Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs.

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Twitching motility-mediated biofilm expansion is a complex, multicellular behavior that enables the active colonization of surfaces by many species of bacteria. In this study we have explored the emergence of intricate network patterns of interconnected trails that form in actively expanding biofilms of Pseudomonas aeruginosa. We have used high-resolution, phase-contrast time-lapse microscopy and developed sophisticated computer vision algorithms to track and analyze individual cell movements during expansion of P. aeruginosa biofilms. We have also used atomic force microscopy to examine the topography of the substrate underneath the expanding biofilm. Our analyses reveal that at the leading edge of the biofilm, highly coherent groups of bacteria migrate across the surface of the semisolid media and in doing so create furrows along which following cells preferentially migrate. This leads to the emergence of a network of trails that guide mass transit toward the leading edges of the biofilm. We have also determined that extracellular DNA (eDNA) facilitates efficient traffic flow throughout the furrow network by maintaining coherent cell alignments, thereby avoiding traffic jams and ensuring an efficient supply of cells to the migrating front. Our analyses reveal that eDNA also coordinates the movements of cells in the leading edge vanguard rafts and is required for the assembly of cells into the "bulldozer" aggregates that forge the interconnecting furrows. Our observations have revealed that large-scale self-organization of cells in actively expanding biofilms of P. aeruginosa occurs through construction of an intricate network of furrows that is facilitated by eDNA.collective behavior | t4p | type IV pili | tfp | swarming

Background The recent resurgence of invasive group A streptococcal disease has been paralleled by the emergence of the M1T1 clone. Recently, invasive disease initiation to has been linked to mutations in the covR/S two-compnent regulator. Here we investigate if a fitness cost is associated with covS mutation that counterbalances hypervirulence. Methods Wild-type M1T1 GAS and an isogenic covS mutant derived from animal passage were compared for adherence to human laryngeal epithelial cells, keratinocytes or fibronectin, biofilm formation, and binding to intact mouse skin. Targeted mutagenesis of capsule expression from both strains was performed for analysis of its unique contribution to the observed phenotypes. Results The covS mutant bacteria showed reduced capacity to bind to epithelial cell layers as a consequence of increased capsule expression. The covS mutant strain also had reduced capacity to bind fibronectin and to form biofilms on plastic and epithelial cell layers. A defect in skin adherence of the covS mutant strain was demonstrated in a murine model. Conclusions Reduced colonization capacity provides a potential explanation as to why the covS mutation conferring hypervirulence has not become fixed in the globally-disseminated M1T1 GAS clone, but rather may arise anew under innate immune selection in individual patients.

bThe Gram-negative human pathogen Pseudomonas aeruginosa tolerates high concentrations of ␤-lactam antibiotics. Despite inhibiting the growth of the organism, these cell wall-targeting drugs exhibit remarkably little bactericidal activity. However, the mechanisms underlying ␤-lactam tolerance are currently unclear. Here, we show that P. aeruginosa undergoes a rapid en masse transition from normal rod-shaped cells to viable cell wall-defective spherical cells when treated with ␤-lactams from the widely used carbapenem and penicillin classes. When the antibiotic is removed, the entire population of spherical cells quickly converts back to the normal bacillary form. Our results demonstrate that these rapid population-wide cell morphotype transitions function as a strategy to survive antibiotic exposure. Taking advantage of these findings, we have developed a novel approach to efficiently kill P. aeruginosa by using carbapenem treatment to induce en masse transition to the spherical cell morphotype and then exploiting the relative fragility and sensitivity of these cells to killing by antimicrobial peptides (AMPs) that are relatively inactive against P. aeruginosa bacillary cells. This approach could broaden the repertoire of antimicrobial compounds used to treat P. aeruginosa and serve as a basis for developing new therapeutic agents to combat bacterial infections. P seudomonas aeruginosa is a major human pathogen and a leading cause of hospital-acquired infections. P. aeruginosa infections are difficult to eradicate and are often fatal, which is in part due to the organism's high intrinsic resistance to a variety of different antimicrobials (1). The mechanisms underlying intrinsic antibiotic resistance in P. aeruginosa are largely well understood. However, an important and as yet unexplained observation is the ability of P. aeruginosa to survive in the presence of high concentrations of the cell wall-targeting ␤-lactam antibiotics.␤-Lactams are a broad class of antibiotics that include penicillin derivatives, cephalosporins, monobactams, and carbapenems. They are the most widely used group of antibiotics in the world (2) and mediate bacterial killing primarily by inhibiting the enzymes, known as penicillin-binding proteins (PBPs), that catalyze the formation of peptidoglycan cross-links in the bacterial cell wall (3). ␤-Lactam drugs typically exhibit bactericidal activity against susceptible organisms (4), and this is often associated with changes in bacterial morphology and the formation of spherical or filamentous cells that are prone to lysis (3,(5)(6)(7)(8).Interestingly, despite inhibiting cell growth, ␤-lactams have been found to have very little bactericidal activity against P. aeruginosa (9-13). This intrinsic tolerance of ␤-lactams likely accounts for the fact that when P. aeruginosa infections are treated, optimal microbiological outcomes occur when ␤-lactam concentrations are maintained at 4 to 6.6 times the MIC throughout the majority of the dosing period (13). Furthermore, ␤-lactam tolerance by P. aer...