Unbalanced predatory communities and a lack of microbial degraders characterize the microbiota of a highly sewage-polluted Eastern-Mediterranean stream.

Wastewater pollution of water resources takes a heavy toll on humans and on the environment. In highly polluted water bodies, self-purification is impaired, as the capacity of the riverine microbes to regenerate the ecosystem is overwhelmed. To date, information on the composition, dynamics and functions of the microbial communities in highly sewage-impacted rivers is limited, in particular in arid and semi-arid environments. In this year-long study of the highly sewage-impacted Al-Nar/Kidron stream in the Barr al-Khalil/Judean Desert east of Jerusalem, we show, using 16S and 18S rRNA gene-based community analysis and targeted qPCR, that both the bacterial and micro-eukaryotic communities, while abundant, exhibited low stability and diversity. Hydrolyzers of organics compounds, as well as nitrogen and phosphorus recyclers were lacking, pointing at reduced potential for regeneration. Furthermore, facultative bacterial predators were almost absent, and the obligate predators Bdellovibrio and like organisms were found at very low abundance. Finally, the micro-eukaryotic predatory community differed from those of other freshwater environments. The lack of essential biochemical functions may explain the stream's inability to self-purify, while the very low levels of bacterial predators and the disturbed assemblages of micro-eukaryote predators present in Al-Nar/Kidron may contribute to community instability and disfunction.

Prey killing without invasion by Bdellovibrio bacteriovorus defective for a MIDAS-family adhesin.

The bacterium Bdellovibrio bacteriovorus is a predator of other Gram-negative bacteria. The predator invades the prey's periplasm and modifies the prey's cell wall, forming a rounded killed prey, or bdelloplast, containing a live B. bacteriovorus. Redundancy in adhesive processes makes invasive mutants rare. Here, we identify a MIDAS adhesin family protein, Bd0875, that is expressed at the predator-prey invasive junction and is important for successful invasion of prey. A mutant strain lacking bd0875 is still able to form round, dead bdelloplasts; however, 10% of the bdelloplasts do not contain B. bacteriovorus, indicative of an invasion defect. Bd0875 activity requires the conserved MIDAS motif, which is linked to catch-and-release activity of MIDAS proteins in other organisms. A proteomic analysis shows that the uninvaded bdelloplasts contain B. bacteriovorus proteins, which are likely secreted into the prey by the Δbd0875 predator during an abortive invasion period. Thus, secretion of proteins into the prey seems to be sufficient for prey killing, even in the absence of a live predator inside the prey periplasm.

Chromosome structure and DNA replication dynamics during the life cycle of the predatory bacterium Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus, an obligate predatory Gram-negative bacterium that proliferates inside and kills other Gram-negative bacteria, was discovered more than 60 years ago. However, we have only recently begun to understand the detailed cell biology of this proficient bacterial killer. Bdellovibrio bacteriovorus exhibits a peculiar life cycle and bimodal proliferation, and thus represents an attractive model for studying novel aspects of bacterial cell biology. The life cycle of B. bacteriovorus consists of two phases: a free-living nonreplicative attack phase and an intracellular reproductive phase. During the reproductive phase, B. bacteriovorus grows as an elongated cell and undergoes binary or nonbinary fission, depending on the prey size. In this review, we discuss: (1) how the chromosome structure of B. bacteriovorus is remodeled during its life cycle; (2) how its chromosome replication dynamics depends on the proliferation mode; (3) how the initiation of chromosome replication is controlled during the life cycle, and (4) how chromosome replication is spatiotemporally coordinated with the proliferation program.

Binary or Nonbinary Fission? Reproductive Mode of a Predatory Bacterium Depends on Prey Size.

