Caenorhabditis elegans Genomic Response to Soil Bacteria Predicts Environment-Specific Genetic Effects on Life History Traits

With the post-genomic era came a dramatic increase in high-throughput technologies, of which transcriptional profiling by microarrays was one of the most popular. One application of this technology is to identify genes that are differentially expressed in response to different environmental conditions. These experiments are constructed under the assumption that the differentially expressed genes are functionally important in the environment where they are induced. However, whether differential expression is predictive of functional importance has yet to be tested. Here we have addressed this expectation by employing Caenorhabditis elegans as a model for the interaction of native soil nematode taxa and soil bacteria. Using transcriptional profiling, we identified candidate genes regulated in response to different bacteria isolated in association with grassland nematodes or from grassland soils. Many of the regulated candidate genes are predicted to affect metabolism and innate immunity suggesting similar genes could influence nematode community dynamics in natural systems. Using mutations that inactivate 21 of the identified genes, we showed that most contribute to lifespan and/or fitness in a given bacterial environment. Although these bacteria may not be natural food sources for C. elegans, we show that changes in food source, as can occur in environmental disturbance, can have a large effect on gene expression, with important consequences for fitness. Moreover, we used regression analysis to demonstrate that for many genes the degree of differential gene expression between two bacterial environments predicted the magnitude of the effect of the loss of gene function on life history traits in those environments

An Inexpensive Infrared Detector to Verify The Delivery of Food Pellets

The reproducibility of experimental outcomes depends on consistent control of independent variables. In food-maintained operant performance, it is of utmost importance that the quantity of food delivered is reliable. To that end, some commercial food pellet dispensers have add-on attachments to sense the delivery of pellets. Not all companies, however, offer such add-ons. Aside from availability, cost and temporary reduction in throughput may be a problem for smaller labs. The present paper outlines our recent development of a simple, inexpensive infrared device to detect and confirm the delivery of pellets. The in-line construction of the detector routes the falling pellet through a barrel so that it passes between an infrared emitter and receiver. The circuitry was designed to be compatible with all commercially available behavioral measurement systems, and so may be retrofit to any existing system. Our tests with the detector so far have shown that it is 100% accurate in detecting pellet delivery. The individual unit cost is approximately 25 dollars. The low cost and versatility of the device offer an easy method to ensure the integrity of food delivery in operant settings

Functional studies of E. faecalis RNase J2 and its role in virulence and fitness.

Post-transcriptional control provides bacterial pathogens a method by which they can rapidly adapt to environmental change. Dual exo- and endonucleolytic activities of RNase J enzymes contribute to Gram-positive RNA processing and decay. First discovered in Bacillus subtilis, RNase J1 plays a key role in mRNA maturation and degradation, while the function of the paralogue RNase J2 is largely unknown. Previously, we discovered that deletion of the Enterococcus faecalis rnjB gene significantly attenuates expression of a major virulence factor involved in enterococcal pathogenesis, the Ebp pili. In this work, we demonstrate that E. faecalis rnjB encodes an active RNase J2, and that the ribonuclease activity of RNase J2 is required for regulation of Ebp pili. To further investigate how rnjB affects E. faecalis gene expression on a global scale, we compared transcriptomes of the E. faecalis strain OG1RF with its isogenic rnjB deletion mutant (ΔrnjB). In addition to Ebp pili regulation, previously demonstrated to have a profound effect on the ability of E. faecalis to form biofilm or establish infection, we identified that rnjB regulates the expression of several other genes involved in bacterial virulence and fitness, including gls24 (a virulence factor important in stress response). We further demonstrated that the E. faecalis RNase J2 deletion mutant is more sensitive to bile salt and greatly attenuated in in vivo organ infection as determined by an IV-sublethal challenge infection mouse model, indicating that E. faecalis RNase J2 plays an important role in E. faecalis virulence

Enterococcus faecalis rnjB Is Required for Pilin Gene Expression and Biofilm Formation ▿

