BUSCADOR RECOLECTA

Functional amyloids are considered as common building block structures of the biofilm matrix in different bacteria. In previous work, we have shown that the staphylococcal surface protein Bap, a member of the Biofilm-Associated Proteins (BAP) family, is processed and the fragments containing the N-terminal region become aggregation-prone and self-assemble into amyloid-like structures. Here, we report that Esp, a Bap-orthologous protein produced by Enterococcus faecalis, displays a similar amyloidogenic behavior. We demonstrate that at acidic pH the N-terminal region of Esp forms aggregates with an amyloid-like conformation, as evidenced by biophysical analysis and the binding of protein aggregates to amyloid-indicative dyes. Expression of a chimeric protein, with its Esp N-terminal domain anchored to the cell wall through the R domain of clumping factor A, showed that the Esp N-terminal region is sufficient to confer multicellular behavior through the formation of an extracellular amyloid-like material. These results suggest that the mechanism of amyloid-like aggregation to build the biofilm matrix might be widespread among BAP-like proteins. This amyloid-based mechanism may not only have strong relevance for bacteria lifestyle but could also contribute to the amyloid burden to which the human physiology is potentially exposed.

Staphylococcus aureus clinical strains are able to produce at least two distinct types of biofilm matrixes: biofilm matrixes made of the polysaccharide intercellular adhesin (PIA) or poly-N-acetylglucosamine (PNAG), whose synthesis is mediated by the icaADBC locus, and biofilm matrixes built of proteins (polysaccharide independent). σB is a conserved alternative sigma factor that regulates the expression of more than 100 genes in response to changes in environmental conditions. While numerous studies agree that σB is required for polysaccharide-independent biofilms, controversy persists over the role of σB in the regulation of PIA/PNAG-dependent biofilm development. Here, we show that genetically unrelated S. aureus σB-deficient strains produced stronger biofilms under both static and flow conditions and accumulated higher levels of PIA/PNAG exopolysaccharide than their corresponding wild-type strains. The increased accumulation of PIA/PNAG in the σB mutants correlated with a greater accumulation of the IcaC protein showed that it was not due to adjustments in icaADBC operon transcription and/or icaADBC mRNA stability. Overall, our results reveal that in the presence of active σB, the turnover of Ica proteins is accelerated, reducing the synthesis of PIA/PNAG exopolysaccharide and consequently the PIA/PNAG-dependent biofilm formation capacity., Work in the Laboratory of Microbial Pathogenesis is funded by the Spanish Ministry of Science, Innovation and Universities (grants SAF2015-74267-JIN and BIO2017-83035-R [Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union])., Peer reviewed

Bacterial genes are typically grouped into operons defined as clusters of adjacent genes encoding for proteins that fill related roles and are transcribed into a single polycistronic mRNA molecule. This simple organization provides an efficient mechanism to coordinate the expression of neighboring genes and is at the basis of gene regulation in bacteria. Here, we report the existence of a higher level of organization in operon structure that we named noncontiguous operon and consists in an operon containing a gene(s) that is transcribed in the opposite direction to the rest of the operon. This transcriptional architecture is exemplified by the genes menE-menC-MW1733-ytkD-MW1731 involved in menaquinone synthesis in the major human pathogen Staphylococcus aureus. We show that menE-menC-ytkD-MW1731 genes are transcribed as a single transcription unit, whereas the MW1733 gene, located between menC and ytkD, is transcribed in the opposite direction. This genomic organization generates overlapping transcripts whose expression is mutually regulated by transcriptional interference and RNase III processing at the overlapping region. In light of our results, the canonical view of operon structure should be revisited by including this operon arrangement in which cotranscription and overlapping transcription are combined to coordinate functionally related gene expression., This work was supported by the Spanish Ministry of Economy and
Competitiveness Grants BIO2014-53530-R and BIO2017-83035-R (Agencia
Española de Investigación/Fondo Europeo de Desarrollo Regional, European
Union). A.T.-A. is supported by the European Research Council under the
European Union’s Horizon 2020 research and innovation programme Grant
Agreement 646869., Peer reviewed

