Production of 3′,3′-cGAMP by a Bdellovibrio bacteriovorus promiscuous GGDEF enzyme,Bd0367, regulates exit from prey by gliding motility

Bacterial second messengers are important for regulating diverse bacterial lifestyles. Cyclic di-GMP (c-di-GMP) is produced by diguanylate cyclase enzymes, named GGDEF proteins, which are widespread across bacteria. Recently, hybrid promiscuous (Hypr) GGDEF proteins have been described in some bacteria, which produce both c-di-GMP and a more recently identified bacterial second messenger, 3′,3′-cyclic-GMP-AMP (cGAMP). One of these proteins was found in the predatory Bdellovibrio bacteriovorus, Bd0367. The bd0367 GGDEF gene deletion strain was found to enter prey cells, but was incapable of leaving exhausted prey remnants via gliding motility on a solid surface once predator cell division was complete. However, it was unclear which signal regulated this process. We show that cGAMP signalling is active within B. bacteriovorus and that, in addition to producing c-di-GMP and some c-di-AMP, Bd0367 is a primary producer of cGAMP in vivo. Site-directed mutagenesis of serine 214 to an aspartate rendered Bd0367 into primarily a c-di-GMP synthase. B. bacteriovorus strain bd0367S214D phenocopies the bd0367 deletion strain by being unable to glide on a solid surface, leading to an inability of new progeny to exit from prey cells post-replication. Thus, this process is regulated by cGAMP. Deletion of bd0367 was also found to be incompatible with wild-type flagellar biogenesis, as a result of an acquired mutation in flagellin chaperone gene homologue fliS, implicating c-di-GMP in regulation of swimming motility. Thus the single Bd0367 enzyme produces two secondary messengers by action of the same GGDEF domain, the first reported example of a synthase that regulates multiple second messengers in vivo. Unlike roles of these signalling molecules in other bacteria, these signal to two separate motility systems, gliding and flagellar, which are essential for completion of the bacterial predation cycle and prey exit by B. bacteriovorus.     

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.     

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.     

Ras GTPase-Like Protein MglA,a Controller of Bacterial Social-Motility in Myxobacteria,Has Evolved to Control Bacterial Predation by Bdellovibrio

Bdellovibrio bacteriovorus invade Gram-negative bacteria in a predatory process requiring Type IV pili (T4P) at a single invasive pole, and also glide on surfaces to locate prey. Ras-like G-protein MglA, working with MglB and RomR in the deltaproteobacterium Myxococcus xanthus, regulates adventurous gliding and T4P-mediated social motility at both M. xanthus cell poles. Our bioinformatic analyses suggested that the GTPase activating protein (GAP)-encoding gene mglB was lost in Bdellovibrio, but critical residues for MglA_Bd GTP-binding are conserved. Deletion of mglA_Bd abolished prey-invasion, but not gliding, and reduced T4P formation. MglA_Bd interacted with a previously uncharacterised tetratricopeptide repeat (TPR) domain protein Bd2492, which we show localises at the single invasive pole and is required for predation. Bd2492 and RomR also interacted with cyclic-di-GMP-binding receptor CdgA, required for rapid prey-invasion. Bd2492, RomR_Bd and CdgA localize to the invasive pole and may facilitate MglA-docking. Bd2492 was encoded from an operon encoding a TamAB-like secretion system. The TamA protein and RomR were found, by gene deletion tests, to be essential for viability in both predatory and non-predatory modes. Control proteins, which regulate bipolar T4P-mediated social motility in swarming groups of deltaproteobacteria, have adapted in evolution to regulate the anti-social process of unipolar prey-invasion in the “lone-hunter” Bdellovibrio. Thus GTP-binding proteins and cyclic-di-GMP inputs combine at a regulatory hub, turning on prey-invasion and allowing invasion and killing of bacterial pathogens and consequent predatory growth of Bdellovibrio.     

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.     

Specialized Peptidoglycan Hydrolases Sculpt the Intra-bacterial Niche of Predatory Bdellovibrio and Increase Population Fitness

Bdellovibrio are predatory bacteria that have evolved to invade virtually all Gram-negative bacteria, including many prominent pathogens. Upon invasion, prey bacteria become rounded up into an osmotically stable niche for the Bdellovibrio, preventing further superinfection and allowing Bdellovibrio to replicate inside without competition, killing the prey bacterium and degrading its contents. Historically, prey rounding was hypothesized to be associated with peptidoglycan (PG) metabolism; we found two Bdellovibrio genes, bd0816 and bd3459, expressed at prey entry and encoding proteins with limited homologies to conventional dacB/PBP4 DD-endo/carboxypeptidases (responsible for peptidoglycan maintenance during growth and division). We tested possible links between Bd0816/3459 activity and predation. Bd3459, but not an active site serine mutant protein, bound β-lactam, exhibited DD-endo/carboxypeptidase activity against purified peptidoglycan and, importantly, rounded up E. coli cells upon periplasmic expression. A ΔBd0816 ΔBd3459 double mutant invaded prey more slowly than the wild type (with negligible prey cell rounding) and double invasions of single prey by more than one Bdellovibrio became more frequent. We solved the crystal structure of Bd3459 to 1.45 Å and this revealed predation-associated domain differences to conventional PBP4 housekeeping enzymes (loss of the regulatory domain III, alteration of domain II and a more exposed active site). The Bd3459 active site (and by similarity the Bd0816 active site) can thus accommodate and remodel the various bacterial PGs that Bdellovibrio may encounter across its diverse prey range, compared to the more closed active site that “regular” PBP4s have for self cell wall maintenance. Therefore, during evolution, Bdellovibrio peptidoglycan endopeptidases have adapted into secreted predation-specific proteins, preventing wasteful double invasion, and allowing activity upon the diverse prey peptidoglycan structures to sculpt the prey cell into a stable intracellular niche for replication.     

