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

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

Analyses of DGGE banding patterns (PCR-amplified 16S rRNA gene fragments) in microcosms established with indigenous prey species and reference strains.

<p>Microcosms were established in DB6 water and samples were taken at various time points. (A) Microcosms spike with <i>Vv</i> and <i>Vv</i>2. (B) Microcosms spiked with <i>Vp</i> and <i>Vibrio sp.</i> (VB). (C) Microcosms inoculated with <i>Pseudoalteromonas sp.</i> (PSAM) and <i>Photobacterium sp.</i> (PHBT). Open circles indicate the excised and sequenced bands.</p

Measurements of environmental parameters of water samples collected to establish microcosm experiments.

<p>Measurements of environmental parameters of water samples collected to establish microcosm experiments.</p

Numbers of <i>Bacteriovorax</i> from the microcosms established with native bacteria and reference strains (<i>Vv</i>,<i>Vp</i>), respectively.

<p>Microcosms were established in DB6 water. Bars indicate standard errors of the mean (N = 3).</p

Analyses of DGGE banding patterns (PCR-amplified 16S rRNA gene fragments) in microcosms amended with freshwater species and the reference strains.

<p>Microcosms were established in DB4 water and samples were taken at various time points. (A) Microcosms amended with <i>Vv</i> and <i>Vp</i> as reference prey. (B) Microcosms established with <i>V. cholera</i> (Vc), <i>E. coli</i> (Ec) and <i>P. putida</i> (Pp). Lanes labeled pre-spike, 48 h, 72 h, 96 h and 120 h indicate the time points at which the samples were removed from the microcosm. Open circles indicate the excised and sequenced bands.</p

Numbers of <i>Bacteriovorax</i> from microcosms amended with three freshwater bacteria and the reference strains (<i>Vv</i>,<i>Vp</i>) respectively.

<p>Microcosms were established in DB4 waters. Samples were taken at various time intervals. Bars indicate standard errors of the mean (N = 3).</p

Identification of bacteria (based on 16S rDNA sequence similarity to the nearest neighbor from NCBI database) in samples retrieved from microcosms established with freshwater bacteria in DB4 water (See Fig. 6 for position of the bands).

<p>Identification of bacteria (based on 16S rDNA sequence similarity to the nearest neighbor from NCBI database) in samples retrieved from microcosms established with freshwater bacteria in DB4 water (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034174#pone-0034174-g006" target="_blank">Fig. 6</a> for position of the bands).</p

Identification of bacteria (based on 16S rDNA sequence similarity to the nearest neighbor from NCBI database) in samples retrieved from microcosms established with indigenous saltwater prey species in DB6 water (See Fig. 7 for position of the bands).

<p>Identification of bacteria (based on 16S rDNA sequence similarity to the nearest neighbor from NCBI database) in samples retrieved from microcosms established with indigenous saltwater prey species in DB6 water (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034174#pone-0034174-g007" target="_blank">Fig. 7</a> for position of the bands).</p

Predominant <i>Bacteriovorax</i> OTUs recovered from the microcosms established with freshwater prey and reference strains.

<p>Microcosms were amended with <i>Vv</i> (A), <i>Vp</i> (B), <i>V. cholera</i> (<b>C</b>), <i>E. coli</i> (D) and <i>P. putida</i> (E). Clusters based on 96.5% 16S rRNA gene sequence similarity are numbered consistently with previous reports <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034174#pone.0034174-Davidov1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034174#pone.0034174-Pineiro1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034174#pone.0034174-Pineiro2" target="_blank">[17]</a>.</p