Most bacteria, including model organisms such as Escherichia coli, Bacillus subtilis, and Caulobacter crescentus, reproduce by binary fission. However, some bacteria belonging to various lineages, including antibiotic-producing Streptomyces and predatory Bdellovibrio, proliferate by nonbinary fission, wherein three or more chromosome copies are synthesized and the resulting multinucleoid filamentous cell subdivides into progeny cells. Here, we demonstrate for the first time that the predatory bacterium Bdellovibrio bacteriovorus reproduces through both binary and nonbinary fission inside different prey bacteria. Switching between the two modes correlates with the prey size. In relatively small prey cells, B. bacteriovorus undergoes binary fission; the FtsZ ring assembles in the midcell, and the mother cell splits into two daughter cells. In larger prey cells, B. bacteriovorus switches to nonbinary fission and creates multiple asynchronously assembled FtsZ rings to produce three or more daughter cells. Completion of bacterial cell cycle critically depends on precise spatiotemporal coordination of chromosome replication with other cell cycle events, including cell division. We show that B. bacteriovorus always initiates chromosome replication at the invasive pole of the cell, but the spatiotemporal choreography of subsequent steps depends on the fission mode and/or the number of progeny cells. In nonbinary dividing filaments producing five or more progeny cells, the last round(s) of replication may also be initiated at the noninvasive pole. Altogether, we find that B. bacteriovorus reproduces through bimodal fission and that extracellular factors, such as the prey size, can shape replication choreography, providing new insights about bacterial life cycles. IMPORTANCE Most eukaryotic and bacterial cells divide by binary fission, where one mother cell produces two progeny cells, or, rarely, by nonbinary fission. All bacteria studied to date use only one of these two reproduction modes. We demonstrate for the first time that a predatory bacterium, Bdellovibrio bacteriovorus, exhibits bimodal fission and the mode of division depends on the size of the prey bacterium inside which B. bacteriovorus grows. This work provides key insights into the mode and dynamics of B. bacteriovorus proliferation in different pathogens that pose a major threat to human health due to their emerging antibiotic resistance (Proteus mirabilis, Salmonella enterica, and Shigella flexneri). The use of predatory bacteria such as B. bacteriovorus is currently regarded as a promising strategy to kill antibiotic-resistant pathogens. We find that B. bacteriovorus employs different chromosome replication choreographies and division modes when preying on those pathogens. Our findings may facilitate the design of efficient pathogen elimination strategies.

An MltA-Like Lytic Transglycosylase Secreted by Bdellovibrio bacteriovorus Cleaves the Prey Septum during Predatory Invasion.

Lytic transglycosylases cut peptidoglycan backbones, facilitating a variety of functions within bacteria, including cell division, pathogenesis, and insertion of macromolecular machinery into the cell envelope. Here, we identify a novel role of a secreted lytic transglycosylase associated with the predatory lifestyle of Bdellovibrio bacteriovorus strain HD100. During wild-type B. bacteriovorus prey invasion, the predator rounds up rod-shaped prey into spherical prey bdelloplasts, forming a spacious niche within which the predator grows. Deleting the MltA-like lytic transglycosylase Bd3285 still permitted predation but resulted in three different, invaded prey cell shapes: spheres, rods, and "dumbbells." Amino acid D321 within the catalytic C-terminal 3D domain of Bd3285 was essential for wild-type complementation. Microscopic analyses revealed that dumbbell-shaped bdelloplasts are derived from Escherichia coli prey undergoing cell division at the moment of Δbd3285 predator invasion. Prelabeling of E. coli prey peptidoglycan prior to predation with the fluorescent D-amino acid HADA showed that the dumbbell bdelloplasts invaded by B. bacteriovorus Δbd3285 contained a septum. Fluorescently tagged Bd3285, expressed in E. coli, localized to the septum of dividing cells. Our data indicate that B. bacteriovorus secretes the lytic transglycosylase Bd3285 into the E. coli periplasm during prey invasion to cleave the septum of dividing prey, facilitating prey cell occupation. IMPORTANCE Antimicrobial resistance is a serious and rapidly growing threat to global health. Bdellovibrio bacteriovorus can prey upon an extensive range of Gram-negative bacterial pathogens and thus has promising potential as a novel antibacterial therapeutic and is a source of antibacterial enzymes. Here, we elucidate the role of a unique secreted lytic transglycosylase from B. bacteriovorus which acts on the septal peptidoglycan of its prey. This improves our understanding of mechanisms that underpin bacterial predation.

Parallel Evolution in Predatory Bdellovibrio sp. NC01 during Long-Term Coculture with a Single Prey Strain.

