Use of microcalorimetry to determine the costs and benefits to Pseudomonas putida strain KT2440 of harboring cadmium efflux genes.

A novel microcalorimetric approach was used to analyze the responses of a metal-tolerant soil bacterium (Pseudomonas putida strain KT2440) to metal resistance gene deletions in cadmium-amended media. As hypothesized, under cadmium stress, the wild-type strain benefited from the resistance genes by entering the exponential growth phase earlier than two knockout strains. In the absence of cadmium, strain KT1, carrying a deletion in the main component (czcA1) of a Cd/Zn chemiosmotic efflux transporter (CzcCBA1), grew more efficiently than the wild type and released ∼700 kJ (per mole of biomass carbon) less heat than the wild-type strain, showing the energetic cost of maintaining CzcCBA1 in the absence of cadmium. A second mutant strain (KT4) carrying a different gene deletion, ΔcadA2, which encodes the main Cd/Pb efflux transporter (a P-type ATPase), did not survive beyond moderate cadmium concentrations and exhibited a decreased growth yield in the absence of cadmium. Therefore, CadA2 plays an essential role in cadmium resistance and perhaps serves an additional function. The results of this study provide direct evidence that heavy metal cation efflux mechanisms facilitate shorter lag phases in the presence of metals and that the maintenance and expression of tolerance genes carry quantifiable energetic costs and benefits.

Habitat heterogeneity and associated microbial community structure in a small-scale floodplain hyporheic flow path.

The Nyack floodplain is located on the Middle Fork of the Flathead River, an unregulated, pristine, fifth-order stream in Montana, USA, bordering Glacier National Park. The hyporheic zone is a nutritionally heterogeneous floodplain component harboring a diverse array of microbial assemblages essential in fluvial biogeochemical cycling, riverine ecosystem productivity, and trophic interactions. Despite these functions, microbial community structure in pristine hyporheic systems is not well characterized. The current study was designed to assess whether physical habitat heterogeneity within the hyporheic zone of the Nyack floodplain was sufficient to drive bacterial beta diversity between three different hyporheic flow path locations. Habitat heterogeneity was assessed by measuring soluble reactive phosphorous, nitrate, dissolved organic carbon, dissolved oxygen, and soluble total nitrogen levels seasonally at surface water infiltration, advection, and exfiltration zones. Significant spatial differences were detected in dissolved oxygen and nitrate levels, and seasonal differences were detected in dissolved oxygen, nitrate, and dissolved organic carbon levels. Denaturing gradient gel electrophoresis (DGGE) and cell counts indicated that bacterial diversity increased with abundance, and DGGE fingerprints covaried with nitrate levels where water infiltrated the hyporheic zone. The ribosomal gene phylogeny revealed that hyporheic habitat heterogeneity was sufficient to drive beta diversity between bacterial assemblages. Phylogenetic (P) tests detected sequence disparity between the flow path locations. Small distinct lineages of Firmicutes, Actinomycetes, Planctomycetes, and Acidobacteria defined the infiltration zone and alpha- and beta-proteobacterial lineages delineated the exfiltration and advection zone communities. These data suggest that spatial habitat heterogeneity drives hyporheic microbial community development and that attempts to understand functional differences between bacteria inhabiting nutritionally heterogeneous hyporheic environments might begin by focusing on the biology of these taxa.

Phylogeny of arbuscular mycorrhizal fungi predicts community composition of symbiosis-associated bacteria.

Many physicochemical and biotic aspects of the soil environment determine the community composition of bacteria. In this study, we examined the effects of arbuscular mycorrhizal fungi, common symbionts of higher plants, on the composition of bacterial communities after long-term (7-8 years) enrichment culture in the presence of a plant host. We showed that the phylogeny of arbuscular mycorrhizal fungal isolates was a highly significant predictor of bacterial community composition, as assessed by cluster analysis, redundancy analysis and linear discriminant analysis of phospholipid fatty acid patterns. Numerous phospholipid fatty acids differed between the phylogenetic groupings; this pattern also held for fungal-origin phospholipid fatty acids and in a combined bacterial/fungal analysis, suggesting that categorizing phospholipid fatty acids into predominantly bacterial and fungal origin did not affect the overall outcome. The mechanisms underlying this observation could include substrate quality (and quantity) effects, interactions mediated by the host plant (e.g. rhizodeposition) and direct biotic interactions between arbuscular mycorrhizal fungi and bacterial populations. Our results suggest that aspects of arbuscular mycorrhizal fungal functions may be partially explained by the symbiosis-accompanying bacterial communities, a possibility that should be explicitly considered in studies examining the roles of arbuscular mycorrhizal fungal species diversity in soil and ecosystem processes.