Pili in Gram-positive bacteria play a major role in the colonization of host tissue and in the development of biofilms. They are promising candidates for vaccines or drug targets since they are highly immunogenic and share common structural and functional features among various Gram-positive pathogens. Numerous publications have helped build a detailed understanding of pilus surface assembly, yet regulation of pilin gene expression has not been well defined. Utilizing a monoclonal antibody developed against the Enterococcus faecalis major pilus protein EbpC, we identified mutants from a transposon (Tn) insertion library which lack surface-exposed Ebp pili. In addition to insertions in the ebp regulon, an insertion in ef1184 (dapA) significantly reduced levels of EbpC. Analysis of in-frame dapA deletion mutants and mutants with the downstream gene rnjB deleted further demonstrated that rnjB was responsible for the deficiency of EbpC. Sequence analysis revealed that rnjB encodes a putative RNase J2. Subsequent quantitative real-time PCR (qRT-PCR) and Northern blotting demonstrated that the ebpABC mRNA transcript level was significantly decreased in the rnjB deletion mutant. In addition, using a reporter gene assay, we confirmed that rnjB affects the expression of the ebpABC operon. Functionally, the rnjB deletion mutant was attenuated in its ability to produce biofilm, similar to that of an ebpABC deletion mutant which lacks Ebp pili. Together, these results demonstrate the involvement of rnjB in E. faecalis pilin gene expression and provide insight into a novel mechanism of regulation of pilus production in Gram-positive pathogens

Processing of the major autolysin of E. faecalis, AtlA, by the zinc-metalloprotease, GelE, impacts AtlA septal localization and cell separation.

AtlA is the major peptidoglycan hydrolase of Enterococcus faecalis involved in cell division and cellular autolysis. The secreted zinc metalloprotease, gelatinase (GelE), has been identified as an important regulator of cellular function through post-translational modification of protein substrates. AtlA is a known target of GelE, and their interplay has been proposed to regulate AtlA function. To study the protease-mediated post-translational modification of AtlA, monoclonal antibodies were developed as research tools. Flow cytometry and Western blot analysis suggests that in the presence of GelE, surface-bound AtlA exists primarily as a N-terminally truncated form whereas in the absence of GelE, the N-terminal domain of AtlA is retained. We identified the primary GelE cleavage site occurring near the transition between the T/E rich Domain I and catalytic region, Domain II via N-terminal sequencing. Truncation of AtlA had no effect on the peptidoglycan hydrolysis activity of AtlA. However, we observed that N-terminal cleavage was required for efficient AtlA-mediated cell division while unprocessed AtlA was unable to resolve dividing cells into individual units. Furthermore, we observed that the processed AtlA has the propensity to localize to the cell septum on wild-type cells whereas unprocessed AtlA in the ΔgelE strain were dispersed over the cell surface. Combined, these results suggest that AtlA septum localization and subsequent cell separation can be modulated by a single GelE-mediated N-terminal cleavage event, providing new insights into the post-translation modification of AtlA and the mechanisms governing chaining and cell separation

Primers used in this study

<p>Primers used in this study</p

Growth and surface protein expression comparison of OG1RF and <i>ΔrnjB</i>.

<p>(A) Growth rates of OG1RF and <i>ΔrnjB</i> in BHI broth were determined by monitoring OD₆₀₀. Surface expression of EbpC (B), EbpA (C) and Ace (D) on OG1RF and <i>ΔrnjB</i> were determined by TEM using anti-EbpC mAb 69, anti-EbpA mAb 9, and anti-Ace mAb 70, respectively.</p

Comparison of transcript level of <i>pyrR</i> gene by qRT-PCR.

<p>The relative transcript levels of <i>pyrR</i> in the rnjB deletion mutant and wild-type OG1RF were determined using 23S rRNA as the internal standard. Experiments were performed in triplicate and error bars represent the standard error of the means.</p