Experimental evolution provides a powerful tool for examining how Bdellovibrio evolves in response to unique selective pressures associated with its predatory lifestyle. We tested how Bdellovibrio sp. NC01 adapts to long-term coculture with Pseudomonas sp. NC02, which is less susceptible to predation compared to other Gram-negative bacteria. Analyzing six replicate Bdellovibrio populations across six time points spanning 40 passages and 2,880 h of coculture, we detected 30 to 40 new mutations in each population that exceeded a frequency of 5%. Nonsynonymous substitutions were the most abundant type of new mutation, followed by small indels and synonymous substitutions. After completing the final passage, we detected 20 high-frequency (>75%) mutations across all six evolved Bdellovibrio populations. Eighteen of these alter protein sequences, and most increased in frequency rapidly. Four genes acquired a high-frequency mutation in two or more evolved Bdellovibrio populations, reflecting parallel evolution and positive selection. The genes encode a sodium/phosphate cotransporter family protein (Bd2221), a metallophosphoesterase (Bd0054), a TonB family protein (Bd0396), and a hypothetical protein (Bd1601). Tested prey range and predation efficiency phenotypes did not differ significantly between evolved Bdellovibrio populations and the ancestor; however, all six evolved Bdellovibrio populations demonstrated enhanced starvation survival compared to the ancestor. These results suggest that, instead of evolving improved killing of Pseudomonas sp. NC02, Bdellovibrio evolved to better withstand nutrient limitation in the presence of this prey strain. The mutations identified here point to genes and functions that may be important for Bdellovibrio adaptation to the different selective pressures of long-term coculture with Pseudomonas. IMPORTANCE Bdellovibrio attack and kill Gram-negative bacteria, including drug-resistant pathogens of animals and plants. This lifestyle is unusual among bacteria, and it imposes unique selective pressures on Bdellovibrio. Determining how Bdellovibrio evolve in response to these pressures is valuable for understanding the mechanisms that govern predation. We applied experimental evolution to test how Bdellovibrio sp. NC01 evolved in response to long-term coculture with a single Pseudomonas strain, which NC01 can kill, but with low efficiency. Our experimental design imposed different selective pressures on the predatory bacteria and tracked the evolutionary trajectories of replicate Bdellovibrio populations. Using genome sequencing, we identified Bdellovibrio genes that acquired high-frequency mutations in two or more populations. Using phenotype assays, we determined that evolved Bdellovibrio populations did not improve their ability to kill Pseudomonas, but rather are better able to survive starvation. Overall, our results point to functions that may be important for Bdellovibrio adaptation.

Biocontrol treatment: Application of Bdellovibrio bacteriovorus HD100 against burn wound infection caused by Pseudomonas aeroginosa in mice.

Owing to the high level of resistance to various antibiotics in bacteria causing burn wound infections, the alternative therapeutics is highly demanded. Bdellovibrio and like organisms (BALOs) seem to be a superb choice. In the present study, Bdellovibrio bacteriovorus HD100 was selected for treating burn wound infection caused by Pseudomonas aeruginosa strain PAO1 in a mouse model. In this experiment, two treatments, meropenem as antibiotic and B. bacteriovorus, were employed. Histopathology indicated an accelerated healing rate in both treatments in comparison with the control. Moreover, quantitative reverse transcription PCR (qRT-PCR) was applied to investigate the expression of tnf-α (tumor necrosis factor alpha), pdgf (platelet-derived growth factor), tgf-ß1 (transforming growth factor beta1), ifn-γ (interferon gamma), vegf (vascular endothelial group factor), and col1 (collagen type 1). The results demonstrated that treating burn wound areas with Bdellovibrio not only decrease the inflammatory phase period, but also may improve the characteristics of proliferative phases of wound healing. In addition, a significant difference was explored between the two treatment groups in the regulation of all genes, except for pdgf revealed a significant up regulation in both treatment groups. The results disclose that Bdellovibrio attenuates P. aeruginosa in burn wounds infections and improves the wound healing process.

Strain-specific predation of Bdellovibrio bacteriovorus on Pseudomonas aeruginosa with a higher range for cystic fibrosis than for bacteremia isolates.

