Predator-Prey Interactions between Halobacteriovorax and Pathogenic Vibrio parahaemolyticus Strains: Geographical Considerations and Influence of Vibrio Hemolysins.

Halobacteriovorax is a genus of naturally occurring marine predatory bacteria that attack, replicate within, and lyse vibrios and other bacteria. This study evaluated the specificity of four Halobacteriovorax strains against important sequence types (STs) of clinically relevant Vibrio parahaemolyticus, including pandemic strains ST3 and ST36. The Halobacteriovorax bacteria were previously isolated from seawater from the Mid-Atlantic, Gulf of Mexico, and Hawaiian coasts of the United States. Specificity screening was performed using a double agar plaque assay technique on 23 well-characterized and genomically sequenced V. parahaemolyticus strains isolated from infected individuals from widely varying geographic locations within the United States. With few exceptions, results showed that Halobacteriovorax bacteria were excellent predators of the V. parahaemolyticus strains regardless of the origins of the predator or prey. Sequence types and serotypes of V. parahaemolyticus did not influence host specificity, nor did the presence or absence of genes for the thermostable direct hemolysin (TDH) or the TDH-related hemolysin, although faint (cloudy) plaques were present when one or both hemolysins were absent in three of the Vibrio strains. Plaque sizes varied depending on both the Halobacteriovorax and Vibrio strains evaluated, suggesting differences in Halobacteriovorax replication and/or growth rates. The very broad infectivity of Halobacteriovorax toward pathogenic strains of V. parahaemolyticus makes Halobacteriovorax a strong candidate for use in commercial processing applications to enhance the safety of seafoods. IMPORTANCE Vibrio parahaemolyticus is a formidable obstacle to seafood safety. Strains pathogenic to humans are numerous and difficult to control, especially within molluscan shellfish. The pandemic spread of ST3 and ST36 has caused considerable concern, but many other STs are also problematic. The present study demonstrates broad predatory activity of Halobacteriovorax strains obtained along U.S. coastal waters from the Mid-Atlantic, Gulf Coast, and Hawaii toward strains of pathogenic V. parahaemolyticus. This broad activity against clinically relevant V. parahaemolyticus strains suggests a role for Halobacteriovorax in mediating pathogenic V. parahaemolyticus levels in seafoods and their environment as well as the potential application of these predators in the development of new disinfection technologies to reduce pathogenic vibrios in molluscan shellfish and other seafoods.

Environmental Regulation of the Distribution and Ecology of Bdellovibrio and Like Organisms.

The impact of key environmental factors, salinity, prey, and temperature, on the survival and ecology of Bdellovibrio and like bacteria (BALOs), including the freshwater/terrestrial, non-halotolerant group and the halophilic Halobacteriovorax strains, has been assessed based on a review of data in the literature. These topics have been studied by numerous investigators for nearly six decades now, and much valuable information has been amassed and reported. The collective data shows that salinity, prey, and temperature play a major role in, not only the growth and survival of BALOs, but also the structure and composition of BALO communities and the distribution of the predators. Salinity is a major determinant in the selection of BALO habitats, distribution, prey bacteria, and systematics. Halophilic BALOs require salt for cellular functions and are found only in saltwater habitats, and prey primarily on saltwater bacteria. To the contrary, freshwater/terrestrial BALOs are non-halotolerant and inhibited by salt concentrations greater than 0.5%, and are restricted to freshwater, soils, and other low salt environments. They prey preferentially on bacteria in the same habitats. The halophilic BALOs are further separated on the basis of their tolerance to various salt concentrations. Some strains are found in low salt environments and others in high salt regions. In situ studies have demonstrated that salinity gradients in estuarine systems govern the type of BALO communities that will persist within a specific gradient. Bacterial prey for BALOs functions more than just being a substrate for the predators and include the potential for different prey species to structure the BALO community at the phylotype level. The pattern of susceptibility or resistance of various bacteria species has been used almost universally to differentiate strains of new BALO isolates. However, the method suffers from a lack of uniformity among different laboratories. The use of molecular methods such as comparative analysis of the 16S rDNA gene and metagenomics have provided more specific approaches to distinguished between isolates. Differences in temperature growth range among different BALO groups and strains have been demonstrated in many laboratory experiments. The temperature optima and growth range for the saltwater BALOs is typically lower than that of the freshwater/terrestrial BALOs. The collective data shows not only that environmental factors have a great impact on BALO ecology, but also how the various factors affect BALO populations in nature.

