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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.

SummaryGram-negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1-dependent manner to Gramnegative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram-negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gramnegative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF-kB and NOD1-dependent responses in vitro. These OMVs entered epithelial cells through lipid rafts thereby inducing NOD1-dependent responses in vitro. Moreover, OMVs delivered intragastrically to miceinduced innate and adaptive immune responses via a NOD1-dependent but TLR-independent mechanism. Collectively, our findings identify OMVs as a generalized mechanism whereby Gramnegative bacteria deliver peptidoglycan to cytosolic NOD1. We propose that OMVs released by bacteria in vivo may promote inflammation and pathology in infected hosts.

Erythrocyte invasion by the merozoite is an obligatory stage in Plasmodium parasite infection and essential to malaria disease progression. Attempts to study this process have been hindered by the poor invasion synchrony of merozoites from the only in vitro culture-adapted human malaria parasite, Plasmodium falciparum. Using fluorescence, three-dimensional structured illumination, and immunoelectron microscopy of filtered merozoites, we analyze cellular and molecular events underlying each discrete step of invasion. Monitoring the dynamics of these events revealed that commitment to the process is mediated through merozoite attachment to the erythrocyte, triggering all subsequent invasion events, which then proceed without obvious checkpoints. Instead, coordination of the invasion process involves formation of the merozoite-erythrocyte tight junction, which acts as a nexus for rhoptry secretion, surface-protein shedding, and actomyosin motor activation. The ability to break down each molecular step allows us to propose a comprehensive model for the molecular basis of parasite invasion.

Tubulin is a major component of the eukaryotic cytoskeleton, controlling cell shape, structure and dynamics, whereas its bacterial homolog FtsZ establishes the cytokinetic ring that constricts during cell division 1,2 . How such different roles of tubulin and FtsZ evolved is unknown. Archaea may hold clues as these organisms share characteristics with Eukarya and Bacteria 3 . Here we report the structure and function of proteins from a distinct family related to tubulin and FtsZ, named CetZ, which co-exists with FtsZ in many archaea. CetZ crystal structures showed the FtsZ/ tubulin superfamily fold, and one crystal form contained sheets of protofilaments, suggesting a structural role. However, inactivation of the CetZs in Haloferax volcanii did not affect cell division. Instead, CetZ1 was required for differentiation of the irregular plate-shaped cells into a rod-shaped cell type that was essential for normal swimming motility. CetZ1 formed dynamic cytoskeletal structures in vivo, relating to its capacity to remodel the cell envelope and direct rod formation. CetZ2 was also implicated in H. volcanii cell shape control. Our findings expand the known roles of the FtsZ/tubulin superfamily to include archaeal cell shape dynamics, suggesting that a cytoskeletal role might predate eukaryotic cell evolution, and they support the premise that a major function of microbial rod-shape is to facilitate swimming.Many archaea have FtsZ that appears to function in cell division 4-8 . However, unlike bacteria, archaeal genomes frequently contain additional genes belonging to the FtsZ/tubulin superfamily 9 . These genes are abundant in the haloarchaea, which dominate hyper-saline lakes globally 10 and are generally noted for their unusual flattened cell morphologies.

When grown as a biofilm in laboratory flow chambers Pseudomonas aeruginosa can develop mushroom-shaped multicellular structures consisting of distinct subpopulations in the cap and stalk portions. We have previously presented evidence that formation of the cap portion of the mushroom-shaped structures in P. aeruginosa biofilms occurs via bacterial migration and depends on type IV pili (Mol Microbiol 50: 61-68). In the present study we examine additional factors involved in the formation of this multicellular substructure. While pilA mutants, lacking type IV pili, are deficient in mushroom cap formation, pilH and chpA mutants, which are inactivated in the type IV pili-linked chemosensory system, showed only minor defects in cap formation. On the contrary, fliM mutants, which are non-flagellated, and cheY mutants, which are inactivated in the flagellum-linked chemotaxis system, were largely deficient in cap formation. Experiments involving DNase treatment of developing biofilms provided evidence that extracellular DNA plays a role in cap formation. Moreover, mutants that are deficient in quorum sensing-controlled DNA release formed microcolonies upon which wild-type bacteria could not form caps. These results constitute evidence that type IV pili, flagellum-mediated motility and quorum sensing-controlled DNA release are involved in the formation of mature multicellular structures in P. aeruginosa biofilms.

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices.

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