This work aimed to evaluate the predatory activity of Bdellovibrio bacteriovorus 109J on clinical isolates of Pseudomonas aeruginosa selected from well-characterized collections of cystic fibrosis (CF) lung colonization (n = 30) and bloodstream infections (BSI) (n = 48) including strains selected by genetic lineage (frequent and rare sequence types), antibiotic resistance phenotype (susceptible and multidrug-resistant isolates), and colony phenotype (mucoid and non-mucoid isolates). The intraspecies predation range (I-PR) was defined as the proportion of susceptible strains within the entire collection. In contrast, the predation efficiency (PE) is the ratio of viable prey cells remaining after predation compared to the initial inoculum. I-PR was significantly higher for CF (67%) than for BSI P. aeruginosa isolates (35%) probably related to an environmental origin of CF strains whereas invasive strains are more adapted to humans. I-PR correlation with bacterial features such as mucoid morphotype, genetic background, or antibiotic susceptibility profile was not detected. To test the possibility of increasing I-PR of BSI isolates, a polyhydroxyalkanoate depolymerase deficient B. bacteriovorus bd2637 mutant was used. Global median I-PR and PE values remained constant for both predators, but 31.2% of 109J-resistant isolates were susceptible to the mutant, and 22.9% of 109J-susceptible isolates showed resistance to predation by the mutant, pointing to a predator-prey specificity process. The potential use of predators in the clinical setting should be based on the determination of the I-PR for each species, and the PE of each particular target strain.

Asymmetric peptidoglycan editing generates cell curvature in Bdellovibrio predatory bacteria.

Peptidoglycan hydrolases contribute to the generation of helical cell shape in Campylobacter and Helicobacter bacteria, while cytoskeletal or periskeletal proteins determine the curved, vibrioid cell shape of Caulobacter and Vibrio. Here, we identify a peptidoglycan hydrolase in the vibrioid-shaped predatory bacterium Bdellovibrio bacteriovorus which invades and replicates within the periplasm of Gram-negative prey bacteria. The protein, Bd1075, generates cell curvature in B. bacteriovorus by exerting LD-carboxypeptidase activity upon the predator cell wall as it grows inside spherical prey. Bd1075 localizes to the outer convex face of B. bacteriovorus; this asymmetric localization requires a nuclear transport factor 2-like (NTF2) domain at the protein C-terminus. We solve the crystal structure of Bd1075, which is monomeric with key differences to other LD-carboxypeptidases. Rod-shaped Δbd1075 mutants invade prey more slowly than curved wild-type predators and stretch invaded prey from within. We therefore propose that the vibrioid shape of B. bacteriovorus contributes to predatory fitness.

Short-Term Dynamics of Bdellovibrio and Like Organisms in Lake Geneva in Response to a Simulated Climatic Extreme Event.

The short time-scale dynamics of three families of Bdellovibrio and like organisms (i.e. Bdellovibrionaceae, Peredibacteraceae, and Bacteriovoracaceae) were studied on the surface waters of Lake Geneva in summer. Using mesocosms deployed nearshore in July 2019, we simulated an extreme climatic event (an input of carbon from the watershed in response to runoff from the catchment, light reduction, and mixing in response to stormy conditions) and aimed to study the impact of both abiotic and biotic factors on their dynamics. The three families of Bdellovibrio and like organisms (BALOs) showed different dynamics during the experiment. Peredibacteraceae was the most abundant group, whereas Bacteriovoracaceae was the least abundant. Compared with the other two families, the abundance of Bdellovibrionaceae did not fluctuate, remaining relatively stable over time. Environmental variables only partially explained the dynamics of these families; in particular, temperature, pH, and chloride concentrations were positively correlated with Bacteriovoracaceae, Bdellovibrionaceae, and Peredibacteraceae abundance, respectively. Prokaryote-like particles (PLPs), such as those with high DNA content (HDNA), were strongly and positively correlated with Peredibacteraceae and Bacteriovoracaceae. In contrast, no relationships were found between Bdellovibrionaceae and PLP abundance, nor between the virus-like particles (VLPs) and the different BALOs. Overall, the experiment revealed that predation was stable in the face of the simulated climatic events. In addition, we observed that Peredibacteraceae and Bacteriovoracaceae share common traits, while Bdellovibrionaceae seems to constitute a distinct category.

Bdellovibrio reynosensis sp. nov., from a Mexico soil sample.

