Project description:Early electron microscopy and more recent studies in our laboratory of Bdellovibrio bacteriovorus cells indicated the presence of narrow fibers at the nonflagellar pole of this unusual predatory bacterium. Analysis of the B. bacteriovorus HD100 genome showed a complete set of genes potentially encoding type IV pili and an incomplete gene set for Flp pili; therefore, the role of type IV pili in the predatory life cycle of B. bacteriovorus HD100 was investigated. Alignment of the predicted PilA protein with known type IV pilins showed the characteristic conserved N terminus common to type IVa pilins. The pilA gene, encoding the type IV pilus fiber protein, was insertionally inactivated in multiple Bdellovibrio replicate cultures, and the effect upon the expression of other pilus genes was monitored by reverse transcriptase PCR. Interruption of pilA in replicate isolates abolished Bdellovibrio predatory capability in liquid prey cultures and on immobilized yellow fluorescent protein-labeled prey, but the mutants could be cultured prey independently. Expression patterns of pil genes involved in the formation of type IV pili were profiled across the predatory life cycle from attack phase predatory Bdellovibrio throughout the intraperiplasmic bdelloplast stages to prey lysis and in prey-independent growth. Taken together, the data show that type IV pili play a critical role in Bdellovibrio predation.
Project description:Bdellovibrio bacteriovorus bacteria are predatory organisms that attack other gram-negative bacteria. Here, we report that Bd0714 is a Nudix dGTPase from B. bacteriovorus HD100 with a substrate specificity similar to that of Escherichia coli MutT and complements an E. coli mutT-deficient strain. We observed different transcription levels of the gene throughout the predator life cycle.
Project description:Attack-Phase Bdellovibrio bacteriovorus Responses to Extracellular Nutrients Are Analogous to Those Seen During Late Intraperiplasmic Growth
Project description:Bdellovibrio bacteriovorus HD100 is an obligate predatory bacterium that attacks and invades Gram-negative bacteria. The predator requires living bacteria to survive as growth and replication take place inside the bacterial prey. It is possible to isolate mutants that grow and replicate outside prey bacteria. Such mutants are designated host or prey independent, and their nutritional requirements vary. Some mutants are saprophytic and require prey extracts for extracellular growth, whereas other mutants grow axenically, which denotes the formation of colonies on complete medium in the absence of any prey components. The initial events leading to prey-independent growth are still under debate, and several genes may be involved. We selected new mutants by three different methods: spontaneous mutation, transposon mutagenesis, and targeted gene knockout. By all approaches we isolated mutants of the hit (host interaction) locus. As the relevance of this locus for the development of prey independence has been questioned, we performed whole-genome sequencing of five prey-independent mutants. Three mutants were saprophytic, and two mutants could grow axenically. Whole-genome analysis revealed that the mutation of a small open reading frame of the hit locus is sufficient for the conversion from predatory to saprophytic growth. Complementation experiments were performed by introduction of a plasmid carrying the wild-type hit gene into saprophytic mutants, and predatory growth could be restored. Whole-genome sequencing of two axenic mutants demonstrated that in addition to the hit mutation the colony formation on complete medium was shown to be influenced by the mutations of two genes involved in RNA processing. Complementation experiments with a wild-type gene encoding an RNA helicase, RhlB, abolished the ability to form colonies on complete medium, indicating that stability of RNA influences axenic growth.
