6 cm depth. A single stress protein, spore protein SP21 (hspA), had a tenfold higher mRNA abundance than any other transcript, and was a hundredfold higher on average than other transcripts. At three out of the four sites, SP21 transcript abundance increased with depth, while total mRNA abundance and richness decreased, indicating a shift in investment from metabolism and other cellular processes to build-up of spore protein SP21. The SP21 gene in MAGs was often flanked by genes involved in membrane-associated stress response. The ability of Woeseiales to shift from sulfur oxidation to nitrite reduction with burial into marine sediments with decreasing access to overlying oxic bottom waters, as well as enter into a dormant state dominated by SP21, may account for its ubiquity and high abundance in marine sediments worldwide, including those of the rapidly shifting Arctic.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Dissimilatory manganese reduction dominates anaerobic carbon oxidation in marine sediments with high manganese oxide concentrations, but the microorganisms responsible for this process are largely ...unknown. In this study, the acetate-utilizing manganese-reducing microbiota in geographically well-separated, manganese oxide-rich sediments from Gullmar Fjord (Sweden), Skagerrak (Norway) and Ulleung Basin (Korea) were analyzed by 16S rRNA-stable isotope probing (SIP). Manganese reduction was the prevailing terminal electron-accepting process in anoxic incubations of surface sediments, and even the addition of acetate stimulated neither iron nor sulfate reduction. The three geographically distinct sediments harbored surprisingly similar communities of acetate-utilizing manganese-reducing bacteria: 16S rRNA of members of the genera Colwellia and Arcobacter and of novel genera within the Oceanospirillaceae and Alteromonadales were detected in heavy RNA-SIP fractions from these three sediments. Most probable number (MPN) analysis yielded up to 10(6) acetate-utilizing manganese-reducing cells cm(-3) in Gullmar Fjord sediment. A 16S rRNA gene clone library that was established from the highest MPN dilutions was dominated by sequences of Colwellia and Arcobacter species and members of the Oceanospirillaceae, supporting the obtained RNA-SIP results. In conclusion, these findings strongly suggest that (i) acetate-dependent manganese reduction in manganese oxide-rich sediments is catalyzed by members of taxa (Arcobacter, Colwellia and Oceanospirillaceae) previously not known to possess this physiological function, (ii) similar acetate-utilizing manganese reducers thrive in geographically distinct regions and (iii) the identified manganese reducers differ greatly from the extensively explored iron reducers in marine sediments.
Humans and animals host diverse communities of microorganisms important to their physiology and health. Despite extensive sequencing-based characterization of host-associated microbiomes, there ...remains a dramatic lack of understanding of microbial functions. Stable-isotope probing (SIP) is a powerful strategy to elucidate the ecophysiology of microorganisms in complex host-associated microbiotas. Here, we suggest that SIP methodologies should be more frequently exploited as part of a holistic functional microbiomics approach. We provide examples of how SIP has been used to study host-associated microbes in vivo and ex vivo. We highlight recent developments in SIP technologies and discuss future directions that will facilitate deeper insights into the function of human and animal microbiomes.
Meta-omic analysis has produced an immense amount of knowledge on the composition and development of human and animal microbiomes, yet most functions encoded in the metagenome are unknown.
Stable-isotope probing (SIP) is a powerful strategy for elucidating the ecophysiology of individual microorganisms in complex host-associated microbiotas.
An array of SIP approaches has been developed that allow testing of hypotheses from meta-omic datasets and discovery of new functions.
SIP is being used to determine the nutrient-utilization strategies of microbes in vivo and ex vivo as well as ecological relationships such as competition, cooperation, and cross-feeding.
Abstract
Taurine-respiring gut bacteria produce H
2
S with ambivalent impact on host health. We report the isolation and ecophysiological characterization of a taurine-respiring mouse gut bacterium.
...Taurinivorans muris
strain LT0009 represents a new widespread species that differs from the human gut sulfidogen
Bilophila wadsworthia
in its sulfur metabolism pathways and host distribution.
T. muris
specializes in taurine respiration in vivo, seemingly unaffected by mouse diet and genotype, but is dependent on other bacteria for release of taurine from bile acids. Colonization of
T. muris
in gnotobiotic mice increased deconjugation of taurine-conjugated bile acids and transcriptional activity of a sulfur metabolism gene-encoding prophage in other commensals, and slightly decreased the abundance of
Salmonella enterica
, which showed reduced expression of galactonate catabolism genes. Re-analysis of metagenome data from a previous study further suggested that
T. muris
can contribute to protection against pathogens by the commensal mouse gut microbiota. Together, we show the realized physiological niche of a key murine gut sulfidogen and its interactions with selected gut microbiota members.
