Role of Type IV Pili in Predation by Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus, as an obligate predator of Gram-negative bacteria, requires contact with the surface of a prey cell in order to initiate the life cycle. After attachment, the predator penetrates the prey cell outer membrane and enters the periplasmic space. Attack phase cells of B. bacteriovorus have polar Type IV pili that are required for predation. In other bacteria, these pili have the ability to extend and retract via the PilT protein. B. bacteriovorus has two pilT genes, pilT1 and pilT2, that have been implicated in the invasion process. Markerless in-frame deletion mutants were constructed in a prey-independent mutant to assess the role of PilT1 and PilT2 in the life cycle. When predation was assessed using liquid cocultures, all mutants produced bdelloplasts of Escherichia coli. These results demonstrated that PilT1 and PilT2 are not required for invasion of prey cells. Predation of the mutants on biofilms of E. coli was also assessed. Wild type B. bacteriovorus 109JA and the pilT1 mutant decreased the mass of the biofilm to 35.4% and 27.9% respectively. The pilT1pilT2 mutant was able to prey on the biofilm, albeit less efficiently with 50.2% of the biofilm remaining. The pilT2 mutant was unable to disrupt the biofilm, leaving 92.5% of the original biofilm after predation. The lack of PilT2 function may impede the ability of B. bacteriovorus to move in the extracellular polymeric matrix and find a prey cell. The role of Type IV pili in the life cycle of B. bacteriovorus is thus for initial recognition of and attachment to a prey cell in liquid cocultures, and possibly for movement within the matrix of a biofilm.     

Bdellovibrio possesses a prey-derived OmpF protein in its outer membrane

Bdellovibrio bacteriovorus 109D andBdellovibrio stolpii derive one of their major outer membrane proteins from the outer membrane of their prey. This prey-derived protein corresponds to the OmpF protein ofEscherichia coli. Bdellovibrios cultivated onSalmonella typhimurium prey acquire theSalmonella OmpF protein; this protein is distinguishable electrophoretically from the OmpF protein ofE. coli. Bdellovibrios containing the prey-derived OmpF protein are sensitive to killing by colicin A but not colicin E1, whereas bdellovibrios without this protein are completely resistant to colicin killing.     

Differential Predation by Bdellovibrio bacteriovorus 109J

Bdellovibrio bacteriovorus is a predatory bacterium that can replicate only inside Gram-negative bacteria. We incubated B. bacteriovorus 109J in a mixture of two prey cells present in equal numbers and enumerated prey cells after 3 h of predation. In multiple prey pairings, B. bacteriovorus preferentially lysed on one prey over the other. When prey were individually incubated with B. bacteriovorus, they were preyed on with different efficiencies. Three prey had only 5–8% of cells remaining after Bdellovibrio predation and the other three prey had 37–43% of cells remaining. Timing of attachment of B. bacteriovorus to prey cells also varied with Bdellovibrio attachment to more preferred prey occurring the fastest. These results suggest that B. bacteriovorus 109J does not randomly infect prey cells but infects and kills some prey more readily than others.     

Visualizing Bdellovibrio bacteriovorus by Using the tdTomato Fluorescent Protein

Bdellovibrio bacteriovorus is a Gram-negative bacterium that belongs to the delta subgroup of proteobacteria and is characterized by a predatory life cycle. In recent years, work has highlighted the potential use of this predator to control bacteria and biofilms. Traditionally, the reduction in prey cells was used to monitor predation dynamics. In this study, we introduced pMQ414, a plasmid that expresses the tdTomato fluorescent reporter protein, into a host-independent strain and a host-dependent strain of B. bacteriovorus 109J. The new construct was used to conveniently monitor predator proliferation in real time, in different growth conditions, in the presence of lytic enzymes, and on several prey bacteria, replicating previous studies that used plaque analysis to quantify B. bacteriovorus. The new fluorescent plasmid also enabled us to visualize the predator in liquid cultures, in the context of a biofilm, and in association with human epithelial cells.     

