Cross inoculation of rumen fluid to improve dry matter disappearance and its effect on bacterial composition using an in vitro batch culture model

Environmental pressures of ruminant production could be reduced by improving digestive efficiency. Previous in vivo attempts to manipulate the rumen microbial community have largely been unsuccessful probably due to the influencing effect of the host. Using an in vitro consecutive batch culture technique, the aim of this study was to determine whether manipulation was possible once the bacterial community was uncoupled from the host. Two cross inoculation experiments were performed. Rumen fluid was collected at time of slaughter from 11 Holstein-Friesian steers from the same herd for Experiment 1, and in Experiment 2 were collected from 11 Charolais cross steers sired by the same bull and raised on a forage only diet on the same farm from birth. The two fluids that differed most in their in vitro dry matter disappearance (IVDMD; “Good,” “Bad”) were selected for their respective experiment. The fluids were also mixed (1:1, “Mix”) and used to inoculate the model. In Experiment 1, the mixed rumen fluid resulted in an IVDMD midway between that of the two rumen fluids from which it was made for the first 24 h batch culture (34, 29, 20 g per 100 g DM for the Good, Mix, and Bad, respectively, P < 0.001) which was reflected in fermentation parameters recorded. No effect of cross inoculation was seen for Experiment 2, where the Mix performed most similarly to the Bad. In both experiments, IVDMD increased with consecutive culturing as the microbial population adapted to the in vitro conditions and differences between the fluids were lost. The improved performance with each consecutive batch culture was associated with reduced bacterial diversity. Increases in the genus Pseudobutyrivibrio were identified, which may be, at least in part, responsible for the improved digestive efficiency observed, whilst Prevotella declined by 50% over the study period. It is likely that along with host factors, there are individual factors within each community that prevent other microbes from establishing. Whilst we were unable to manipulate the bacterial community, uncoupling the microbiota from the host resulted in changes in the community, becoming less diverse with time, likely due to environmental heterogeneity, and more efficient at digesting DM

Direct entry by RNase E is a major pathway for the degradation and processing of RNA in Escherichia coli

Escherichia coli endoribonuclease E has a major influence on gene expression. It is essential for the maturation of ribosomal and transfer RNA as well as the rapid degradation of messenger RNA. The latter ensures that translation closely follows programming at the level of transcription. Recently, one of the hallmarks of RNase E, i.e. its ability to bind via a 5'-monophosphorylated end, was shown to be unnecessary for the initial cleavage of some polycistronic tRNA precursors. Here we show using RNA-seq analyses of ribonuclease-deficient strains in vivo and a 5'-sensor mutant of RNase E in vitro that, contrary to current models, 5'-monophosphate-independent, 'direct entry' cleavage is a major pathway for degrading and processing RNA. Moreover, we present further evidence that direct entry is facilitated by RNase E binding simultaneously to multiple unpaired regions. These simple requirements may maximize the rate of degradation and processing by permitting multiple sites to be surveyed directly without being constrained by 5'-end tethering. Cleavage was detected at a multitude of sites previously undescribed for RNase E, including ones that regulate the activity and specificity of ribosomes. A potentially broad role for RNase G, an RNase E paralogue, in the trimming of 5'-monophosphorylated ends was also revealed

The recognition of structured elements by a conserved groove distant from domains associated with catalysis is an essential determinant of RNase E

RNase E is an endoribonuclease found in many bacteria, including important human pathogens. Within Escherichia coli, it has been shown to have a major role in both the maturation of all classes of RNA involved in translation and the initiation of mRNA degradation. Thus, knowledge of the major determinants of RNase E cleavage is central to our understanding and manipulation of bacterial gene expression. We show here that the binding of RNase E to structured RNA elements is crucial for the processing of tRNA, can activate catalysis and may be important in mRNA degradation. The recognition of structured elements by RNase E is mediated by a recently discovered groove that is distant from the domains associated with catalysis. The functioning of this groove is shown here to be essential for E. coli cell viability and may represent a key point of evolutionary divergence from the paralogous RNase G family, which we show lack amino acid residues conserved within the RNA-binding groove of members of the RNase E family. Overall, this work provides new insights into the recognition and cleavage of RNA by RNase E and provides further understanding of the basis of RNase E essentiality in E. coli

Rapid cleavage of RNA by RNase E in the absence of 5′ monophosphate stimulation

The best characterized pathway for the initiation of mRNA degradation in Escherichia coli involves the removal of the 5′‐terminal pyrophosphate to generate a monophosphate group that stimulates endonucleolytic cleavage by RNase E. We show here however, using well‐characterized oligonucleotide substrates and mRNA transcripts, that RNase E can cleave certain RNAs rapidly without requiring a 5′‐monophosphorylated end. Moreover, the minimum substrate requirement for this mode of cleavage, which can be categorized as ‘direct’ or ‘internal’ entry, appears to be multiple single‐stranded segments in a conformational context that allows their simultaneous interaction with RNase E. While previous work has alluded to the existence of a 5′ end‐independent mechanism of mRNA degradation, the relative simplicity of the requirements identified here for direct entry suggests that it could represent a major means by which mRNA degradation is initiated in E. coli and other organisms that contain homologues of RNase E. Our results have implications for the interplay of translation and mRNA degradation and models of gene regulation by small non‐coding RNAs

