In Vivo Titration of Folate Pathway Enzymes.

How enzymes behave in cells is likely different from how they behave in the test tube. Previous in vitro studies find that osmolytes interact weakly with folate. Removal of the osmolyte from the solvation shell of folate is more difficult than removal of water, which weakens binding of folate to its enzyme partners. To examine if this phenomenon occurs in vivo, osmotic stress titrations were performed with Escherichia coli Two strategies were employed: resistance to an antibacterial drug and complementation of a knockout strain by the appropriate gene cloned into a plasmid that allows tight control of expression levels as well as labeling by a degradation tag. The abilities of the knockout and complemented strains to grow under osmotic stress were compared. Typically, the knockout strain could grow to high osmolalities on supplemented medium, while the complemented strain stopped growing at lower osmolalities on minimal medium. This pattern was observed for an R67 dihydrofolate reductase clone rescuing a ΔfolA strain, for a methylenetetrahydrofolate reductase clone rescuing a ΔmetF strain, and for a serine hydroxymethyltransferase clone rescuing a ΔglyA strain. Additionally, an R67 dihydrofolate reductase clone allowed E. coli DH5α to grow in the presence of trimethoprim until an osmolality of ∼0.81 is reached, while cells in a control titration lacking antibiotic could grow to 1.90 osmol.IMPORTANCEE. coli can survive in drought and flooding conditions and can tolerate large changes in osmolality. However, the cell processes that limit bacterial growth under high osmotic stress conditions are not known. In this study, the dose of four different enzymes in E. coli was decreased by using deletion strains complemented by the gene carried in a tunable plasmid. Under conditions of limiting enzyme concentration (lower than that achieved by chromosomal gene expression), cell growth can be blocked by osmotic stress conditions that are normally tolerated. These observations indicate that E. coli has evolved to deal with variations in its osmotic environment and that normal protein levels are sufficient to buffer the cell from environmental changes. Additional factors involved in the osmotic pressure response may include altered protein concentration/activity levels, weak solute interactions with ligands which can make it more difficult for proteins to bind their substrates/inhibitors/cofactors in vivo, and/or viscosity effects.

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.

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.

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.

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.