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.     

Cyclic di-GMP-dependent Signaling Pathways in the Pathogenic Firmicute Listeria monocytogenes

We characterized key components and major targets of the c-di-GMP signaling pathways in the foodborne pathogen Listeria monocytogenes, identified a new c-di-GMP-inducible exopolysaccharide responsible for motility inhibition, cell aggregation, and enhanced tolerance to disinfectants and desiccation, and provided first insights into the role of c-di-GMP signaling in listerial virulence. Genome-wide genetic and biochemical analyses of c-di-GMP signaling pathways revealed that L. monocytogenes has three GGDEF domain proteins, DgcA (Lmo1911), DgcB (Lmo1912) and DgcC (Lmo2174), that possess diguanylate cyclase activity, and three EAL domain proteins, PdeB (Lmo0131), PdeC (Lmo1914) and PdeD (Lmo0111), that possess c-di-GMP phosphodiesterase activity. Deletion of all phosphodiesterase genes (ΔpdeB/C/D) or expression of a heterologous diguanylate cyclase stimulated production of a previously unknown exopolysaccharide. The synthesis of this exopolysaccharide was attributed to the pssA-E (lmo0527-0531) gene cluster. The last gene of the cluster encodes the fourth listerial GGDEF domain protein, PssE, that functions as an I-site c-di-GMP receptor essential for exopolysaccharide synthesis. The c-di-GMP-inducible exopolysaccharide causes cell aggregation in minimal medium and impairs bacterial migration in semi-solid agar, however, it does not promote biofilm formation on abiotic surfaces. The exopolysaccharide also greatly enhances bacterial tolerance to commonly used disinfectants as well as desiccation, which may contribute to survival of L. monocytogenes on contaminated food products and in food-processing facilities. The exopolysaccharide and another, as yet unknown c-di-GMP-dependent target, drastically decrease listerial invasiveness in enterocytes in vitro, and lower pathogen load in the liver and gallbladder of mice infected via an oral route, which suggests that elevated c-di-GMP levels play an overall negative role in listerial virulence.     

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.     

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.     

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.     

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.     

Reward for Bdellovibrio bacteriovorus for preying on a polyhydroxyalkanoate producer

Bdellovibrio bacteriovorus HD100 is an obligate predator that invades and grows within the periplasm of Gram‐negative bacteria, including mcl‐polyhydroxyalkanoate (PHA) producers such as Pseudomonas putida. We investigated the impact of prey PHA content on the predator fitness and the potential advantages for preying on a PHA producer. Using a new procedure to control P. putida KT2442 cell size we demonstrated that the number of Bdellovibrio progeny depends on the prey biomass and not on the viable prey cell number or PHA content. The presence of mcl‐PHA hydrolysed products in the culture supernatant after predation on P. putida KT42Z, a PHA producing strain lacking PhaZ depolymerase, confirmed the ability of Bdellovibrio to degrade the prey's PHA. Predator motility was higher when growing on PHA accumulating prey. External addition of PHA polymer (latex suspension) to Bdellovibrio preying on the PHA minus mutant P. putida KT42C1 restored predator movement, suggesting that PHA is a key prey component to sustain predator swimming speed. High velocities observed in Bdellovibrio preying on the PHA producing strain were correlated to high intracellular ATP levels of the predator. These effects brought Bdellovibrio fitness benefits as predation on PHA producers was more efficient than predation on non‐producing bacteria.     

Spatially Organized Films from Bdellovibrio bacteriovorus Prey Lysates

Bdellovibrio bacteriovorus is a Gram-negative predator of other Gram-negative bacteria. Interestingly, Bdellovibrio bacteriovorus 109J cells grown in coculture with Escherichia coli ML-35 prey develop into a spatially organized two-dimensional film when located on a nutrient-rich surface. From deposition of 10 μl of a routine cleared coculture of B. bacteriovorus and E. coli cells, the cells multiply into a macroscopic community and segregate into an inner, yellow circular region and an outer, off-white region. Fluorescence in situ hybridization and atomic force microscopy measurements confirm that the mature film is spatially organized into two morphologically distinct Bdellovibrio populations, with primarily small, vibroid cells in the center and a complex mixture of pleomorphic cells in the outer radii. The interior region cell population exhibits the hunting phenotype while the outer region cell subpopulation does not. Crowding and high nutrient availability with limited prey appear to favor diversification of the B. bacteriovorus population into two distinct, thriving subpopulations and may be beneficial to the persistence of B. bacteriovorus in biofilms.     

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.     

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.     

Systematic Nomenclature for GGDEF and EAL Domain-Containing Cyclic Di-GMP Turnover Proteins of Escherichia coli

In recent years, Escherichia coli has served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely used E. coli K-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 “degenerate” enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenic E. coli strains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling in E. coli, we now propose a general and systematic dgc and pde nomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains of E. coli in future studies.