Heavy metal tolerance genes alter cellular thermodynamics in Pseudomonas putida and river Pseudomonas spp. and influence amebal predation.

Predation rates were measured for two Acanthamoeba castellanii strains feeding on metal-tolerant and metal-sensitive strains of Pseudomonas putida and compared with cellular thermodynamic data. Predation rates by A. castellanii strain ATCC 30010 correlated with cell volume of the prey. To explore whether this observation could be environmentally relevant, pseudomonad species were isolated from a pristine and a metal-contaminated river and were paired based on phylogenetic and physiological relatedness. Then, cellular thermodynamics and predation rates were measured on the most similar pseudomonad pair. Under cadmium stress, the strain from contaminated river sediments, Pseudomonas sp. CF150, exited metabolic dormancy faster than its pair from pristine sediments, Pseudomonas sp. N9, but consumed available resources less efficiently (more energy was lost as heat). Predation rates by both strains of ameba were greater on Pseudomonas sp. CF150 than on Pseudomonas sp. N9 at the highest cadmium concentration.

Pedobacter nyackensis sp. nov., Pedobacter alluvionis sp. nov. and Pedobacter borealis sp. nov., isolated from Montana flood-plain sediment and forest soil.

Three Gram-negative, rod-shaped, non-spore-forming eubacterial strains were isolated in western Montana, USA, and subjected to taxonomic studies. Strains NWG-II14(T) and NWER-II11(T) were isolated from hyporheic sediments of a large alluvial flood plain, whereas strain G-1(T) was isolated from a conifer forest soil. On the basis of 16S rRNA gene sequence similarity, strains NWG-II14(T), NWER-II11(T) and G-1(T) were shown to belong to the family Sphingobacteriaceae and are most closely related to various species of the genus Pedobacter. The results of molecular, physiological and biochemical tests allowed genotypic and phenotypic differentiation of these three strains from 23 Pedobacter species with validly published names. The three isolates therefore represent novel species, for which the names Pedobacter nyackensis sp. nov. (type strain NWG-II14(T) =DSM 19625(T) =LMG 24260(T)), Pedobacter alluvionis sp. nov. (type strain NWER-II11(T) =DSM 19624(T) =LMG 24258(T)) and Pedobacter borealis sp. nov. (type strain G-1(T) =DSM 19626(T) =LMG 24259(T)) are proposed.

Hyporheic microbial community development is a sensitive indicator of metal contamination.

Accurate natural resource damage assessment necessitates monitoring organisms or communities that respond most sensitively to contaminants. Observational studies have demonstrated a correlation between fluvial heavy metal deposition and hyporheic microbial community structure. To establish a causal relationship between sediment metal content and the structure of colonizing bacterial communities, we performed a controlled field experiment River sediments of 1.75-2.36 mm in diameter with five different contaminant concentrations were collected from an environmental metal contamination gradient. Sediments were sterilized and then recolonized by incubation in the hyporheic zone of an uncontaminated river (i.e., a common garden experiment was performed). A significant correlation between hyporheic microbial community structure and heavy metal contamination (R2 = 0.81) was observed. The abundance of two phylogenetic groups was highly correlated with the level of heavy metal contamination (Group I, R2 = 0.96; Group III, R2 = 0.96, most closely affiliated with the alpha- and gamma-proteobacteria, respectively). Microbial community structural responses were detected at metal concentrations an order of magnitude lower than those previously reported to impact benthic macroinvertebrate communities. We conclude that hyporheic microbial communities could offer the most sensitive method for assessing natural resource damage in lotic ecosystems in response to fluvial heavy metal deposition.

Relationship between communities and processes; new insights from a field study of a contaminated ecosystem.

We used a 93-year-old mine waste contamination gradient in alluvial soil to explore the relationship between ecosystem level functioning and community structure in a chronically stressed ecosystem. The sensitivity of broad functional parameters (in situ soil respiration, microbial biomass, above and below ground plant biomass) and microbial diversity [phospholipid fatty acid (PLFA) abundance and richness] were compared. Functional responses were linear with respect to contaminants while thresholds were detected in the community structural response to contamination along the gradient. For example, in situ soil respiration was negatively and linearly correlated to contamination concentration (R = -0.783, P < 0.01), but changes in microbial community structure only became evident where contaminant concentrations were greater than 28 times above background levels. Our results suggest that functional redundancy does not prevent depression of ecosystem function in the long-term.