Discrete Cyclic di-GMP-Dependent Control of Bacterial Predation versus Axenic Growth in Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a Delta-proteobacterium that oscillates between free-living growth and predation on Gram-negative bacteria including important pathogens of man, animals and plants. After entering the prey periplasm, killing the prey and replicating inside the prey bdelloplast, several motile B. bacteriovorus progeny cells emerge. The B. bacteriovorus HD100 genome encodes numerous proteins predicted to be involved in signalling via the secondary messenger cyclic di-GMP (c-di-GMP), which is known to affect bacterial lifestyle choices. We investigated the role of c-di-GMP signalling in B. bacteriovorus, focussing on the five GGDEF domain proteins that are predicted to function as diguanylyl cyclases initiating c-di-GMP signalling cascades. Inactivation of individual GGDEF domain genes resulted in remarkably distinct phenotypes. Deletion of dgcB (Bd0742) resulted in a predation impaired, obligately axenic mutant, while deletion of dgcC (Bd1434) resulted in the opposite, obligately predatory mutant. Deletion of dgcA (Bd0367) abolished gliding motility, producing bacteria capable of predatory invasion but unable to leave the exhausted prey. Complementation was achieved with wild type dgc genes, but not with GGAAF versions. Deletion of cdgA (Bd3125) substantially slowed predation; this was restored by wild type complementation. Deletion of dgcD (Bd3766) had no observable phenotype. In vitro assays showed that DgcA, DgcB, and DgcC were diguanylyl cyclases. CdgA lacks enzymatic activity but functions as a c-di-GMP receptor apparently in the DgcB pathway. Activity of DgcD was not detected. Deletion of DgcA strongly decreased the extractable c-di-GMP content of axenic Bdellovibrio cells. We show that c-di-GMP signalling pathways are essential for both the free-living and predatory lifestyles of B. bacteriovorus and that obligately predatory dgcC- can be made lacking a propensity to survive without predation of bacterial pathogens and thus possibly useful in anti-pathogen applications. In contrast to many studies in other bacteria, Bdellovibrio shows specificity and lack of overlap in c-di-GMP signalling pathways.     

Incorporation of Substrate Cell Lipid A Components into the Lipopolysaccharide of Intraperiplasmically Grown Bdellovibrio bacteriovorus

The composition of Bdellovibrio bacteriovorus lipopolysaccharide (LPS) was determined for cells grown axenically and intraperiplasmically on Escherichia coli or Pseudomonas putida. The LPS of axenically grown bdellovibrios contained glucose and fucosamine as the only detectable neutral sugar and amino sugar, and nonadecenoic acid (19:1) as the predominant fatty acid. Additional fatty acids, heptose, ketodeoxyoctoic acid, and phosphate were also detected. LPS from bdellovibrios grown intraperiplasmically contained components characteristic of both axenically grown bdellovibrios and the substrate cells. Substrate cell-derived LPS fatty acids made up the majority of the bdellovibrio LPS fatty acids and were present in about the same proportions as in the substrate cell LPS. Glucosamine derived from E. coli LPS amounted to about one-third of the hexosamine residues in intraperiplasmically grown bdellovibrio LPS. However, galactose, characteristic of the E. coli outer core and O antigen, was not detected in the bdellovibrio LPS, suggesting that only lipid A components of the substrate cell were incorporated. Substrate cell-derived and bdellovibrio-synthesized LPS materials were conserved in the B. bacteriovorus outer membrane for at least two cycles of intraperiplasmic growth. When bdellovibrios were grown on two different substrate cells successively, lipid A components were taken up from the second while the components incorporated from the lipid A of the first were conserved in the bdellovibrio LPS. The data show that substrate cell lipid A components were incorporated into B. bacteriovorus lipid A during intraperiplasmic growth with little or no change, and that these components, fatty acids and hexosamines, comprised a substantial portion of bdellovibrio lipid A.     

The First Bite— Profiling the Predatosome in the Bacterial Pathogen Bdellovibrio

Bdellovibrio bacteriovorus is a Gram-negative bacterium that is a pathogen of other Gram-negative bacteria, including many bacteria which are pathogens of humans, animals and plants. As such Bdellovibrio has potential as a biocontrol agent, or living antibiotic. B. bacteriovorus HD100 has a large genome and it is not yet known which of it encodes the molecular machinery and genetic control of predatory processes. We have tried to fill this knowledge-gap using mixtures of predator and prey mRNAs to monitor changes in Bdellovibrio gene expression at a timepoint of early-stage prey infection and prey killing in comparison to control cultures of predator and prey alone and also in comparison to Bdellovibrio growing axenically (in a prey-or host independent “HI” manner) on artificial media containing peptone and tryptone. From this we have highlighted genes of the early predatosome with predicted roles in prey killing and digestion and have gained insights into possible regulatory mechanisms as Bdellovibrio enter and establish within the prey bdelloplast. Approximately seven percent of all Bdellovibrio genes were significantly up-regulated at 30 minutes of infection- but not in HI growth- implicating the role of these genes in prey digestion. Five percent were down-regulated significantly, implicating their role in free-swimming, attack-phase physiology. This study gives the first post- genomic insight into the predatory process and reveals some of the important genes that Bdellovibrio expresses inside the prey bacterium during the initial attack.     

