Overlapping transcription and bacterial RNA removal

The precise understanding of the biology of a living cell requires the identification and quantification of the molecular components necessary to sustain life. One such element is RNA. Two independent high-throughput strategies are available to identify the entire collection of RNA molecules produced by a cell population, which is currently known as the transcriptome. One technique relies on microarray technology (tiling arrays), whereas the second one relies on sequencing the RNA pool (RNA-seq) (1). Both techniques offer the advantage that the identification of the RNA content is not biased by protein-based genome annotation. The application of these methods to the transcriptome analysis in bacteria has uncovered the existence of a large amount of RNA molecules that overlap at least in some portion with protein-encoding RNA transcripts, generating perfect sense/antisense RNA duplexes (2⇓⇓–5). However, because transcriptome studies have been performed using microgram amounts of RNA purified from millions of bacterial cells instead of RNA purified from a single bacterium, the presence of overlapping sense/antisense RNAs from a genomic region does not necessarily mean that both sense and antisense transcripts are simultaneously present in the same bacteria. Hence, it might be possible that a subgroup in the bacterial population synthesized the sense transcript, another subgroup synthesized the antisense transcript, and consequently overlapping transcripts would never be together in the same cell. A report in PNAS by Lybecker et al. (6) provides clear evidences that both sense and antisense transcripts can be present simultaneously within the same bacterial cell. Using a monoclonal antibody that recognizes double-stranded RNA molecules (dsRNA) irrespectively of the nucleotide sequence, the authors perform immunoprecipitation assays to pull down dsRNA molecules (IP-dsRNA) from a total RNA sample extracted from Escherichia coli, and identified the purified dsRNA by RNA-seq

Amyloid structures as biofilm matrix scaffolds

Recent insights into bacterial biofilm matrix structures have induced a paradigm shift toward the recognition of amyloid fibers as common building block structures that confer stability to the exopolysaccharide matrix. Here we describe the functional amyloid systems related to biofilm matrix formation in both Gram-negative and Gram-positive bacteria and recent knowledge regarding the interaction of amyloids with other biofilm matrix components such as extracellular DNA (eDNA) and the host immune system. In addition, we summarize the efforts to identify compounds that target amyloid fibers for therapeutic purposes and recent developments that take advantage of the amyloid structure to engineer amyloid fibers of bacterial biofilm matrices for biotechnological applications.This work, including the efforts of Jaione Valle, was funded by Ministerio de Economía y Competitividad (MINECO) (AGL2011-23954). This work, including the efforts of Íñigo Lasa, was funded by Ministerio de Economía y Competitividad (MINECO) (BIO2011-30503-C02-02 and BIO2014-53530-R)

The role of ArlRS and VraSR in regulating ceftaroline hypersusceptibility in methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus infections are a global health problem. New control strategies, including fifth-generation cephalosporins such as ceftaroline, have been developed, however rare sporadic resistance has been reported. Our study aimed to determine whether disruption of two-component environmental signal systems detectably led to enhanced susceptibility to ceftaroline in S. aureus CA-MRSA strain MW2 at sub-MIC concentrations where cells normally continue to grow. A collection of sequential mutants in all fifteen S. aureus non-essential two-component systems (TCS) was first screened for ceftaroline sub-MIC susceptibility, using the spot population analysis profile method. We discovered a role for both ArlRS and VraSR TCS as determinants responsible for MW2 survival in the presence of sub-MIC ceftaroline. Subsequent analysis showed that dual disruption of both arlRS and vraSR resulted in a very strong ceftaroline hypersensitivity phenotype. Genetic complementation analysis confirmed these results and further revealed that arlRS and vraSR likely regulate some common pathway(s) yet to be determined. Our study shows that S. aureus uses particular TCS environmental sensing systems for this type of defense and illustrates the proof of principle that if these TCS were inhibited, the efficacy of certain antibiotics might be considerably enhanced.This work was supported by the Swiss National Science Foundation grants (AR 310030-169404), (WLK 10030-146540 and 10030-192784). MV was supported by FNS (Fonds National Suisse) through project funding 10030-146540. The funders had no role in study design, data collection, and interpretation or the decision to submit the work for publication

Functional analysis of intergenic regulatory regions of genes encoding surface adhesins in Staphylococcus aureus isolates from periprosthetic joint infections

Staphylococcus aureus is a leading cause of prosthetic joint infections (PJI). Surface adhesins play an important role in the primary attachment to plasma proteins that coat the surface of prosthetic devices after implantation. Previous efforts to identify a genetic component of the bacterium that confers an enhanced capacity to cause PJI have focused on gene content, kmers, or single-nucleotide polymorphisms (SNPs) in coding sequences. Here, using a collection of S. aureus strains isolated from PJI and wounds, we investigated whether genetic variations in the regulatory region of genes encoding surface adhesins lead to differences in their expression levels and modulate the capacity of S. aureus to colonize implanted prosthetic devices. The data revealed that S. aureus isolates from the same clonal complex (CC) contain a specific pattern of SNPs in the regulatory region of genes encoding surface adhesins. As a consequence, each clonal lineage shows a specific profile of surface proteins expression. Co-infection experiments with representative isolates of the most prevalent CCs demonstrated that some lineages have a higher capacity to colonize implanted catheters in a murine infection model, which correlated with a greater ability to form a biofilm on coated surfaces with plasma proteins. Together, results indicate that differences in the expression level of surface adhesins may modulate the propensity of S. aureus strains to cause PJI. Given the high conservation of surface proteins among staphylococci, our work lays the framework for investigating how diversification at intergenic regulatory regions affects the capacity of S. aureus to colonize the surface of medical implants.This work was financially supported by the Spanish Ministry of Science and Innovation grant PID2020-113494RB-I00 to I.L. (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union), the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 754412 [MoRE2020 - Region Väs-tra Götaland], and the IngaBritt and Arne Lundberg Foundation (LU2021-0048). L.M.L was supported by the European Union's H2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No 801586 (IberusTalent)