In the last decade, the implementation of high-throughput methods for RNA profiling has uncovered that a large part of the bacterial genome is transcribed well beyond the boundaries of known genes. Therefore, the transcriptional space of a gene very often invades the space of a neighbouring gene, creating large regions of overlapping transcription. The biological significance of these findings was initially regarded with scepticism. However, mounting evidence suggests that overlapping transcription between neighbouring genes conforms to regulatory purposes and provides new strategies for coordinating bacterial gene expression. In this MicroReview, considering the discoveries made in a pioneering transcriptome analysis performed on Listeria monocytogenes as a starting point, we discuss the progress in understanding the biological meaning of overlapping transcription that has given rise to the excludon concept. We also discuss new conditional transcriptional termination events that create antisense RNAs depending on the metabolite concentrations and new genomic arrangements, known as noncontiguous operons, which contain an interspersed gene that is transcribed in the opposite direction to the rest of the operon., This review is a tribute to part of the research legacy of P. Cossart's lab, to whom the authors have had the honour of belonging. This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (ERC Consolidator Grant Agreement No. 646869) to A.T‐A. and Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union (BIO2017‐83035‐R) to I.L.

Functional amyloids are considered as common building block structures of the biofilm matrix in different bacteria. In previous work, we have shown that the staphylococcal surface protein Bap, a member of the Biofilm-Associated Proteins (BAP) family, is processed and the fragments containing the N-terminal region become aggregation-prone and self-assemble into amyloid-like structures. Here, we report that Esp, a Bap-orthologous protein produced by Enterococcus faecalis, displays a similar amyloidogenic behavior. We demonstrate that at acidic pH the N-terminal region of Esp forms aggregates with an amyloid-like conformation, as evidenced by biophysical analysis and the binding of protein aggregates to amyloid-indicative dyes. Expression of a chimeric protein, with its Esp N-terminal domain anchored to the cell wall through the R domain of clumping factor A, showed that the Esp N-terminal region is sufficient to confer multicellular behavior through the formation of an extracellular amyloid-like material. These results suggest that the mechanism of amyloid-like aggregation to build the biofilm matrix might be widespread among BAP-like proteins. This amyloid-based mechanism may not only have strong relevance for bacteria lifestyle but could also contribute to the amyloid burden to which the human physiology is potentially exposed., This research was supported by grants RTI2018-096011-B-I00 and BIO2017-83035-R from the Spanish Ministry of Science, Innovation and Universities, and Proyecto Intramural Incorporación-2018 CSIC.

One of the major components of the staphylococcal biofilm is surface proteins that assemble as scaffold components of the biofilm matrix. Among the different surface proteins able to contribute to biofilm formation, this review is dedicated to the Biofilm Associated Protein (Bap). Bap is part of the accessory genome of Staphylococcus aureus but orthologs of Bap in other staphylococcal species belong to the core genome. When present, Bap promotes adhesion to abiotic surfaces and induces strong intercellular adhesion by self-assembling into amyloid like aggregates in response to the levels of calcium and the pH in the environment. During infection, Bap enhances the adhesion to epithelial cells where it binds directly to the host receptor Gp96 and inhibits the entry of the bacteria into the cells. To perform such diverse range of functions, Bap comprises several domains, and some of them include several motifs associated to distinct functions. Based on the knowledge accumulated with the Bap protein of S. aureus, this review aims to summarize the current knowledge of the structure and properties of each domain of Bap and their contribution to Bap functionality., Work in the laboratories of JV and IL is supported by grants RTI2018-096011-B-I00 and BIO2017-83035-R from Spanish Ministry of Science, Innovation and Universities. Work in the laboratory of XF is supported by grants from the National Natural Science Foundation of China (No. 31872712) and the Beijing Advanced Innovation Center for Structural Biology., Peer reviewed