Bdellovibrio Predation in the Presence of Decoys: Three-Way Bacterial Interactions Revealed by Mathematical and Experimental Analyses

Bdellovibrio bacteriovorus is a small, gram-negative, motile bacterium that preys upon other gram-negative bacteria, including several known human pathogens. Its predation efficiency is usually studied in pure cultures containing solely B. bacteriovorus and a suitable prey. However, in natural environments, as well as in any possible biomedical uses as an antimicrobial, Bdellovibrio is predatory in the presence of diverse decoys, including live nonsusceptible bacteria, eukaryotic cells, and cell debris. Here we gathered and mathematically modeled data from three-member cultures containing predator, prey, and nonsusceptible bacterial decoys. Specifically, we studied the rate of predation of planktonic late-log-phase Escherichia coli S17-1 prey by B. bacteriovorus HD100, both in the presence and in the absence of Bacillus subtilis nonsporulating strain 671, which acted as a live bacterial decoy. Interestingly, we found that although addition of the live Bacillus decoy did decrease the rate of Bdellovibrio predation in liquid cultures, this addition also resulted in a partially compensatory enhancement of the availability of prey for predation. This effect resulted in a higher final yield of Bdellovibrio than would be predicted for a simple inert decoy. Our mathematical model accounts for both negative and positive effects of predator-prey-decoy interactions in the closed batch environment. In addition, it informs considerations for predator dosing in any future therapeutic applications and sheds some light on considerations for modeling the massively complex interactions of real mixed bacterial populations in nature.     

Development of a Novel Genetic System To Create Markerless Deletion Mutants of Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a species of unique obligate predatory bacteria that utilize gram-negative bacteria as prey. Their life cycle alternates between a motile extracellular phase and a growth phase within the prey cell periplasm. The mechanism of prey cell invasion and the genetic networks and regulation during the life cycle have not been elucidated. The obligate predatory nature of the B. bacteriovorus life cycle suggests the use of this bacterium in potential applications involving pathogen control but adds complexity to the development of practical genetic systems that can be used to determine gene function. This work reports the development of a genetic technique for allelic exchange or gene inactivation by construction of in-frame markerless deletion mutants including the use of a counterselectable marker in B. bacteriovorus. A suicide plasmid carrying the sacB gene for counterselection was used to inactivate the strB gene in B. bacteriovorus HD100 by an in-frame deletion. Despite the inactivation of the strB gene, B. bacteriovorus was found to retain resistance to high concentrations of streptomycin. The stability of a plasmid for use in complementation experiments was also investigated, and it was determined that pMMB206 replicates autonomously in B. bacteriovorus. Development of this practical genetic system now facilitates the study of B. bacteriovorus at the molecular level and will aid in understanding the regulatory networks and gene function in this fascinating predatory bacterium.     

A small predatory core genome in the divergent marine Bacteriovorax marinus SJ and the terrestrial Bdellovibrio bacteriovorus

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.     

The Lifestyle Switch Protein Bd0108 of Bdellovibrio bacteriovorus Is an Intrinsically Disordered Protein

Bdellovibrio bacteriovorus is a δ-proteobacterium that preys upon Salmonella spp., E. coli, and other Gram-negative bacteria. Bdellovibrio can grow axenically (host-independent, HI, rare and mutation-driven) or subsist via a predatory lifecycle (host-dependent, HD, the usual case). Upon contact with prey, B. bacteriovorus enters the host periplasm from where it slowly drains the host cytosol of nutrients for its own replication. At the core of this mechanism is a retractile pilus, whose architecture is regulated by the protein Bd0108 and its interaction with the neighboring gene product Bd0109. Deletion of bd0108 results in negligible pilus formation, whereas an internal deletion (the one that instigates host-independence) causes mis-regulation of pilus length. These mutations, along with a suite of naturally occurring bd0108 mutant strains, act to control the entry to HI growth. To further study the molecular mechanism of predatory regulation, we focused on the apparent lifecycle switch protein Bd0108. Here we characterize the solution structure and dynamics of Bd0108 using nuclear magnetic resonance (NMR) spectroscopy complemented with additional biophysical methods. We then explore the interaction between Bd0108 and Bd0109 in detail utilizing isothermal titration calorimetry (ITC) and NMR spectroscopy. Together our results demonstrate that Bd0108 is an intrinsically disordered protein (IDP) and that the interaction with Bd0109 is of low affinity. Furthermore, we observe that Bd0108 retains an IDP nature while binding Bd0109. From our data we conclude that Bdellovibrio bacteriovorus utilizes an intrinsically disordered protein to regulate its pilus and control predation signaling.     