Relative Contributions of Halobacteriovorax and Bacteriophage to Bacterial Cell Death under Various Environmental Conditions.

The role of protists and bacteriophages in bacterial predation in the microbial food web has been well studied. There is mounting evidence that Bdellovibrio and like organisms (BALOs) also contribute to bacterial mortality and, in some cases, more so than bacteriophages. A full understanding of the ecologic function of the microbial food web requires recognition of all major predators and the magnitude of each predator's contribution. Here we investigated the contribution of Halobacteriovorax, one of the BALOs, and bacteriophages when incubated with their common prey, Vibrio vulnificus, in a seawater microcosm. We observed that Halobacteriovorax was the greatest responder to the prey, increasing 18-fold with a simultaneous 4.4-log-unit reduction of V. vulnificus at 40 h, whereas the bacteriophage population showed no significant increase. In subsequent experiments to formulate a medium that would support the predatory activities and replication of both predators, low-nutrient media favored the predation and replication of the Halobacteriovorax, whereas higher-nutrient media enhanced phage growth. The greatest prey reduction and replication of both Halobacteriovorax and phage were observed in media with moderate nutrient levels. Additional experiments show that the predatory activities of both predators were influenced by environmental conditions, specifically, temperature and salinity. The two predators combined exerted greater control on V. vulnificus, a synergism that may be exploited for practical applications to reduce bacterial populations. These findings suggest that along with bacteriophage and protists, Halobacteriovorax has the potential to have a prominent role in bacterial mortality and cycling of nutrients, two vital ecologic functions.IMPORTANCE Although much has been reported about the marine microbial food web and the role of micropredators, specifically viruses and protists, the contribution of Bdellovibrio-like predators has largely been ignored, posing a major gap in understanding food web processes. A complete scenario of the microbial food web cannot be developed until the roles of all major micropredators and the magnitude of their contributions to bacterial mortality, structuring of microbial communities, and cycling of nutrients are assessed. Here we show compelling evidence that Halobacteriovorax, a predatory bacterium, is a significant contributor to bacterial death and, in some cases, may rival viruses as agents of bacterial mortality. These results advance current understanding of the microbial loop and top-down control on the bacterial community.

Analysis of gene gain and loss in the evolution of predatory bacteria.

Predatory bacteria are ubiquitously distributed in nature in including in aquatic environments, sewage, intestinal tracts of animals and humans, rhizophere and, soils. However, our understanding of their evolutionary history is limited. Results of recent studies have shown that acquiring novel genes is a major force driving bacterial evolution. Therefore, to gain a better understanding of the impact of gene gain and loss in the evolution of bacterial predators, this study employed comparative genomic approaches to identify core-set gene families and species-specific gene families, and model gene gain and loss events among 11 genomes that represented diverse lineages. In total, 1977 gene families were classified. Of these 509 (pattern 11111111111) were present all of the 11 species. Among the non-core set gene families, 52 were present only in saltwater bacteria predators and had no ortholog in the other genomes. Similarly 109 and 44 were present only in the genomes of Micavibrio spp. and Bdellovibrio spp., respectively. In this study, the gain loss mapping engine GLOOME was selected to analyze and estimate the expectations and probabilities of both gain and loss events in the predatory bacteria. In total, 354 gene families were involved in significant gene gain events, and 407 gene families were classified into gene loss events with high supported value. Moreover, 18 families from the core set gene family were identified as putative genes under positive selection. The results of this study suggest that acquisition of particular genes that encode functional proteins in metabolism and cellular processes and signaling, especially ABC systems, may help bacterial predators adapt to surrounding environmental changes and present different predation strategies for survival in their habitats.

Halobacteriovorax, an underestimated predator on bacteria: potential impact relative to viruses on bacterial mortality.