A novel predatory bacterium, strain LBG001T, has been isolated from Reynosa, Mexico. The 16S rRNA shares approximately 97 % sequence identity with many reported strains in the genus Bdellovibrio including the type strain Bdellovibrio bacteriovorus HD100T. Phylogenetic trees based on the 16S rRNA gene and on 30 concatenated housekeeping genes or core genes showed that LBG001T is on a separate branch from the B. bacteriovorus group. LBG0001T has a genome size of 3 582 323 bp with a G+C content of 43.1 mol %. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values with other members of the genus Bdellovibrio (<79, <72 and <17 %, respectively) qualifies the strain to represent a new species in the genus. Strain LBG001T formed visible plaques on all 10 tested Gram-negative bacterial species. The phenotypic characteristics, phylogenetic analysis and genomic taxonomic studies support the classification of the strain as representing a new species for which the name Bdellovibrio reynosensis sp. nov. is proposed. The type strain is LBG001T(=ATCC TSD-288T =CM-CNRG 0932T).

Community and single cell analyses reveal complex predatory interactions between bacteria in high diversity systems.

A fundamental question in community ecology is the role of predator-prey interactions in food-web stability and species coexistence. Although microbial microcosms offer powerful systems to investigate it, interrogating the environment is much more arduous. Here, we show in a 1-year survey that the obligate predators Bdellovibrio and like organisms (BALOs) can regulate prey populations, possibly in a density-dependent manner, in the naturally complex, species-rich environments of wastewater treatment plants. Abundant as well as rarer prey populations are affected, leading to an oscillating predatory landscape shifting at various temporal scales in which the total population remains stable. Shifts, along with differential prey range, explain co-existence of the numerous predators through niche partitioning. We validate these sequence-based findings using single-cell sorting combined with fluorescent hybridization and community sequencing. Our approach should be applicable for deciphering community interactions in other systems.

Effects of Acanthamoeba castellanii on the dissolved oxygen and the microbial community under the experimental aquatic model.

Acanthamoeba castellanii is a protist that has a high predation efficiency for bacteria in a number of monoxenic culture experiments. However, the role of A. castellanii in the microbial community is still unknown because of the lack of studies on multiple-species interactions. The aim of this study was to investigate the change of bacterial composition after A. castellanii emerges in a water environment. We added A. castellanii to an environmental water sample and incubated it for two days. Then, we performed 16S ribosomal RNA sequencing techniques to analyze the changes in bacterial composition. In this study, A. castellanii slightly increased the relative abundance of a few opportunistic pathogens, such as Legionella, Roseomonas, and Haemophilus. This result may be related to the training ground hypothesis. On the other hand, the growth of some bacteria was inhibited, such as Cyanobacteria and Firmicutes. Although A. castellanii did not drastically change the whole bacterial community, we surprisingly found the dissolved oxygen concentration was increased after incubation with A. castellanii. We applied environmental water at the laboratory scale to investigate the interactions among A. castellanii, complex microbial communities and the environment. We identified the bacteria that are sensitive to A. castellanii and further found the novel relationship between dissolved oxygen and microbial interaction. Our results helped to clarify the role of A. castellanii in microbial communities.

New 16S rRNA primers to uncover Bdellovibrio and like organisms diversity and abundance.

Appropriate use and specific primers are important in assessing the diversity and abundance of microbial groups of interest. Bdellovibrio and like organisms (BALOs), that refer to obligate Gram-negative bacterial predators of other Gram-negative bacteria, evolved in terms of taxonomy and classification over the past two decades. Hence, some former primers have become inadequate while others are yet to be designed, for both PCR (especially with the advent of NGS) and qPCR approaches. Thus, to study BALOs' abundance and diversity in a variety of aquatic ecosystems, we designed in silico specific primer sets for each BALO genera and tested them in vitro on a variety of cultures and environmental samples. Also, we performed Sanger and Nano Miseq sequencing to reveal the exact degree of specificity of the most promising primers set. Here we report our success in designing specific primers for some BALOs genera, i.e. Bdellovibrio (PCR), Bacteriovorax (qPCR), Peredibacter (PCR).

A novel method to determine antibiotic sensitivity in Bdellovibrio bacteriovorus reveals a DHFR-dependent natural trimethoprim resistance.