Determining rates of change and evaluating group-level resiliency differences in hyporheic microbial communities in response to fluvial heavy-metal deposition.

Prior field studies by our group have demonstrated a relationship between fluvial deposition of heavy metals and hyporheic-zone microbial community structure. Here, we determined the rates of change in hyporheic microbial communities in response to heavy-metal contamination and assessed group-level differences in resiliency in response to heavy metals. A controlled laboratory study was performed using 20 flowthrough river mesocosms and a repeated-measurement factorial design. A single hyporheic microbial community was exposed to five different levels of an environmentally relevant metal treatment (0, 4, 8, 16, and 30% sterilized contaminated sediments). Community-level responses were monitored at 1, 2, 4, 8, and 12 weeks via denaturing gradient gel electrophoresis and quantitative PCR using group-specific primer sets for indigenous populations most closely related to the alpha-, beta-, and gamma-proteobacteria. There was a consistent, strong curvilinear relationship between community composition and heavy-metal contamination (R(2) = 0.83; P < 0.001), which was evident after only 7 days of metal exposure (i.e., short-term response). The abundance of each phylogenetic group was negatively affected by the heavy-metal treatments; however, each group recovered from the metal treatments to a different extent and at a unique rate during the course of the experiment. The structure of hyporheic microbial communities responded rapidly and at contamination levels an order of magnitude lower than those shown to elicit a response in aquatic macroinvertebrate assemblages. These studies indicate that hyporheic microbial communities are a sensitive and useful indicator of heavy-metal contamination in streams.

Differences in hyporheic-zone microbial community structure along a heavy-metal contamination gradient.

The hyporheic zone of a river is nonphotic, has steep chemical and redox gradients, and has a heterotrophic food web based on the consumption of organic carbon entrained from downwelling surface water or from upwelling groundwater. The microbial communities in the hyporheic zone are an important component of these heterotrophic food webs and perform essential functions in lotic ecosystems. Using a suite of methods (denaturing gradient gel electrophoresis, 16S rRNA phylogeny, phospholipid fatty acid analysis, direct microscopic enumeration, and quantitative PCR), we compared the microbial communities inhabiting the hyporheic zone of six different river sites that encompass a wide range of sediment metal loads resulting from large base-metal mining activity in the region. There was no correlation between sediment metal content and the total hyporheic microbial biomass present within each site. However, microbial community structure showed a significant linear relationship with the sediment metal loads. The abundances of four phylogenetic groups (groups I, II, III, and IV) most closely related to alpha-, beta-, and gamma-proteobacteria and the cyanobacteria, respectively, were determined. The sediment metal content gradient was positively correlated with group III abundance and negatively correlated with group II abundance. No correlation was apparent with regard to group I or IV abundance. This is the first documentation of a relationship between fluvially deposited heavy-metal contamination and hyporheic microbial community structure. The information presented here may be useful in predicting long-term effects of heavy-metal contamination in streams and provides a basis for further studies of metal effects on hyporheic microbial communities.

Seasonal dynamics of shallow-hyporheic-zone microbial community structure along a heavy-metal contamination gradient.

Heavy metals contaminate numerous freshwater streams and rivers worldwide. Previous work by this group demonstrated a relationship between the structure of hyporheic microbial communities and the fluvial deposition of heavy metals along a contamination gradient during the fall season. Seasonal variation has been documented in microbial communities in numerous terrestrial and aquatic environments, including the hyporheic zone. The current study was designed to assess whether relationships between hyporheic microbial community structure and heavy-metal contamination vary seasonally by monitoring community structure along a heavy-metal contamination gradient for more than a year. No relationship between total bacterial abundance and heavy metals was observed (R(2) = 0.02, P = 0.83). However, denaturing gradient gel electrophoresis pattern analysis indicated a strong and consistent linear relationship between the difference in microbial community composition (populations present) and the difference in the heavy metal content of hyporheic sediments throughout the year (R(2) = 0.58, P < 0.001). Correlations between heavy-metal contamination and the abundance of four specific phylogenetic groups (most closely related to the alpha, beta, and gamma-proteobacteria and cyanobacteria) were apparent only during the fall and early winter, when the majority of organic matter is deposited into regional streams. These seasonal data suggest that the abundance of susceptible populations responds to heavy metals primarily during seasons when the potential for growth is highest.