Two-component systems (TCS) are modular signal transduction pathways that allow cells to adapt to prevailing environmental conditions by modifying cellular physiology. Staphylococcus aureus has 16 TCSs to adapt to the diverse microenvironments encountered during its life cycle, including host tissues and implanted medical devices. S. aureus is particularly prone to cause infections associated to medical devices, whose surfaces coated by serum proteins constitute a particular environment. Identification of the TCSs involved in the adaptation of S. aureus to colonize and survive on the surface of implanted devices remains largely unexplored. Here, using an in vivo catheter infection model and a collection of mutants in each non-essential TCS of S. aureus, we investigated the requirement of each TCS for colonizing the implanted catheter. Among the 15 mutants in non-essential TCSs, the arl mutant exhibited the strongest deficiency in the capacity to colonize implanted catheters. Moreover, the arl mutant was the only one presenting a major deficit in PNAG production, the main exopolysaccharide of the S. aureus biofilm matrix whose synthesis is mediated by the icaADBC locus. Regulation of PNAG synthesis by ArlRS occurred through repression of IcaR, a transcriptional repressor of icaADBC operon expression. Deficiency in catheter colonization was restored when the arl mutant was complemented with the icaADBC operon. MgrA, a global transcriptional regulator downstream ArlRS that accounts for a large part of the arlRS regulon, was unable to restore PNAG expression and catheter colonization deficiency of the arlRS mutant. These findings indicate that ArlRS is the key TCS to biofilm formation on the surface of implanted catheters and that activation of PNAG exopolysaccharide production is, among the many traits controlled by the ArlRS system, a major contributor to catheter colonization., JV was supported by SAF2015-74267-JIN. This research was supported by grants BIO2014-53530-R, BIO2017-83035-R and RTC-2015-3184-1 from the Spanish Ministry of Economy and Competitivity.

© 2021 Dorado-Morales et al., Plasmids have largely contributed to the spread of antimicrobial resistance genes among Staphylococcus strains. Knowledge about the fitness cost that plasmids confer on clinical staphylococcal isolates and the coevolutionary dynamics that drive plasmid maintenance is still scarce. In this study, we aimed to analyze the initial fitness cost of plasmids in the bacterial pathogen Staphylococcus aureus and the plasmid-host adaptations that occur over time. For that, we first designed a CRISPR (clustered regularly interspaced palindromic repeats)-based tool that enables the removal of native S. aureus plasmids and then transferred three different plasmids isolated from clinical S. aureus strains to the same-background clinical cured strain. One of the plasmids, pUR2940, obtained from a livestock-associated methicillin-resistant S. aureus (LA-MRSA) ST398 strain, imposed a significant fitness cost on both its native and the new host. Experimental evolution in a nonselective medium resulted in a high rate pUR2940 loss and selected for clones with an alleviated fitness cost in which compensatory adaptation occurred via deletion of a 12.8-kb plasmid fragment, contained between two ISSau10 insertion sequences and harboring several antimicrobial resistance genes. Overall, our results describe the relevance of plasmid-borne insertion sequences in plasmid rearrangement and maintenance and suggest the potential benefits of reducing the use of antibiotics both in animal and clinical settings for the loss of clinical multidrug resistance plasmids., This work was financially supported by the Spanish Ministry of Science, Innovation and Universities grants BIO2014-53530-R and BIO2017-83035-R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I.L. and C.S. P.D.-M. was supported by an F.P.I. (BES-2015-072859) contract from the Spanish Ministry of Science, Innovation and Universities.

Bacterial genomes are pervasively transcribed, generating a wide variety of antisense
RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the
transcription termination process. Previous transcriptome analyses revealed that the lexA gene from
Staphylococcus aureus, which encodes the main SOS response regulator, is affected by the presence
of an asRNA. Here, we show that the lexA antisense RNA (lexA-asRNA) is generated by a TRE
on the intrinsic terminator (TTsbrB) of the sbrB gene, which is located downstream of lexA, in the
opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TTsbrB
structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in
the hairpin of TTsbrB prevented lexA-asRNA transcription. The level of lexA-asRNA directly correlated
with cellular stress since the expressions of sbrB and lexA-asRNA depend on the stress transcription
factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TTsbrB,
suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis
of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections
with downstream genes and, ultimately, transcriptomic variability among S. aureus strains., This work was supported by the European Research Council (ERC) under the European
Union’s Horizon 2020 research and innovation program (grant No. 646869 to A.T.-A.) and by the
Spanish Ministry of Science and Innovation grants (BIO2017-83035-R to I.L. and PID2019-105216GBI00
to A.T.-A.). Funding for open access charge was provided by the CSIC Open Access Publication
Support Initiative, Unit of Information Resources for Research (URICI).