Assessment of the Mobilizable Vector Plasmids pSUP202 and pSUP404.2 as Genetic Tools for the Predatory Bacterium <Emphasis Type="Italic">Bdellovibrio bacteriovorus</Emphasis>

Bdellovibrio and like organisms (BALOs) form the group of predatory bacteria which require Gram-negative bacteria as prey. Genetic studies with Bdellovibrio bacteriovorus can be performed with vectors which are introduced into the predator via conjugation. The usefulness of the two vectors pSUP202 and pSUP404.2 as genetic tools were assessed. Both vectors were transferable into B. bacteriovorus by conjugative matings with an Escherichia coli K12 strain as donor. The transfer frequency was higher for vector pSUP404.2 (approx. 10⁻¹–10⁻⁴) as for pSUP202 (approx. 10⁻⁵–10⁻⁶). Vector pSUP202 with a pMB1 origin is unstable in the predatory bacterium, whereas pSUP404.2 is stably maintained in the absence of selective antibiotics. pSUP404.2 harbors two plasmid replicons, the p15A ori and the RSF1010 replication region The copy number of pSUP404.2 was determined by quantitative PCR in B. bacteriovorus and averages seven copies per genome. pSUP404.2 harbors two resistance genes (chloramphenicol and kanamycin) which can be used for cloning either by selection for transconjugants or by insertional inactivation.     

The Predatory Bacterium Bdellovibrio bacteriovorus Aspartyl-tRNA Synthetase Recognizes tRNAAsn as a Substrate

The predatory bacterium Bdellovibrio bacteriovorus preys on other Gram-negative bacteria and was predicted to be an asparagine auxotroph. However, despite encoding asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, B. bacteriovorus also contains the amidotransferase GatCAB. Deinococcus radiodurans, and Thermus thermophilus also encode both of these aminoacyl-tRNA synthetases with GatCAB. Both also code for a second aspartyl-tRNA synthetase and use the additional aspartyl-tRNA synthetase with GatCAB to synthesize asparagine on tRNA^Asn. Unlike those two bacteria, B. bacteriovorus encodes only one aspartyl-tRNA synthetase. Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNA^Asn formation that GatCAB can then amidate to asparaginyl-tRNA^Asn. This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNA^Asn formation with the asparagine synthesized in a tRNA-dependent manner. Thus, in contrast to a previous prediction, B. bacteriovorus codes for a biosynthetic route for asparagine. Analysis of bacterial genomes suggests a significant number of other bacteria may also code for both routes for Asn-tRNA^Asn synthesis with only a limited number encoding a second aspartyl-tRNA synthetase.     

Measurement of Predation and Biofilm Formation under Different Ambient Oxygen Conditions Using a Simple Gasbag-Based System

Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are Gram-negative bacteria characterized by predatory behavior. The aim of this study was to evaluate the ability of the predators to prey in different oxygen environments. When placed on an orbital shaker, a positive association between the rate of aeration and predation was observed. To further examine the effects of elevated ambient oxygen levels on predation, a simple gasbag system was developed. Using the system, we were able to conduct experiments at ambient oxygen levels of 3% to 86%. When placed in gasbags and inflated with air, 50% O₂, and 100% O₂, positive predation was seen on both planktonic and biofilm-grown prey cells. However, in low-oxygen environments, predatory bacteria were able to attack only prey cells grown as biofilms. To further evaluate the gasbag system, biofilm development of Gram-positive and Gram-negative microorganisms was also measured. Although the gasbag system was found to be suitable for culturing bacteria that require a low-oxygen environment, it was not capable of supporting, with its current configuration, the growth of obligate anaerobes in liquid or agar medium.     