The first small-molecule inhibitors of members of the ribonuclease E family

The Escherichia coli endoribonuclease RNase E is central to the processing and degradation of all types of RNA and as such is a pleotropic regulator of gene expression. It is essential for growth and was one of the first examples of an endonuclease that can recognise the 5′-monophosphorylated ends of RNA thereby increasing the efficiency of many cleavages. Homologues of RNase E can be found in many bacterial families including important pathogens, but no homologues have been identified in humans or animals. RNase E represents a potential target for the development of new antibiotics to combat the growing number of bacteria that are resistant to antibiotics in use currently. Potent small molecule inhibitors that bind the active site of essential enzymes are proving to be a source of potential drug leads and tools to dissect function through chemical genetics. Here we report the use of virtual high-throughput screening to obtain small molecules predicted to bind at sites in the N-terminal catalytic half of RNase E. We show that these compounds are able to bind with specificity and inhibit catalysis of Escherichia coli and Mycobacterium tuberculosis RNase E and also inhibit the activity of RNase G, a paralogue of RNase E

PomBase – the scientific resource for fission yeast

The fission yeast Schizosaccharomyces pombe has become well established as a model species for studying conserved cell-level biological processes, especially the mechanics and regulation of cell division. PomBase integrates the S. pombe genome sequence with traditional genetic, molecular and cell biological experimental data as well as the growing body of large datasets generated by emerging high-throughput methods. This chapter provides insight into the curation philosophy and data organization at PomBase, and provides a guide to using PomBase for infrequent visitors and anyone considering exploring S. pombe in their research

Interpopulation differences in expression of candidate genes for salinity tolerance in winter migrating anadromous brown trout (Salmo trutta L.)

<p>Abstract</p> <p>Background</p> <p>Winter migration of immature brown trout (<it>Salmo trutta</it>) into freshwater rivers has been hypothesized to result from physiologically stressful combinations of high salinity and low temperature in the sea.</p> <p>Results</p> <p>We sampled brown trout from two Danish populations entering different saline conditions and quantified expression of the <it>hsp70 </it>and <it>Na/K-ATPases α 1b </it>genes following acclimation to freshwater and full-strength seawater at 2°C and 10°C. An interaction effect of low temperature and high salinity on expression of both <it>hsp70 </it>and <it>Na/K-ATPase α 1b </it>was found in trout from the river entering high saline conditions, while a temperature independent up-regulation of both genes in full-strength seawater was found for trout entering marine conditions with lower salinities.</p> <p>Conclusion</p> <p>Overall our results support the hypothesis that physiologically stressful conditions in the sea drive sea-run brown trout into freshwater rivers in winter. However, our results also demonstrate intra-specific differences in expression of important stress and osmoregulative genes most likely reflecting adaptive differences between trout populations on a regional scale, thus strongly suggesting local adaptations driven by the local marine environment.</p

On-line analysis and in situ pH monitoring of mixed acid fermentation by Escherichia coli using combined FTIR and Raman techniques

We introduce an experimental setup allowing continuous monitoring of bacterial fermentation processes by simultaneous optical density (OD) measurements, long-path FTIR headspace monitoring of CO2, acetaldehyde and ethanol, and liquid Raman spectroscopy of acetate, formate, and phosphate anions, without sampling. We discuss which spectral features are best suited for detection, and how to obtain partial pressures and concentrations by integrations and least squares fitting of spectral features. Noise equivalent detection limits are about 2.6 mM for acetate and 3.6 mM for formate at 5 min integration time, improving to 0.75 mM for acetate and 1.0 mM for formate at 1 h integration. The analytical range extends to at least 1 M with a standard deviation of percentage error of about 8%. The measurement of the anions of the phosphate buffer allows the spectroscopic, in situ determination of the pH of the bacterial suspension via a modified Henderson-Hasselbalch equation in the 6–8 pH range with an accuracy better than 0.1. The 4 m White cell FTIR measurements provide noise equivalent detection limits of 0.21 μbar for acetaldehyde and 0.26 μbar for ethanol in the gas phase, corresponding to 3.2 μM acetaldehyde and 22 μM ethanol in solution, using Henry’s law. The analytical dynamic range exceeds 1 mbar ethanol corresponding to 85 mM in solution. As an application example, the mixed acid fermentation of Escherichia coli is studied. The production of CO2, ethanol, acetaldehyde, acids such as formate and acetate, and the changes in pH are discussed in the context of the mixed acid fermentation pathways. Formate decomposition into CO2 and H2 is found to be governed by a zeroth-order kinetic rate law, showing that adding exogenous formate to a bioreactor with E. coli is expected to have no beneficial effect on the rate of formate decomposition and biohydrogen production