Project description:UnlabelledOver the course of the last 3 decades the role of the second messenger cyclic di-GMP (c-di-GMP) as a master regulator of bacterial physiology was determined. Although the control over c-di-GMP levels via synthesis and breakdown and the allosteric regulation of c-di-GMP over receptor proteins (effectors) and riboswitches have been extensively studied, relatively few effectors have been identified and most are of unknown functions. The obligate predatory bacterium Bdellovibrio bacteriovorus has a peculiar dimorphic life cycle, in which a phenotypic transition from a free-living attack phase (AP) to a sessile, intracellular predatory growth phase (GP) is tightly regulated by specific c-di-GMP diguanylate cyclases. B. bacteriovorus also bears one of the largest complement of defined effectors, almost none of known functions, suggesting that additional proteins may be involved in c-di-GMP signaling. In order to uncover novel c-di-GMP effectors, a c-di-GMP capture-compound mass-spectroscopy experiment was performed on wild-type AP and host-independent (HI) mutant cultures, the latter serving as a proxy for wild-type GP cells. Eighty-four proteins were identified as candidate c-di-GMP binders. Of these proteins, 65 did not include any recognized c-di-GMP binding site, and 3 carried known unorthodox binding sites. Putative functions could be assigned to 59 proteins. These proteins are included in metabolic pathways, regulatory circuits, cell transport, and motility, thereby creating a potentially large c-di-GMP network. False candidate effectors may include members of protein complexes, as well as proteins binding nucleotides or other cofactors that were, respectively, carried over or unspecifically interacted with the capture compound during the pulldown. Of the 84 candidates, 62 were found to specifically bind the c-di-GMP capture compound in AP or in HI cultures, suggesting c-di-GMP control over the whole-cell cycle of the bacterium. High affinity and specificity to c-di-GMP binding were confirmed using microscale thermophoresis with a hypothetical protein bearing a PilZ domain, an acyl coenzyme A dehydrogenase, and a two-component system response regulator, indicating that additional c-di-GMP binding candidates may be bona fide novel effectors.ImportanceIn this study, 84 putative c-di-GMP binding proteins were identified in B. bacteriovorus, an obligate predatory bacterium whose lifestyle and reproduction are dependent on c-di-GMP signaling, using a c-di-GMP capture compound precipitation approach. This predicted complement covers metabolic, energy, transport, motility and regulatory pathways, and most of it is phase specific, i.e., 62 candidates bind the capture compound at defined modes of B. bacteriovorus lifestyle. Three of the putative binders further demonstrated specificity and high affinity to c-di-GMP via microscale thermophoresis, lending support for the presence of additional bona fide c-di-GMP effectors among the pulled-down protein repertoire.

Project description:Bdellovibrio bacteriovorus is a small Gram-negative, obligate predatory bacterium that is largely found in wet, aerobic environments (e.g., soil). This bacterium attacks and invades other Gram-negative bacteria, including animal and plant pathogens. The intriguing life cycle of B. bacteriovorus consists of two phases: a free-living nonreplicative attack phase, in which the predatory bacterium searches for its prey, and a reproductive phase, in which B. bacteriovorus degrades a host's macromolecules and reuses them for its own growth and chromosome replication. Although the cell biology of this predatory bacterium has gained considerable interest in recent years, we know almost nothing about the dynamics of its chromosome replication. Here, we performed a real-time investigation into the subcellular localization of the replisome(s) in single cells of B. bacteriovorus Our results show that in B. bacteriovorus, chromosome replication takes place only during the reproductive phase and exhibits a novel spatiotemporal arrangement of replisomes. The replication process starts at the invasive pole of the predatory bacterium inside the prey cell and proceeds until several copies of the chromosome have been completely synthesized. Chromosome replication is not coincident with the predator cell division, and it terminates shortly before synchronous predator filament septation occurs. In addition, we demonstrate that if this B. bacteriovorus life cycle fails in some cells of Escherichia coli, they can instead use second prey cells to complete their life cycle.IMPORTANCE New strategies are needed to combat multidrug-resistant bacterial infections. Application of the predatory bacterium Bdellovibrio bacteriovorus, which kills other bacteria, including pathogens, is considered promising for combating bacterial infections. The B. bacteriovorus life cycle consists of two phases, a free-living, invasive attack phase and an intracellular reproductive phase, in which this predatory bacterium degrades the host's macromolecules and reuses them for its own growth. To understand the use of B. bacteriovorus as a "living antibiotic," it is first necessary to dissect its life cycle, including chromosome replication. Here, we present a real-time investigation into subcellular localization of chromosome replication in a single cell of B. bacteriovorus This process initiates at the invasion pole of B. bacteriovorus and proceeds until several copies of the chromosome have been completely synthesized. Interestingly, we demonstrate that some cells of B. bacteriovorus require two prey cells sequentially to complete their life cycle.

Dataset Information

Quinolone Signals Related to Pseudomonas Quinolone Signal-Quorum Sensing Inhibits the Predatory Activity of Bdellovibrio bacteriovorus.

Publications

Quinolone Signals Related to <i>Pseudomonas</i> Quinolone Signal-Quorum Sensing Inhibits the Predatory Activity of <i>Bdellovibrio bacteriovorus</i>.

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