Characterizing the flagellar filament and the role of motility in bacterial prey-penetration by Bdellovibrio bacteriovorus.
ABSTRACT: The predatory bacterium Bdellovibrio bacteriovorus swims rapidly by rotation of a single, polar flagellum comprised of a helical filament of flagellin monomers, contained within a membrane sheath and powered by a basal motor complex. Bdellovibrio collides with, enters and replicates within bacterial prey, a process previously suggested to firstly require flagellar motility and then flagellar shedding upon prey entry. Here we show that flagella are not always shed upon prey entry and we study the six fliC flagellin genes of B. bacteriovorus, finding them all conserved and expressed in genome strain HD100 and the widely studied lab strain 109J. Individual inactivation of five of the fliC genes gave mutant Bdellovibrio that still made flagella, and which were motile and predatory. Inactivation of the sixth fliC gene abolished normal flagellar synthesis and motility, but a disordered flagellar sheath was still seen. We find that this non-motile mutant was still able to predate when directly applied to lawns of YFP-labelled prey bacteria, showing that flagellar motility is not essential for prey entry but important for efficient encounters with prey in liquid environments.
Project description:The predatory bacterium Bdellovibrio bacteriovorus uses flagellar motility to locate regions rich in Gram-negative prey bacteria, colliding and attaching to prey and then ceasing flagellar motility. Prey are then invaded to form a "bdelloplast" in a type IV pilus-dependent process, and prey contents are digested, allowing Bdellovibrio growth and septation. After septation, Bdellovibrio flagellar motility resumes inside the prey bdelloplast prior to its lysis and escape of Bdellovibrio progeny. Bdellovibrio can also grow slowly outside prey as long flagellate host-independent (HI) cells, cultured on peptone-rich media. The B. bacteriovorus HD100 genome encodes three pairs of MotAB flagellar motor proteins, each of which could potentially form an inner membrane ion channel, interact with the FliG flagellar rotor ring, and produce flagellar rotation. In 2004, Flannagan and coworkers (R. S. Flannagan, M. A. Valvano, and S. F. Koval, Microbiology 150:649-656, 2004) used antisense RNA and green fluorescent protein (GFP) expression to downregulate a single Bdellovibrio motA gene and reported slowed release from the bdelloplast and altered motility of the progeny. Here we inactivated each pair of motAB genes and found that each pair contributes to motility, both predatorily, inside the bdelloplast and during HI growth; however, each pair was dispensable, and deletion of no pair abolished motility totally. Driving-ion studies with phenamil, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and different pH and sodium conditions indicated that all Mot pairs are proton driven, although the sequence similarities of each Mot pair suggests that some may originate from halophilic species. Thus, Bdellovibrio is a "dedicated motorist," retaining and expressing three pairs of mot genes.
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. Bdellovibrio bacteriovorus HD100 was incubated for 30 min at 30Â°C in HEPES buffer supplemented with 0,1, and 2 mM indole. RNA was then extracted from each sample and purified. 100 ng of RNA from each sample were used for microarray experiment. For zero and 1 mM indole treatments, three independant samples were tested while for 2 mM indole treatment, two samples were tested. A total of 8 arrays were used.
Project description:Bdellovibrio bacteriovorus is an obligate predator of bacteria that grows and divides within the periplasm of its prey. Functions involved in the early steps of predation have been identified and characterized, but mediators of prey invasion are still poorly detailed. By combining omics data available for Bdellovibrio and like organisms (BALO's), we identified 43 genes expressed in B. bacteriovorus during the early interaction with prey. These included genes in a tight adherence (TAD) operon encoding for two type IVb fimbriae-like pilin proteins (flp1 and flp2), and their processing and export machinery. Two additional flp genes (flp3 and flp4) were computationally identified at other locations along the chromosome, defining the largest and most diverse type IVb complement known in bacteria to date. Only flp1, flp2 and flp4 were expressed; their respective gene knock-outs resulted in a complete loss of the predatory ability without losing the ability to adhere to prey cells. Additionally, we further demonstrate differential regulation of the flp genes as the TAD operon of BALOs with different predatory strategies is controlled by a flagellar sigma factor FliA, while flp4 is not. Finally, we show that FliA, a known flagellar transcriptional regulator in other bacteria, is an essential Bdellovibrio gene.
Project description:Bdellovibrio bacteriovorus is a predatory deltaproteobacterium that encounters individual Gram-negative prey bacteria with gliding or swimming motility, and then is able to invade such prey cells via type IVa pilus-dependent mechanisms. Movement control (pili or gliding) in other deltaproteobacteria, such as the pack hunting Myxococcus xanthus, uses a response regulator protein, RomRMx (which dynamically relocalises between the cell poles) and a GTPase, MglAMx, previously postulated as an interface between the FrzMx chemosensory system and gliding or pilus-motility apparatus, to produce regulated bidirectional motility. In contrast, B. bacteriovorus predation is a more singular encounter between a lone predator and prey; contact is always via the piliated, non-flagellar pole of the predator, involving MglABd, but no Frz system. In this new study, tracking fluorescent RomRBd microscopically during predatory growth shows that it does not dynamically relocalise, in contrast to the M. xanthus protein; instead having possible roles in growth events. Furthermore, transcriptional start analysis, site-directed mutagenesis and bacterial two-hybrid interaction studies, indicate an evolutionary loss of RomRBd activation (via receiver domain phosphorylation) in this lone hunting bacterium, demonstrating divergence from its bipolar role in motility in pack-hunting M. xanthus and further evolution that may differentiate lone from pack predators.