Temperature has a fundamental impact on the metabolic rates of microorganisms and strongly influences microbial ecology and biogeochemical cycling in the environment. In this study, we examined the ...catabolic temperature response of natural communities of sulfate-reducing microorganisms (SRM) in polar, temperate and tropical marine sediments. In short-term sediment incubation experiments with (35)S-sulfate, we demonstrated how the cardinal temperatures for sulfate reduction correlate with mean annual sediment temperatures, indicating specific thermal adaptations of the dominant SRM in each of the investigated ecosystems. The community structure of putative SRM in the sediments, as revealed by pyrosequencing of bacterial 16S rRNA gene amplicons and phylogenetic assignment to known SRM taxa, consistently correlated with in situ temperatures, but not with sediment organic carbon concentrations or C:N ratios of organic matter. Additionally, several species-level SRM phylotypes of the class Deltaproteobacteria tended to co-occur at sites with similar mean annual temperatures, regardless of geographic distance. The observed temperature adaptations of SRM imply that environmental temperature is a major controlling variable for physiological selection and ecological and evolutionary differentiation of microbial communities.
Summary
The Black Sea, with its highly sulfidic water column, is the largest anoxic basin in the world. Within its sediments, the mineralization of organic matter occurs essentially through sulfate ...reduction and methanogenesis. In this study, the sulfate‐reducing community was investigated in order to understand how these microorganisms are distributed relative to the chemical zonation: in the upper sulfate zone, at the sulfate–methane transition zone, and deeply within the methane zone. Total bacteria were quantified by real‐time PCR of 16S rRNA genes whereas sulfate‐reducing microorganisms (SRM) were quantified by targeting their metabolic key gene, the dissimilatory (bi)sulfite reductase (dsrA). Sulfate‐reducing microorganisms were predominant in the sulfate zone but occurred also in the methane zone, relative proportion was maximal around the sulfate–methane transition, c. 30%, and equally high in the sulfate and methane zones, 5–10%. The dsrAB clone library from the sulfate–methane transition zone, showed mostly sequences affiliated with the Desulfobacteraceae. While, the dsrAB clone libraries from the upper, sulfate‐rich zone and the deep, sulfate‐poor zone were dominated by similar, novel deeply branching sequences which might represent Gram‐positive spore‐forming sulfate‐ and/or sulfite‐reducing microorganisms. We thus hypothesize that terminal carbon mineralization in surface sediments of the Black Sea is largely due to the sulfate reduction activity of previously hidden SRM. Although these novel SRM were also abundant in sulfate‐poor, methanogenic areas of the Black Sea sediment, their activities and possibly very versatile metabolic capabilities remain subject of further study.
is an abundant inhabitant of the intestinal mucus layer of rodents and other animals and has been suggested to be a pathobiont, a commensal that plays a role in disease. In order to gain insights ...into its lifestyle, we analyzed the genome and transcriptome of
ASF 457 and performed physiological experiments to test traits predicted by its genome. Although described as a mucus inhabitant,
has limited capacity for degrading host-derived mucosal glycans and other complex polysaccharides. Additionally,
reduces nitrate and expresses systems for scavenging oxygen and reactive oxygen species
, which may account for its localization close to the mucosal tissue and expansion during inflammation. Also of note,
harbors a type VI secretion system and putative effector proteins and can modify gene expression in mucosal tissue, suggesting intimate interactions with its host and a possible role in inflammation. The
genome has been shaped by extensive horizontal gene transfer, primarily from intestinal
- and
, indicating that horizontal gene transfer has played a key role in defining its niche in the gut ecosystem.
Shifts in gut microbiota composition have been associated with intestinal inflammation, but it remains unclear whether inflammation-associated bacteria are commensal or detrimental to their host. Here, we studied the lifestyle of the gut bacterium
, which is associated with inflammation in widely used mouse models. We found that
has specialized systems to handle oxidative stress during inflammation. Additionally, it expresses secretion systems and effector proteins and can modify the mucosal gene expression of its host. This suggests that
undergoes intimate interactions with its host and may play a role in inflammation. The insights presented here aid our understanding of how commensal gut bacteria may be involved in altering susceptibility to disease.
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