Search

High-throughput RNA sequencing technology has found the 5′ untranslated region of sarA to contain two putative small RNAs (sRNAs), designated teg49 and teg48. Northern blot analysis disclosed that teg49 and teg48 were detectable within the P3-P1 and P1 sarA promoter regions, respectively. Focusing on teg49, we found that this sRNA, consisting of 196 nucleotides, is transcribed in the same direction as the sarA P3 transcript. The expression of both P3 and teg49 transcripts is dependent on sigB and cshA, which encodes a DEAD box RNA helicase. Within the sRNA teg49, there are two putative hairpin-loop structures, HP1 and HP2. Transversion mutation of the HP1 loop produced a smaller amount of sarA P3 and P2 transcripts and SarA protein than the corresponding HP1 stem and the HP2 stem and loop mutations, leading to lower RNAII transcription and derepression of aur transcription. The HP1 loop mutant also exhibited less biofilm formation than the parental and complemented strains. Complementation with shuttle plasmid pEPSA5 carrying teg49 was able to reestablish sarA P3 and P2 transcription and augment RNAII expression in the HP1 loop mutant. We thus conclude that teg49, embedded within the extended promoter regions of sarA, is modulated by sigB and cshA and plays an important trans-acting role in modulating the transcription and ensuing expression of sarA.

Background: Lung transplantation (LT) is a recognized treatment for end-stage pulmonary disease. Bacteria from the recipient nasopharynx seed the new lungs leading to infections and allograft damage. Understanding the characteristics and topological variations of the microbiota may be important to apprehend the pathophysiology of allograft dysfunction. Objectives: To examine the characteristics and relationship of bacterial compositions between conducting and respiratory zones of the allograft. Methods: We performed 16S rRNA gene sequencing on bronchial aspirates (BAs) and bronchoalveolar lavages (BALs) collected in pairs in 19 patients at several time-points post-LT. Results: The respiratory zone was characterized independently of the time post-LT by a higher bacterial richness than the conducting zone (p = 0.041). The phyla Firmicutes and Proteobacteria dominated both sampling zones, with an inverse correlation between these two phyla (Spearman r = -0.830). Samples of the same pair, as well as pairs from the same individual clustered together (Pseudo-F = 3.8652, p < 0.01). Microbiota of BA and BAL were more closely related in samples from the same patient than each sample type across different patients, with variation in community structure being mainly inter-individual (p < 0.01). Both number of antibiotics administered (p < 0.01) and time interval post-LT (p < 0.01) contributed to the variation in global microbiota structure. Longitudinal analysis of BA-BAL pairs of two patients showed dynamic wave like fluctuations of the microbiota. Conclusions: Our results show that post-transplant respiratory zones harbor higher bacterial richness, but overall similar bacterial profiles as compared to conductive zones. They further support an individual microbial signature following LT.

The critical role of noncoding small RNAs (sRNAs) in the bacterial response to changing conditions is increasingly recognized. However, a specific role for sRNAs during antibiotic exposure has not been investigated in Staphylococcus aureus . Here, we used Illumina RNA-Seq to examine the sRNA response of multiresistant sequence type 239 (ST239) S. aureus after exposure to four antibiotics (vancomycin, linezolid, ceftobiprole, and tigecycline) representing the major classes of antimicrobials used to treat methicillin-resistant S. aureus (MRSA) infections. We identified 409 potential sRNAs and then compared global sRNA and mRNA expression profiles at 2 and 6 h, without antibiotic exposure and after exposure to each antibiotic, for a vancomycin-susceptible strain (JKD6009) and a vancomycin-intermediate strain (JKD6008). Exploration of this data set by multivariate analysis using a novel implementation of nonnegative matrix factorization (NMF) revealed very different responses for mRNA and sRNA. Where mRNA responses clustered with strain or growth phase conditions, the sRNA responses were predominantly linked to antibiotic exposure, including sRNA responses that were specific for particular antibiotics. A remarkable feature of the antimicrobial response was the prominence of antisense sRNAs to genes encoding proteins involved in protein synthesis and ribosomal function. This study has defined a large sRNA repertoire in epidemic ST239 MRSA and shown for the first time that a subset of sRNAs are part of a coordinated transcriptional response to specific antimicrobial exposures in S. aureus . These data provide a framework for interrogating the role of staphylococcal sRNAs in antimicrobial resistance and exploring new avenues for sRNA-based antimicrobial therapies.

Staphylococcus aureus RN4220, a cloning intermediate, is sometimes used in virulence, resistance, and metabolic studies. Using whole-genome sequencing, we showed that RN4220 differs from NCTC8325 and contains a number of genetic polymorphisms that affect both virulence and general fitness, implying a need for caution in using this strain for such studies.

Functional transcriptomics studies have resulted in interesting insights into Staphylococcus aureus diversity and pathogenicity. Here we review the principles, advantages and disadvantages of recent technical developments in the field of transcriptomics and their potential impact on S. aureus research.