Rational probe design for efficient rRNA depletion and improved metatranscriptomic analysis of human microbiomes.

The microbiota that colonize the human gut and other tissues are dynamic, varying both in composition and functional state between individuals and over time. Gene expression measurements can provide insights into microbiome composition and function. However, efficient and unbiased removal of microbial ribosomal RNA (rRNA) presents a barrier to acquiring metatranscriptomic data. Here we describe a probe set that achieves efficient enzymatic rRNA removal of complex human-associated microbial communities. We demonstrate that the custom probe set can be further refined through an iterative design process to efficiently deplete rRNA from a range of human microbiome samples. Using synthetic nucleic acid spike-ins, we show that the rRNA depletion process does not introduce substantial quantitative error in gene expression profiles. Successful rRNA depletion allows for efficient characterization of taxonomic and functional profiles, including during the development of the human gut microbiome. The pan-human microbiome enzymatic rRNA depletion probes described here provide a powerful tool for studying the transcriptional dynamics and function of the human microbiome.

Rifampicin potentiation of aminoglycoside activity against cystic fibrosis isolates of Pseudomonas aeruginosa.

OBJECTIVES: Rifampicin potentiates the activity of aminoglycosides (AGs) versus Pseudomonas aeruginosa by targeting the AmgRS two-component system. In this study we examine the impact of rifampicin on the AG susceptibility of cystic fibrosis (CF) lung isolates of P. aeruginosa and the contribution of AmgRS to AG resistance in these isolates. METHODS: amgR deletion derivatives of clinical isolates were constructed using standard gene replacement technology. Susceptibility to AGs ± rifampicin (at ½ MIC) was assessed using a serial 2-fold dilution assay. RESULTS: Rifampicin showed a variable ability to potentiate AG activity versus the CF isolates, enhancing AG susceptibility between 2- and 128-fold. Most strains showed potentiation for at least two AGs, with only a few strains showing no AG potentiation by rifampicin. Notably, loss of amgR increased AG susceptibility although rifampicin potentiation of AG activity was still observed in the ΔamgR derivatives. CONCLUSIONS: AmgRS contributes to AG resistance in CF isolates of P. aeruginosa and rifampicin shows a variable ability to potentiate AG activity against these, highlighting the complexity of AG resistance in such isolates.

EsrC, an envelope stress-regulated repressor of the mexCD-oprJ multidrug efflux operon in Pseudomonas aeruginosa.

mexCD-oprJ is an envelope stress-inducible multidrug efflux operon of Pseudomonas aeruginosa. A gene encoding a homologue of the NfxB repressor of this operon, PA4596, occurs downstream of oprJ and was proposed as a second repressor of this efflux operon. Inactivation of this gene had no impact on mexCD-oprJ expression in cells not exposed to envelope stress although its loss under envelope stress conditions yielded a > 10-fold increase in mexCD-oprJ expression. Consistent with PA4596 functioning as a mexCD-oprJ repressor, the purified protein was able to bind to a DNA fragment carrying the mexCD-oprJ promoter region. Expression of PA4596 was induced under conditions of envelope stress dependent on the AlgU envelope stress sigma factor, consistent with PA4596 operating under envelope stress conditions where it possibly serves to moderate envelope stress-inducible mexCD-oprJ expression. nfxB mutants showed elevated PA4596 expression and purified NfxB bound to DNA encompassing the PA4596 upstream region, an indication that NfxB functions as a repressor of PA4596 expression. Elimination of PA4596 in P. aeruginosa lacking nfxB and hyperexpressing mexCD-oprJ had no additional impact on mexCD-oprJ expression, regardless of the presence of envelope stress, suggesting that PA4596 repressor activity may be dependent on NfxB. This envelope stress-regulated repressor of mexCD-oprJ has been renamed esrC.