Predation on bacteria and accompanying mortality are important mechanisms in controlling bacterial populations and recycling of nutrients through the microbial loop. The agents most investigated and seen as responsible for bacterial mortality are viruses and protists. However, a body of evidence suggests that predatory bacteria such as the Halobacteriovorax (formerly Bacteriovorax), a Bdellovibrio-like organism, contribute substantially to bacterial death. Until now, conclusive evidence has been lacking. The goal of this study was to better understand the contributors to bacterial mortality by addressing the poorly understood role of Halobacteriovorax and how their role compares with that of viruses. The results revealed that when a concentrated suspension of Vibrio parahaemolyticus was added into microcosms of estuarine waters, the native Halobacteriovorax were the predators that responded first and most rapidly. Their numbers increased by four orders of magnitude, whereas V. parahaemolyticus prey numbers decreased by three orders of magnitude. In contrast, the extant virus population showed little increase and produced little change in the prey density. An independent experiment with stable isotope probing confirmed that Halobacteriovorax were the predators primarily responsible for the mortality of the V. parahaemolyticus. The results show that Halobacteriovorax have the potential to be significant contributors to bacterial mortality, and in such cases, predation by Halobacteriovorax may be an important mechanism of nutrient recycling. These conclusions add another dimension to bacterial mortality and the recycling of nutrients.

Draft genome sequences for the obligate bacterial predators Bacteriovorax spp. of four phylogenetic clusters.

Bacteriovorax is the halophilic genus of the obligate bacterial predators, Bdellovibrio and like organisms. The predators are known for their unique biphasic life style in which they search for and attack their prey in the free living phase; penetrate, grow, multiply and lyse the prey in the intraperiplasmic phase. Bacteriovorax isolates representing four phylogenetic clusters were selected for genomic sequencing. Only one type strain genome has been published so far from the genus Bacteriovorax. We report the genomes from non-type strains isolated from aquatic environments. Here we describe and compare the genomic features of the four strains, together with the classification and annotation.

Genome-wide comparative analysis of ABC systems in the Bdellovibrio-and-like organisms.

Bdellovibrio-and-like organisms (BALOs) are gram-negative, predatory bacteria with wide variations in genome sizes and GC content and ecological habitats. The ATP-binding cassette (ABC) systems have been identified in several prokaryotes, fungi and plants and have a role in transport of materials in and out of cells and in cellular processes. However, knowledge of the ABC systems of BALOs remains obscure. A total of 269 putative ABC proteins were identified in BALOs. The genes encoding these ABC systems occupy nearly 1.3% of the gene content in freshwater Bdellovibrio strains and about 0.7% in their saltwater counterparts. The proteins found belong to 25 ABC system families based on their structural characteristics and functions. Among these, 16 families function as importers, 6 as exporters and 3 are involved in various cellular processes. Eight of these 25 ABC system families were deduced to be the core set of ABC systems conserved in all BALOs. All Bacteriovorax strains have 28 or less ABC systems. On the contrary, the freshwater Bdellovibrio strains have more ABC systems, typically around 51. In the genome of Bdellovibrio exovorus JSS (CP003537.1), 53 putative ABC systems were detected, representing the highest number among all the BALO genomes examined in this study. Unexpected high numbers of ABC systems involved in cellular processes were found in all BALOs. Phylogenetic analysis suggests that the majority of ABC proteins can be assigned into many separate families with high bootstrap supports (>50%). In this study, a general framework of sequence-structure-function connections for the ABC systems in BALOs was revealed providing novel insights for future investigations.

454 Pyrosequencing reveals diversity of Bdellovibrio and like organisms in fresh and salt water.

Bdellovibrio-and-like organisms (BALOs) are Gram-negative, predatory bacteria that inhabit terrestrial, freshwater and saltwater environments. They have been detected primarily by culture-dependent methods which have limitations. In this study, diversity and community structure of BALOs in freshwater and saltwater samples were characterized by 16S rRNA gene pyrosequencing with specific BALO group primers. Novel Bacteriovorax 16S rDNA sequences were found both in saltwater enrichment cultures and in situ environmental samples, but no new operational taxonomic units were detected in the freshwater samples. The results revealed unexpected diversity of BALOs and advance understanding of the similarities and differences between Bdellovibrio and Bacteriovorax diversity and distribution in the environment.