Project description:Predatory Bdellovibrio bacteriovorus are natural antimicrobial organisms, killing other bacteria by whole-cell invasion. Self-protection against prey-metabolizing enzymes is important for the evolution of predation. Initial prey entry involves the predator's peptidoglycan DD-endopeptidases, which decrosslink cell walls and prevent wasteful entry by a second predator. Here we identify and characterize a self-protection protein from B. bacteriovorus, Bd3460, which displays an ankyrin-based fold common to intracellular pathogens of eukaryotes. Co-crystal structures reveal Bd3460 complexation of dual targets, binding a conserved epitope of each of the Bd3459 and Bd0816 endopeptidases. Complexation inhibits endopeptidase activity and cell wall decrosslinking in vitro. Self-protection is vital - ΔBd3460 Bdellovibrio deleteriously decrosslink self-peptidoglycan upon invasion, adopt a round morphology, and lose predatory capacity and cellular integrity. Our analysis provides the first mechanistic examination of self-protection in Bdellovibrio, documents protection-multiplicity for products of two different genomic loci, and reveals an important evolutionary adaptation to an invasive predatory bacterial lifestyle.
Project description:Bdellovibrio bacteriovorus is a famously fast, flagellate predatory bacterium, preying upon Gram-negative bacteria in liquids; how it interacts with prey on surfaces such as in medical biofilms is unknown. Here we report that Bdellovibrio bacteria "scout" for prey bacteria on solid surfaces, using slow gliding motility that is present in flagellum-negative and pilus-negative strains.
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. Bdellovibrio bacteriovorus HD100 was incubated for 30 min at 30°C in HEPES buffer supplemented with 0,1, and 2 mM indole. RNA was then extracted from each sample and purified. 100 ng of RNA from each sample were used for microarray experiment. For zero and 1 mM indole treatments, three independant samples were tested while for 2 mM indole treatment, two samples were tested. A total of 8 arrays were used.
Project description:Bacteriovorax marinus SJ is a predatory delta-proteobacterium isolated from a marine environment. The genome sequence of this strain provides an interesting contrast to that of the terrestrial predatory bacterium Bdellovibrio bacteriovorus HD100. Based on their predatory lifestyle, Bacteriovorax were originally designated as members of the genus Bdellovibrio but subsequently were re-assigned to a new genus and family based on genetic and phenotypic differences. B. marinus attaches to gram-negative bacteria, penetrates through the cell wall to form a bdelloplast, in which it replicates, as shown using microscopy. Bacteriovorax is distinct, as it shares only 30% of its gene products with its closest sequenced relatives. Remarkably, 34% of predicted genes over 500?nt in length were completely unique with no significant matches in the databases. As expected, Bacteriovorax shares several characteristic loci with the other delta-proteobacteria. A geneset shared between Bacteriovorax and Bdellovibrio that is not conserved among other delta-proteobacteria such as Myxobacteria (which destroy prey bacteria externally via lysis), or the non-predatory Desulfo-bacteria and Geobacter species was identified. These 291 gene orthologues common to both Bacteriovorax and Bdellovibrio may be the key indicators of host-interaction predatory-specific processes required for prey entry. The locus from Bdellovibrio bacteriovorus is implicated in the switch from predatory to prey/host-independent growth. Although the locus is conserved in B. marinus, the sequence has only limited similarity. The results of this study advance understanding of both the similarities and differences between Bdellovibrio and Bacteriovorax and confirm the distant relationship between the two and their separation into different families.
Project description:Evolution equipped Bdellovibrio bacteriovorus predatory bacteria to invade other bacteria, digesting and replicating, sealed within them thus preventing nutrient-sharing with organisms in the surrounding environment. Bdellovibrio were previously described as "obligate predators" because only by mutations, often in gene bd0108, are 1 in ~1x10(7) of predatory lab strains of Bdellovibrio converted to prey-independent growth. A previous genomic analysis of B. bacteriovorus strain HD100 suggested that predatory consumption of prey DNA by lytic enzymes made Bdellovibrio less likely than other bacteria to acquire DNA by lateral gene transfer (LGT). However the Doolittle and Pan groups predicted, in silico, both ancient and recent lateral gene transfer into the B. bacteriovorus HD100 genome.To test these predictions, we isolated a predatory bacterium from the River Tiber- a good potential source of LGT as it is rich in diverse bacteria and organic pollutants- by enrichment culturing with E. coli prey cells. The isolate was identified as B. bacteriovorus and named as strain Tiberius. Unusually, this Tiberius strain showed simultaneous prey-independent growth on organic nutrients and predatory growth on live prey. Despite the prey-independent growth, the homolog of bd0108 did not have typical prey-independent-type mutations. The dual growth mode may reflect the high carbon content of the river, and gives B. bacteriovorus Tiberius extended non-predatory contact with the other bacteria present. The HD100 and Tiberius genomes were extensively syntenic despite their different cultured-terrestrial/freshly-isolated aquatic histories; but there were significant differences in gene content indicative of genomic flux and LGT. Gene content comparisons support previously published in silico predictions for LGT in strain HD100 with substantial conservation of genes predicted to have ancient LGT origins but little conservation of AT-rich genes predicted to be recently acquired.The natural niche and dual predatory, and prey-independent growth of the B. bacteriovorus Tiberius strain afforded it extensive non-predatory contact with other marine and freshwater bacteria from which LGT is evident in its genome. Thus despite their arsenal of DNA-lytic enzymes; Bdellovibrio are not always predatory in natural niches and their genomes are shaped by acquiring whole genes from other bacteria.
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.