Bdellovibrio bacteriovorus is a small Gram-negative bacterium and an obligate predator of other Gram-negative bacteria. Prey resistance to B. bacteriovorus attack is rare and transient. This consideration together with its safety and low immunogenicity makes B. bacteriovorus a valid alternative to antibiotics, especially in the treatment of multidrug resistant pathogens. In this study we developed a novel technique to estimate B. bacteriovorus sensitivity against antibiotics in order to make feasible the development and testing of co-therapies with antibiotics that would increase its antimicrobial efficacy and at the same time reduce the development of drug resistance. Results from tests performed with this technique show that among all tested antibiotics, trimethoprim has the lowest antimicrobial effect on B. bacteriovorus. Additional experiments revealed that the mechanism of trimethoprim resistance in B. bacteriovorus depends on the low affinity of this compound for the B. bacteriovorus dihydrofolate reductase (Bd DHFR).

From the Inside Out: an Epibiotic Bdellovibrio Predator with an Expanded Genomic Complement.

Bdellovibrio and like organisms are abundant environmental parasitoids of prokaryotes that show diverse predation strategies. The vast majority of studied Bdellovibrio bacteria and like organisms deploy intraperiplasmic replication inside the prey cell, while few isolates with smaller genomes consume their prey from the outside in an epibiotic manner. The novel parasitoid "Candidatus Bdellovibrio qaytius" was isolated from a eutrophic freshwater pond in British Columbia, where it was a continual part of the microbial community. "Ca Bdellovibrio qaytius" was found to preferentially prey on the betaproteobacterium Paraburkholderia fungorum without entering the periplasm. Despite its epibiotic replication strategy, "Ca Bdellovibrio" encodes a large genomic complement more similar to that of complex periplasmic predators. Functional genomic annotation further revealed several biosynthesis pathways not previously found in epibiotic predators, indicating that "Ca Bdellovibrio" represents an intermediate phenotype and at the same time narrowing down the genomic complement specific to epibiotic predators. In phylogenetic analysis, "Ca Bdellovibrio qaytius" occupies a widely distributed, but poorly characterized, basal cluster within the genus Bdellovibrio This suggests that epibiotic predation might be a common predation type in nature and that epibiotic predation could be the ancestral predation type in the genus.IMPORTANCEBdellovibrio and like organisms are bacteria that prey on other bacteria and are widespread in the environment. Most of the known Bdellovibrio species enter the space between the inner and outer prey membrane, where they consume their prey cells. However, one Bdellovibrio species has been described that consumes its prey from the outside. Here, we describe "Ca Bdellovibrio qaytius," a novel member of the genus Bdellovibrio that also remains outside the prey cell throughout its replication cycle. Unexpectedly, the genome of "Ca Bdellovibrio" is much more similar to the genomes of intracellular predators than to the species with a similar life cycle. Since "Ca Bdellovibrio" is also a basal representative of this genus, we hypothesize that extracellular predation could be the ancestral predation strategy.

Bdellovibrio and Like Organisms Are Predictors of Microbiome Diversity in Distinct Host Groups.

Biodiversity is generally believed to be a main determinant of ecosystem functioning. This principle also applies to the microbiome and could consequently contribute to host health. According to ecological theory, communities are shaped by top predators whose direct and indirect interactions with community members cause stability and diversity. Bdellovibrio and like organisms (BALOs) are a neglected group of predatory bacteria that feed on Gram-negative bacteria and can thereby influence microbiome composition. We asked whether BALOs can predict biodiversity levels in microbiomes from distinct host groups and environments. We demonstrate that genetic signatures of BALOs are commonly found within the 16S rRNA reads from diverse host taxa. In many cases, their presence, abundance, and especially richness are positively correlated with overall microbiome diversity. Our findings suggest that BALOs can act as drivers of microbial alpha-diversity and should therefore be considered candidates for the restoration of microbiomes and the prevention of dysbiosis.

Variation in genome content and predatory phenotypes between Bdellovibrio sp. NC01 isolated from soil and B. bacteriovorus type strain HD100.