Project description:Predation of Chromobacterium piscinae by Bdellovibrio bacteriovorus HD100 was inhibited in dilute nutrient broth (DNB) but not in HEPES. Experiments showed that the effector responsible was present in the medium, as cell-free supernatants retained the ability to inhibit predation, and that the effector was not toxic to B. bacteriovorus Violacein, a bisindole secondary metabolite produced by C. piscinae, was not responsible. Further characterization of C. piscinae found that this species produces sufficient concentrations of cyanide (202 µM) when grown in DNB to inhibit the predatory activity of B. bacteriovorus, but that in HEPES, the cyanide concentrations were negligible (19 µM). The antagonistic role of cyanide was further confirmed, as the addition of hydroxocobalamin, which chelates cyanide, allowed predation to proceed. The activity of cyanide against B. bacteriovorus was found to be twofold, depending on the life cycle stage of this predator. For the attack-phase predatory cells, cyanide caused the cells to lose motility and tumble, while for intraperiplasmic predators, development and lysis of the prey cell were halted. These findings suggest that cyanogenesis in nature may be employed by the bacterial strains that produce this compound to prevent and reduce their predation by B. bacteriovorusIMPORTANCE Bacterial predators actively attack, kill, and enter the periplasm of susceptible Gram-negative bacteria, where they consume the prey cell components. To date, the activity of B. bacteriovorus HD100 has been demonstrated against more than 100 human pathogens. As such, this strain and others are being considered as potential alternatives or supplements to conventional antibiotics. However, the production of secondary metabolites by prey bacteria is known to mitigate, and even abolish, predation by bacterivorous nematodes and protists. With the exception of indole, which was shown to inhibit predation, the effects of bacterial secondary metabolites on B. bacteriovorus and its activities have not been considered. Consequently, we undertook this study to better understand the mechanisms that bacterial strains employ to inhibit predation by B. bacteriovorus HD100. We report here that cyanogenic bacterial strains can inhibit predation and show that cyanide affects both attack-phase predators and those within prey, i.e., in the bdelloplast.
Project description:Bdellovibrio bacteriovorus 109J is a predatory bacterium which lives by predating on other Gram-negative bacteria to obtain the nutrients it needs for replication and survival. Here, we evaluated the effects a bacterial signaling molecule called diffusible signaling factor (DSF) have on B. bacteriovorus 109J transcriptomics.
Project description:Bdellovibrio, δ-proteobacteria, including B. bacteriovorus (Bba) and B. exovorus (Bex), are obligate predators of other Gram-negative bacteria. While Bba grows in the periplasm of the prey cell, Bex grows externally. We have analyzed and compared the transport proteins of these 2 organisms based on the current contents of the Transporter Classification Database (TCDB; www.tcdb.org). Bba has 103 transporters more than Bex, 50% more secondary carriers, and 3 times as many MFS carriers. Bba has far more metabolite transporters than Bex as expected from its larger genome, but there are 2 times more carbohydrate uptake and drug efflux systems, and 3 times more lipid transporters. Bba also has polyamine and carboxylate transporters lacking in Bex. Bba has more than twice as many members of the Mot-Exb family of energizers, but both may have energizers for gliding motility. They use entirely different types of systems for iron acquisition. Both contain unexpectedly large numbers of pseudogenes and incomplete systems, suggesting that they are undergoing genome size reduction. Interestingly, all 5 outer-membrane receptors in Bba are lacking in Bex. The 2 organisms have similar numbers and types of peptide and amino acid uptake systems as well as protein and carbohydrate secretion systems. The differences observed correlate with and may account, in part, for the different lifestyles of these 2 bacterial predators.
Project description:Bdellovibrio bacteriovorus HD100 is a predatory bacterium which attacks a wide range of gram negative bacterial pathogens and is proposed to be a potential living antibiotic. In the current study, we evaluated the effects of indole, a bacterial signaling molecule commonly produced within the gut, on the predatory ability of B. bacteriovorus HD100. Indole significantly delayed predation on E. coli MG1655 and S. enterica KACC 11595 at physiological concentrations (0.25 to 1 mM) and completely inhibited predation when present at 2 mM. Microscopic analysis revealed that indole blocked the predator from attacking the prey. Furthermore, indole was not toxic to the predator but slowed down its motility. Microarray and RT-qPCR analyses confirmed this as the gene group showing the greatest down-regulation in the presence of 1 and 2 mM indole was flagellar assembly and motility genes. Aside from this group, indole also caused a wide spectrum changes in gene expression including the general down-regulation of genes involved in ribosome assembly and RNA translation. Furthermore, indole addition to the predatory culture after the entrance of B. bacteriovorus into the prey periplasm slowed down bdelloplast lysis. In conclusion, indole is an important gut-related signaling molecule that can have significant impacts on the predation efficiency and predator behavior. These findings should be taken into consideration especially if B. bacteriovorus is to be applied as a probiotic or living antibiotic.