Predatory Bacteriovorax communities ordered by various prey species

Abstract

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.

Figure 1. Kinetics of the lysis of freshwater prey species and reference strains (Vv,Vp) by Bacteriovorax.

Both test (with predators) and control (without predators) microcosms were established in DB4 water and measurements of cell density in both were taken by OD. Bars indicate standard errors of the mean of three replicates; in some cases bars are too small to be visible.

Figure 2. Numbers of Bacteriovorax from microcosms amended with three freshwater bacteria and the reference strains (Vv,Vp) respectively.

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

Figure 3. Kinetics of the lysis of indigenous saltwater prey by Bacteriovorax over time.

Both test (with predators) and control (without predators) microcosms were established in DB6 water and cell density was measured by OD. Bars indicate standard errors of the mean (N = 3).

Figure 4. Numbers of Bacteriovorax from the microcosms established with native bacteria and reference strains (Vv,Vp), respectively.

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

Figure 5. Predominant Bacteriovorax OTUs recovered from the microcosms established with freshwater prey and reference strains.

Microcosms were amended with Vv (A), Vp (B), V. cholera (C), E. coli (D) and P. putida (E). Clusters based on 96.5% 16S rRNA gene sequence similarity are numbered consistently with previous reports , , .

Figure 6. Analyses of DGGE banding patterns (PCR-amplified 16S rRNA gene fragments) in microcosms amended with freshwater species and the reference strains.

Microcosms were established in DB4 water and samples were taken at various time points. (A) Microcosms amended with Vv and Vp as reference prey. (B) Microcosms established with V. cholera (Vc), E. coli (Ec) and P. putida (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.

Figure 7. Analyses of DGGE banding patterns (PCR-amplified 16S rRNA gene fragments) in microcosms established with indigenous prey species and reference strains.

Microcosms were established in DB6 water and samples were taken at various time points. (A) Microcosms spike with Vv and Vv2. (B) Microcosms spiked with Vp and Vibrio sp. (VB). (C) Microcosms inoculated with Pseudoalteromonas sp. (PSAM) and Photobacterium sp. (PHBT). Open circles indicate the excised and sequenced bands.