Defining phenotypic and associated genotypic variation among Bdellovibrio may further our understanding of how this genus attacks and kills different Gram-negative bacteria. We isolated Bdellovibrio sp. NC01 from soil. Analysis of 16S rRNA gene sequences and average amino acid identity showed that NC01 belongs to a different species than the type species bacteriovorus. By clustering amino acid sequences from completely sequenced Bdellovibrio and comparing the resulting orthologue groups to a previously published analysis, we defined a 'core genome' of 778 protein-coding genes and identified four protein-coding genes that appeared to be missing only in NC01. To determine how horizontal gene transfer (HGT) may have impacted NC01 genome evolution, we performed genome-wide comparisons of Bdellovibrio nucleotide sequences, which indicated that eight NC01 genomic regions were likely acquired by HGT. To investigate how genome variation may impact predation, we compared protein-coding gene content between NC01 and the B. bacteriovorus type strain HD100, focusing on genes implicated as important in successful killing of prey. Of these, NC01 is missing ten genes that may play roles in lytic activity during predation. Compared to HD100, NC01 kills fewer tested prey strains and kills Escherichia coli ML35 less efficiently. NC01 causes a smaller log reduction in ML35, after which the prey population recovers and the NC01 population decreases. In addition, NC01 forms turbid plaques on lawns of E. coli ML35, in contrast to clear plaques formed by HD100. Linking phenotypic variation in interactions between Bdellovibrio and Gram-negative bacteria with underlying Bdellovibrio genome variation is valuable for understanding the ecological significance of predatory bacteria and evaluating their effectiveness in clinical applications.

A Riboflavin Transporter in Bdellovibrio exovorous JSS.

The ImpX transporters of the drug/metabolite transporter superfamily were first proposed to transport riboflavin (RF; vitamin B2) based on findings of a cis-regulatory RNA element responding to flavin mononucleotide (an FMN riboswitch). Bdellovibrio exovorous JSS has a homolog belonging to this superfamily. It has 10 TMSs and shows 30% identity to the previously characterized ImpX transporter from Fusobacterium nucleatum. However, the ImpX homolog is not regulated by an FMN-riboswitch. In order to test the putative function of the ImpX homolog from B. exovorous (BexImpX), we cloned and heterologously expressed its gene. We used functional complementation, growth inhibition experiments, direct uptake experiments and inhibition studies, suggesting a high degree of specificity for RF uptake. The EC50 for growth with RF was estimated to be in the range 0.5-1 µM, estimated from the half-maximal RF concentration supporting the growth of a RF auxotrophic Escherichia coli strain, but the Khalf for RF uptake was 20 µM. Transport experiments suggested that the energy source is the proton motive force but that NaCl stimulates uptake. Thus, members of the ImpX family members are capable of RF uptake, not only in RF prototrophic species such as F.  nucleatum, but also in the B2 auxotrophic species, B. exovorous.

Diversity, Dynamics, and Distribution of Bdellovibrio and Like Organisms in Perialpine Lakes.

Microbes drive a variety of ecosystem processes and services, but many of them remain largely unexplored because of a lack of knowledge on both the diversity and functionality of some potentially crucial microbiological compartments. This is the case with and within the group of bacterial predators collectively known as Bdellovibrio and like organisms (BALOs). Here, we report the abundance, distribution, and diversity of three families of these obligate predatory Gram-negative bacteria in three perialpine lakes (Lakes Annecy, Bourget, and Geneva). The study was conducted at different depths (near-surface versus 45 or 50 m) from August 2015 to January 2016. Using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and cloning-sequencing approaches, we show that the diversity of BALOs is relatively low and very specific to freshwaters or even the lakes themselves. While the Peredibacteraceae family was represented mainly by a single species (Peredibacter starrii), it could represent up to 7% of the total bacterial cell abundances. Comparatively, the abundances of the two other families (Bdellovibrionaceae and Bacteriovoracaceae) were significantly lower. In addition, the distributions in the water column were very different between the three groups, suggesting various life strategies/niches, as follows: Peredibacteraceae dominated near the surface, while Bdellovibrionaceae and Bacteriovoracaceae were more abundant at greater depths. Statistical analyses revealed that BALOs seem mainly to be driven by depth and temperature. Finally, this original study was also the opportunity to design new quantitative PCR (qPCR) primers for Peredibacteraceae quantification.IMPORTANCE This study highlights the abundance, distribution, and diversity of a poorly known microbial compartment in natural aquatic ecosystems, the Bdellovibrio and like organisms (BALOs). These obligate bacterial predators of other bacteria may have an important functional role. This study shows the relative quantitative importance of the three main families of this group, with the design of a new primer pair, and their diversity. While both the diversity and the abundances of these BALOs were globally low, it is noteworthy that the abundance of the Peredibacteraceae could reach important values.