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The Staphylococcal Bap proteins sense environmental signals (such as pH, [Ca2+]) to build amyloid scaffold biofilm matrices via unknown mechanisms. We here report the crystal structure of the aggregation‐prone region of Staphylococcusaureus Bap which adopts a dumbbell‐shaped fold. The middle module (MM) connecting the N‐terminal and C‐terminal lobes consists of a tandem of novel double‐Ca2+‐binding motifs involved in cooperative interaction networks, which undergoes Ca2+‐dependent order–disorder conformational switches. The N‐terminal lobe is sufficient to mediate amyloid aggregation through liquid–liquid phase separation and maturation, and subsequent biofilm formation under acidic conditions. Such processes are promoted by disordered MM at low [Ca2+] but inhibited by ordered MM stabilized by Ca2+ binding, with inhibition efficiency depending on structural integrity of the interaction networks. These studies illustrate a novel protein switch in pathogenic bacteria and provide insights into the mechanistic understanding of Bap proteins in modulation of functional amyloid and biofilm formation, which could be implemented in the anti‐biofilm drug design. SYNOPSIS: Staphylococcal Bap protein self‐assembles into functional amyloid aggregates to build the biofilm matrix in response to environmental signals. Here, the crystal structure of aggregation‐prone BSP region of Staphylococcus aureus Bap and biochemical analyses reveal the role of liquid‐liquid phase separation into bacterial functional amyloid formation. The middle module of the dumbbell‐shaped BSP monomer consists of a tandem of novel double‐Ca2+‐binding motifs.The BSP middle module undergoes Ca2+‐dependent disorder‐to‐order conformational switches and pH‐induced conformational changes.The BSP N‐terminal lobe mediates amyloid aggregation through liquid‐liquid phase separation.Individual BSP structural domains cooperate to promote S. aureus biofilm formation under acidic conditions and low Ca2+ concentration. [ABSTRACT FROM AUTHOR]

Summary Two-component systems (TCSs) are key regulatory pathways allowing bacteria to adapt their genetic expression to environmental changes. Bacitracin, a cyclic dodecylpeptide antibiotic, binds to undecaprenyl pyrophosphate, the lipid carrier for cell wall precursors, effectively inhibiting peptidoglycan biosynthesis. We have identified a novel and previously uncharacterized TCS in the major human pathogen Staphylococcus aureus that we show to be essential for bacitracin and nisin resistance: the BraS/BraR system ( Bacitracin resistance associated; SA2417/SA2418). The braRS genes are located immediately upstream from genes encoding an ABC transporter, accordingly designated BraDE. We have shown that the BraSR/BraDE module is a key bacitracin and nisin resistance determinant in S. aureus. In the presence of low antibiotic concentrations, BraSR activate transcription of two operons encoding ABC transporters: braDE and vraDE. We identified a highly conserved imperfect palindromic sequence upstream from the braDE and vraDE promoter sequences, essential for their transcriptional activation by BraSR, suggesting it is the likely BraR binding site. We demonstrated that the two ABC transporters play distinct and original roles in antibiotic resistance: BraDE is involved in bacitracin sensing and signalling through BraSR, whereas VraDE acts specifically as a detoxification module and is sufficient to confer bacitracin and nisin resistance when produced on its own. We show that these processes require functional BraD and VraD nucleotide-binding domain proteins, and that the large extracellular loop of VraE confers its specificity in bacitracin resistance. This is the first example of a TCS associated with two ABC transporters playing separate roles in signal transduction and antibiotic resistance. [ABSTRACT FROM AUTHOR]

The formation of biofilm results in a major lifestyle switch that is thought to affect the expression of multiple genes and operons. We used DNA arrays to study the global effect of biofilm formation on gene expression in mature Escherichia coli K-12 biofilm. We show that, when biofilm is compared with the exponential growth phase, 1.9% of the genes showed a consistent up- or downregulation by a factor greater than two, and that 10% of the E. coli genome is significantly differentially expressed. The functions of the genes induced in these conditions correspond to stress response as well as energy production, envelope biogenesis and unknown functions. We provide evidence that the expression of stress envelope response genes, such as the psp operon or elements of the cpx and rpoE pathways, is a general feature of E. coli mature biofilms. We also compared biofilm with the stationary growth phase and showed that the biofilm lifestyle, although sharing similarities with the stationary growth phase, triggers the expression of specific sets of genes. Using gene disruption of 54 of the most biofilm-induced genes followed by a detailed phenotypic study, we validated the biological relevance of our analysis and showed that 20 of these genes are required for the formation of mature biofilm. This group includes 11 genes of previously unknown function. These results constitute a comprehensive analysis of the global transcriptional response triggered in mature E. coli biofilms and provide insights into its physiological signature. [ABSTRACT FROM AUTHOR]

Summary Staphylococcus aureus biofilm formation is associated with the production of the polysaccharide intercellular adhesin (PIA/PNAG), the product of the ica operon. The staphylococcal accessory regulator, SarA, is a central regulatory element that controls the production of S. aureus virulence factors. By screening a library of Tn917 insertions in a clinical S. aureus strain, we identified SarA as being essential for biofilm development. Non-polar mutations of sar A in four genetically unrelated S. aureus strains decreased PIA/PNAG production and completely impaired biofilm development, both in steady state and flow conditions via an agr -independent mechanism. Accordingly, real-time PCR showed that the mutation in the sar A gene resulted in downregulation of the ica operon transcription. We also demonstrated that complete deletion of σB did not affect PIA/PNAG production and biofilm formation, although it slightly decreased ica operon transcription. Furthermore, the sar A-σB double mutant showed a significant decrease of ica expression but an increase of PIA/PNAG production and biofilm formation compared to the sar A single mutant. We propose that SarA activates S. aureus development of biofilm by both enhancing the ica operon transcription and suppressing the transcription of either a protein involved in the turnover of PIA/PNAG or a repressor of its synthesis, whose expression would be σB -dependent. [ABSTRACT FROM AUTHOR]

Summary We report here a new screening method based on the fluorescence of colonies on calcofluor agar plates to identify transposon insertion mutants of Salmonella enteritidis that are defective in biofilm development. The results not only confirmed the requirement of genes already described for the modulation of multicellular behaviour in Salmonella typhimurium and other species, but also revealed new aspects of the biofilm formation process, such as two new genetic elements, named as bcs ABZC and bcs EFG operons, required for the synthesis of an exopolysaccharide, digestible with cellulase. Non-polar mutations of bcs C and bcs E genes and complementation experiments demonstrated that both operons are respon-sible for cellulose biosynthesis in both S. enteritidis and S. typhimurium . Using two different growth media, ATM and LB, we showed that the biofilm produced by S. enteritidis is made of different constituents, suggesting that biofilm composition and regulation depends on environmental conditions. Bacterial adherence and invasion assays of eukaryotic cells and in vivo virulence studies of cellulose-deficient mutants indicated that, at least under our experimental conditions, the production of cellulose is not involved in the virulence of S. enteritidis . However, cellulose-deficient mutants were more sensitive to chlorine treatments, suggesting that cellulose production and biofilm formation may be an important factor for the survival of S. enteritidis on surface environments. [ABSTRACT FROM AUTHOR]