A model of prey bacteria,predator bacteria,and bacteriophage in continuous culture

Mathematical models are presented for one-, two- and three -trophic-level microbial systems in continuous culture, based on the respective interactions of Escherichia coli and limiting glucose; glucose, E. coli, and the predaceous bacterium Bdellovibrio bacteriovorus; and glucose, E. coli, B. bacteriovorus, and a virus parasitic on B. bacteriovorus. These models contain explicit time lags to represent reproductive latent periods, and the importance of these to the behavior of the models is demonstrated. The models' behavior was investigated by algebraic analysis and by computer simulation using literature values for the parameters in the equations; equilibrium conditions and stability properties for the various microbial systems are presented over a wide range of potential environmental conditions. A sensitivity analysis indicates which parameters must be most accurately known in order to validate the model experimentally. The results delineate the range of experimental conditions permitting the study of all these continuous culture systems in the laboratory and generate predictions about persistence of such multi-level microbial systems in natures     

Electron Microscopic Observations on the Penetration of Bdellovibrio bacteriovorus into Gram-negative Bacterial Hosts

The progressive stages in Bdellovibrio bacteriovorus penetration into two strains of Escherichia coli were examined by use of electron microscopic techniques. The initial change observed in the ultrastructure of the host following parasitic attack was the swelling of the cell envelope at the site of attachment. The Bdellovibrio then appeared to pierce the center of this swelling, forming a pore in the outer wall layers of the host. The edges of this entry pore constricted the Bdellovibrio throughout its penetration into the host cell. Although partial disruption of the cytoplasmic membrane was always apparent, the parasite did not appear to actively penetrate through this barrier. An attempt is made to correlate the fine structural changes involved in penetration with the physiological data that have accumulated to date.     

Bacterial predator–prey dynamics in microscale patchy landscapes

Soil is a microenvironment with a fragmented (patchy) spatial structure in which many bacterial species interact. Here, we explore the interaction between the predatory bacterium Bdellovibrio bacteriovorus and its prey Escherichia coli in microfabricated landscapes. We ask how fragmentation influences the prey dynamics at the microscale and compare two landscape geometries: a patchy landscape and a continuous landscape. By following the dynamics of prey populations with high spatial and temporal resolution for many generations, we found that the variation in predation rates was twice as large in the patchy landscape and the dynamics was correlated over shorter length scales. We also found that while the prey population in the continuous landscape was almost entirely driven to extinction, a significant part of the prey population in the fragmented landscape persisted over time. We observed significant surface-associated growth, especially in the fragmented landscape and we surmise that this sub-population is more resistant to predation. Our results thus show that microscale fragmentation can significantly influence bacterial interactions.     

Structural and Biochemical Analysis of a Unique Phosphatase from Bdellovibrio bacteriovorus Reveals Its Structural and Functional Relationship with the Protein Tyrosine Phosphatase Class of Phytase

Bdellovibrio bacteriovorus is an unusual δ-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases) are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP) class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.     

Susceptibility of Biofilms to Bdellovibrio bacteriovorus Attack

Biofilms are communities of microorganisms attached to a surface, and the growth of these surface attached communities is thought to provide microorganisms with protection against a range of biotic and abiotic agents. The capability of the gram-negative predatory bacterium Bdellovibrio bacteriovorus to control and reduce an existing Escherichia coli biofilm was evaluated in a static assay. A reduction in biofilm biomass was observed as early as 3 h after exposure to the predator, and an 87% reduction in crystal violet staining corresponding to a 4-log reduction in biofilm cell viability was seen after a 24-h exposure period. We observed that an initial titer of Bdellovibrio as low as 10² PFU/well or an exposure to the predator as short as 30 min is sufficient to reduce a preformed biofilm. The ability of B. bacteriovorus to reduce an existing biofilm was confirmed by scanning electron microscopy. The reduction in biofilm biomass obtained after the first 24 h of exposure to the predator remained unchanged even after longer exposure periods and reinoculation of the samples with fresh Bdellovibrio; however, no genetically stable resistant population of the host bacteria could be detected. Our data suggest that growth in a biofilm does not prevent predation by Bdellovibrio but allows a level of survival from attack greater than that observed for planktonic cells. In flow cell experiments B. bacteriovorus was able to decrease the biomass of both E. coli and Pseudomonas fluorescens biofilms as determined by phase-contrast and epifluorescence microscopy.