Reclassification of Bacteriovorax marinus as Halobacteriovorax marinus gen. nov., comb. nov. and Bacteriovorax litoralis as Halobacteriovorax litoralis comb. nov.; description of Halobacteriovoraceae fam. nov. in the class Deltaproteobacteria.

The taxonomic status of saltwater Bdellovibrio-like prokaryotic predators has been revised to assign species to Halobacteriovorax gen. nov. A reclassification of Bacteriovorax marinus as Halobacteriovorax marinus comb. nov. (type strain ATCC BAA-682(T) = DSM 15412(T)) and Bacteriovorax litoralis as Halobacteriovorax litoralis comb. nov. (type strain ATCC BAA-684(T) = DSM 15409(T)) is proposed. This revision is necessary because a previous proposal to retain saltwater isolates as species of Bacteriovorax and reclassify Bacteriovorax stolpii as Bacteriolyticum stolpii was not approved. The type species of a genus cannot be reassigned to another genus. Bacteriovorax stolpii is thus retained as the type species of Bacteriovorax and Halobacteriovorax marinus is the type species of Halobacteriovorax and of Halobacteriovoraceae fam. nov.

Functional diversity of bacterioplankton in three North Florida freshwater lakes over an annual cycle.

The phylogenetic diversity of freshwater bacterioplankton is widely known; however, there is minimal information on the functional diversity of the bacterial communities in these systems. Understanding the functional diversity of freshwater bacterial communities is important because heterotrophic bacteria can be impacted by anthropogenic perturbation, which in turn can alter biogeochemical cycling. The objective of this study was to use Biolog EcoPlates to acquire spatial and temporal community-level physiological profiles (CLPPs) for three freshwater lakes of different trophic levels and to assess the phylogenetic affiliation of the bacteria responsible for utilizing the various carbon guilds within them by denaturing gradient gel electrophoresis (DGGE). CLPP results showed that bacterial communities utilized the carbon guilds similarly between sites within the three lakes. However, when the metabolic profile of each lake was compared, Lake Bradford and Moore Lake were more similar to one another than to Lake Munson, the eutrophic lake. Additionally, although the bacteria that utilized the five carbon guilds included representatives from the classes α-, ß-, γ-Proteobacteria, Flavobacteria and Sphingobacteria, Lake Munson had the largest number of Flavobacteria and γ-Proteobacteria in comparison to Moore Lake and Lake Bradford. Overall, Biolog analysis was useful in identifying differences in the functional diversity of bacterial communities between lakes of different trophic statuses and can be used as a tool to assess ecosystem health.

Niche partition of Bacteriovorax operational taxonomic units along salinity and temporal gradients in the Chesapeake Bay reveals distinct estuarine strains.

The predatory Bacteriovorax are Gram-negative bacteria ubiquitous in saltwater systems that prey upon other Gram-negative bacteria in a similar manner to the related genus Bdellovibrio. Among the phylogenetically defined clusters of Bacteriovorax, cluster V has only been isolated from estuaries suggesting that it may be a distinct estuarine phylotype. To assess this hypothesis, the spatial and temporal distribution of cluster V and other Bacteriovorax phylogenetic assemblages along the salinity gradient of Chesapeake Bay were determined. Cluster V was expected to be found in significantly greater numbers in low to moderate salinity waters compared to high salinity areas. The analyses of water and sediment samples from sites in the bay revealed cluster V to be present at the lower salinity and not high salinity sites, consistent with it being an estuarine phylotype. Cluster IV had a similar distribution pattern and may also be specifically adapted to estuaries. While the distribution of clusters V and IV were similar for salinity, they were distinct on temperature gradients, being found in cooler and in warmer temperatures, respectively. The differentiation of phylotype populations along the salinity and temporal gradients in Chesapeake Bay revealed distinct niches inhabited by different phylotypes of Bacteriovorax and unique estuarine phylotypes.

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.

Sharing of prey: coinfection of a bacterium by a virus and a prokaryotic predator.

UNLABELLED: Rarely, if ever, has a single bacterial cell been confirmed to simultaneously host two fundamentally different predators. Two such predators are viruses and the predatory prokaryotes known as Bdellovibrio and like organisms. Viruses or bacteriophage are particles requiring prey cells in an active metabolic state to complete their life cycle. The Bdellovibrio and like organisms, unlike viruses, are bacteria that can efficiently infect and grow in prey which are in stationary phase. In this study, electron microscopic examination revealed an unprecedented coinfection by the two agents of Vibrio vulnificus, introducing a new bacterial predation paradigm. Rather than the viruses and Bdellovibrio and like organisms competing for a single prey cell, both can survive in the same cell and successfully reproduce themselves. This is an especially valuable mechanism when the prey is in short supply, and the survival of the predators may be at stake. IMPORTANCE: This article describes the coinfection of a prokaryotic prey or host cell by both a bacteriophage (phage) and the predatory bacterium of the group Bdellovibrio and like organisms (BALOs). Such coinfection has not been previously reported and therefore introduces a new paradigm for predation of bacteria. This finding invites new studies on the interactions of BALOs, phage, and prey in predation. Predation is an important mechanism in nature for helping to keep bacterial populations in check and also plays a major role in the cycling of nutrients through the microbial loop. How dual infection by phage and BALOs imposes on these and other functions of predation is fertile ground for future studies and serves as a keystone reference on bacterial predation and mortality.

Predatory Bacteriovorax communities ordered by various prey species.

The role of predation in altering microbial communities has been studied for decades but few examples are known for bacterial predators. Bacteriovorax are halophilic prokaryotes that prey on susceptible gram-negative bacteria. We recently reported novel observations on the differential selection of Bacteriovorax phylotypes by two different prey, Vibrio parahaemolyticus and Vibrio vulnificus. However, the conclusion is restricted by the limited number of prey tested. In this study, we have conducted two independent investigations involving eight species of prey bacteria while using V. vulnificus and V. parahaemolytics as reference strains. Water samples collected from Dry Bar, Apalachicola Bay were used to establish microcosms which were respectively spiked with prey strains Vibrio cholerae, Escherichia coli or Pseudomonas putida to examine the response of native Bacteriovorax to freshwater bacteria. Indigenous Vibrio sp., Pseudoalteromonas sp., Photobacterium sp. and a clinical strain of V. vulnificus were also tested for the impact of saltwater prey on the Bacteriovorax community. At 24 hour intervals, optical density of the microcosm samples and the abundance of Bacteriovorax were measured over five days. The predominant Bacteriovorax plaques were selected and analyzed by 16S rRNA gene amplification and sequencing. In addition, the impacts of prey on predator population and bacterial community composition were investigated using culture independent denaturing gradient gel electrophoresis. Strikingly, Cluster IV was found consistently as the predominant phylotype produced by the freshwater prey. For all saltwater prey, subgroups of Bacteriovorax phylotype IX were the major predators recovered. The results suggest that prey is an important factor along with temperature, salinity and other environmental parameters in shaping Bacteriovorax communities in aquatic systems.

Prey bacteria shape the community structure of their predators.

Although predator-prey interactions among higher organisms have been studied extensively, only few examples are known for microbes other than protists and viruses. Among the bacteria, the most studied obligate predators are the Bdellovibrio and like organisms (BALOs) that prey on many other bacteria. In the macroscopical world, both predator and prey influence the population size of the other's community, and may have a role in selection. However, selective pressures among prey and predatory bacteria have been rarely investigated. In this study, Bacteriovorax, a predator within the group of BALOs, in environmental waters were fed two prey bacteria, Vibrio vulnificus and Vibrio parahaemolyticus. The two prey species yielded distinct Bacteriovorax populations, evidence that selective pressures shaped the predator community and diversity. The results of laboratory experiments confirmed the differential predation of Bacteriovorax phylotypes on the two bacteria species. Not only did Bacteriovorax Cluster IX exhibit the versatility to be the exclusive efficient predator on Vibrio vulnificus, thereby, behaving as a specialist, but was also able to prey with similar efficiency on Vibrio parahaemolyticus, indicative of a generalist. Therefore, we proposed a designation of versatilist for this predator. This initiative should provide a basis for further efforts to characterize the predatory patterns of bacterial predators. The results of this study have revealed impacts of the prey on Bacteriovorax predation and in structuring the predator community, and advanced understanding of predation behavior in the microbial world.

Soil functional diversity analysis of a bauxite-mined restoration chronosequence.

Soil microorganisms are sensitive to environmental perturbations such that changes in microbial community structure and function can provide early signs of anthropogenic disturbances and even predict restoration success. We evaluated the bacterial functional diversity of un-mined and three chronosequence sites at various stages of rehabilitation (0, 10, and 20 years old) located in the Mocho Mountains of Jamaica. Samples were collected during the dry and wet seasons and analyzed for metal concentrations, microbial biomass carbon, bacterial numbers, and functional responses of soil microbiota using community-level physiological profile (CLPP) assays. Regardless of the season, un-mined soils consisted of higher microbial biomass and numbers than any of the rehabilitated sites. Additionally, the number and rate of substrates utilized and substrate evenness (the distribution of color development between the substrates) were significantly greater in the un-mined soils with carbohydrates being preferentially utilized than amino acids, polymers, carboxylic acids, and esters. To some extent, functional responses varied with the seasons but the least physiological activity was shown by the site rehabilitated in 1987 indicating long-term perturbation to this ecosystem. Small subunit ribosomal DNA (SSUrDNA)-denaturing gradient-gel electrophoresis analyses on the microbiota collected from the most preferred CLPP substrates followed by taxonomic analyses showed Proteobacteria, specifically the gamma-proteobacteria, as the most functionally active phyla, indicating a propensity of this phyla to out-compete other groups under the prevailing conditions. Additionally, multivariate statistical analyses, Shannon's diversity, and evenness indices, principal component analysis, biplot and un-weighted-pair-group method with arithmetic averages dendrograms further confirmed that un-mined sites were distinctly different from the rehabilitated soils.

Survival response of Bacteriovorax in surface biofilm versus suspension when stressed by extremes in environmental conditions.

The Bacteriovorax, previously in the genus Bdellovibrio, are prokaryotes that prey upon many Gram-negative bacteria. They are ubiquitous in salt-water environments and have been reported to have a strong association with biofilms. The purpose of this study was to test the hypothesis that this association affords protection for the Bacteriovorax and enhances their survival in nature when exposed to extreme environmental conditions. Experiments were designed to compare their survival in biofilms versus in suspension when exposed to extremes in salinity and temperature. Natural mixed-population biofilms generated in moderate-salinity (16 per thousand) Patuxent River water and containing Bacteriovorax were exposed to drastic changes in salinity by placing in low-salinity (1 per thousand) river water and salt-free (no measurable salinity) distilled water for up to 14 days. In a separate trial, the biofilm was exposed to extremes in temperature, 5 degrees C and 35 degrees C, for up to 12 weeks in aquarium mesocosms. Simultaneously, suspensions of the Bacteriovorax were exposed to the same extremes in salinity and temperature as biofilms. The results revealed that the Bacteriovorax typically were able to survive for a week or longer while in association with biofilms than when in suspension. These results are consistent with observations from nature and establish that biofilms are important in the survival and ecology of the Bacteriovorax.

Increased diversity of predacious Bdellovibrio-like organisms (blos) as a function of eutrophication in Kumaon Lakes of India.

Predation by Bdellovibrio-like organisms (BLOs) results in bacterial community succession in aquatic ecosystems. The effects of nutrient loading on the distribution and phylogeny of BLOs remain largely unknown. To this end, we present our findings on BLO diversity from four north-Indian lakes that are variable in their trophic status; Nainital is eutrophic, both, Bhimtal and Naukuchiatal are mesotrophic and Sattal remains oligotrophic, respectively. Initially, total heterotrophic bacteria and BLOs were quantified by most probable number (MPN) analyses using Pseudomonas putida and Escherichia coli as prey bacteria. Total bacterial numbers were at least two-logs higher in the eutrophic lake samples compared with oligotrophic lake. Similarly, BLO numbers were approximately 39-fold higher using Pseudomonas sp., which is likely the preferred prey within these lakes. Conversely, significant differences were not observed between mesotrophic and oligotrophic BLO numbers when E. coli was used as the prey. PCR-RFLP of small subunit rDNA (SSU rDNA) of BLOs, followed by cloning, sequencing, and taxonomic categorization revealed distinct differences such that, eutrophic lake consisted of higher BLO diversity compared with mesotrophic and oligotrophic lake, most likely due to both, higher numbers and availability of a diverse population of prey bacteria resulting from nutrient loading in this ecosystem.

Impact of sideways and bottom-up control factors on bacterial community succession over a tidal cycle.

In aquatic systems, bacterial community succession is a function of top-down and bottom-up factors, but little information exists on "sideways" controls, such as bacterial predation by Bdellovibrio-like organisms (BLOs), which likely impacts nutrient cycling within the microbial loop and eventual export to higher trophic groups. Here we report transient response of estuarine microbiota and BLO spp. to tidal-associated dissolved organic matter supply in a river-dominated estuary, Apalachicola Bay, Florida. Both dissolved organic carbon and dissolved organic nitrogen concentrations oscillated over the course of the tidal cycle with relatively higher concentrations observed at low tide. Concurrent with the shift in dissolved organic matter (DOM) supply at low tide, a synchronous increase in numbers of bacteria and predatorial BLOs were observed. PCR-restriction fragment length polymorphism of small subunit rDNA, cloning, and sequence analyses revealed distinct shifts such that, at low tide, significantly higher phylotype abundances were observed from gamma-Proteobacteria, delta-Proteobacteria, Bacteroidetes, and high G+C gram-positive bacteria. Conversely, diversity of alpha-Proteobacteria, beta-Proteobacteria, and Chlamydiales-Verrucomicrobia group increased at high tides. To identify metabolically active BLO guilds, tidal microcosms were spiked with six (13)C-labeled bacteria as potential prey and studied using an adaptation of stable isotope probing. At low tide, representative of higher DOM and increased prey but lower salinity, BLO community also shifted such that mesohaline clusters I and VI were more active; with an increased salinity at high tide, halotolerant clusters III, V, and X were predominant. Eventually, (13)C label was identified from higher micropredators, indicating that trophic interactions within the estuarine microbial food web are potentially far more complex than previously thought.

Development and evaluation of a quantitative real-time PCR assay for the detection of saltwater Bacteriovorax.

Bdellovibrio-and-like-organisms (BALOs) are small, Gram-negative predatory bacteria with the ability to prey on a wide variety of Gram-negative bacteria, and which may have a significant ecological role. Detection and quantification of BALOs by culture-dependent methods are complicated, as their reproduction is dependent upon the use of appropriate prey. For this reason, a sensitive and specific molecular detection method was developed. This paper describes a SYBR Green-based real-time PCR (quantitative PCR) assay that combines the use of a specific 16S rDNA primer with a universal primer for quantitative detection of halophilic Bacteriovorax. 16S rDNA sequences from 174 BALO strains, including both halophilic and freshwater, were aligned and a consensus region was identified that is unique to the halophilic Bacteriovorax strains. A specific primer was designed and analysed for specificity. The PCR conditions were optimized to obtain high specificity and sensitivity. The specificity was evaluated by testing a series of halophilic Bacteriovorax samples and prey specimens, including both pure cultures and environmental saltwater samples. A linear and reproducible standard curve was obtained over a range of 10(1)-10(6) gene copies and the detection limit was determined to be 10 copies of 16S rRNA gene per reaction. The results presented in this study validate the procedure as a rapid, sensitive and accurate method for the detection and quantification of halophilic Bacteriovorax in environmental saltwater samples. It is anticipated that this culture-independent method will facilitate future investigations of the distribution and population dynamics of these interesting predatory bacteria, leading to a better understanding of their ecological role.