Sergio E. Morales

Bacterial gene abundances as indicators of greenhouse gas emission in soils

Nitrogen fixing and denitrifying bacteria, respectively, control bulk inputs and outputs of nitrogen in soils, thereby mediating nitrogen-based greenhouse gas emissions in an ecosystem. Molecular techniques were used to evaluate the relative abundances of nitrogen fixing, denitrifying and two numerically dominant ribotypes (based on the ⩾97% sequence similarity at the 16S rRNA gene) of bacteria in plots representing 10 agricultural and other land-use practices at the Kellogg biological station long-term ecological research site. Quantification of nitrogen-related functional genes (nitrite reductase, nirS; nitrous oxide reductase, nosZ; and nitrogenase, nifH) as well as two dominant 16S ribotypes (belonging to the phyla Acidobacteria, Thermomicrobia) allowed us to evaluate the hypothesis that microbial community differences are linked to greenhouse gas emissions under different land management practices. Our results suggest that the successional stages of the ecosystem are strongly linked to bacterial functional group abundance, and that the legacy of agricultural practices can be sustained over decades. We also link greenhouse gas emissions with specific compositional responses in the soil bacterial community and assess the use of denitrifying gene abundances as proxies for determining nitrous oxide emissions from soils.

Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival

Recent physiological and ecological studies have challenged the long-held belief that microbial metabolism of molecular hydrogen (H2) is a niche process. To gain a broader insight into the importance of microbial H2 metabolism, we comprehensively surveyed the genomic and metagenomic distribution of hydrogenases, the reversible enzymes that catalyse the oxidation and evolution of H2. The protein sequences of 3286 non-redundant putative hydrogenases were curated from publicly available databases. These metalloenzymes were classified into multiple groups based on (1) amino acid sequence phylogeny, (2) metal-binding motifs, (3) predicted genetic organisation and (4) reported biochemical characteristics. Four groups (22 subgroups) of [NiFe]-hydrogenase, three groups (6 subtypes) of [FeFe]-hydrogenases and a small group of [Fe]-hydrogenases were identified. We predict that this hydrogenase diversity supports H2-based respiration, fermentation and carbon fixation processes in both oxic and anoxic environments, in addition to various H2-sensing, electron-bifurcation and energy-conversion mechanisms. Hydrogenase-encoding genes were identified in 51 bacterial and archaeal phyla, suggesting strong pressure for both vertical and lateral acquisition. Furthermore, hydrogenase genes could be recovered from diverse terrestrial, aquatic and host-associated metagenomes in varying proportions, indicating a broad ecological distribution and utilisation. Oxygen content (pO2) appears to be a central factor driving the phylum- and ecosystem-level distribution of these genes. In addition to compounding evidence that H2 was the first electron donor for life, our analysis suggests that the great diversification of hydrogenases has enabled H2 metabolism to sustain the growth or survival of microorganisms in a wide range of ecosystems to the present day. This work also provides a comprehensive expanded system for classifying hydrogenases and identifies new prospects for investigating H2 metabolism.

Empirical Testing of 16S rRNA Gene PCR Primer Pairs Reveals Variance in Target Specificity and Efficacy Not Suggested by In Silico Analysis

ABSTRACT Phylogenetic and “fingerprinting” analyses of the 16S rRNA genes of prokaryotes have been a mainstay of microbial ecology during the last two decades. However, many methods and results from studies that rely on the 16S rRNA gene for detection and quantification of specific microbial taxa have seemingly received only cursory or even no validation. To directly examine the efficacy and specificity of 16S rRNA gene-based primers for phylum-, class-, and operational taxonomic unit-specific target amplification in quantitative PCR, we created a collection of primers based solely on an extensive soil bacterial 16S rRNA gene clone library containing ∼5,000 sequences from a single soil sample (i.e., a closed site-specific library was used to create PCR primers for use at this site). These primers were initially tested in silico prior to empirical testing by PCR amplification of known target sequences and of controls based on disparate phylogenetic groups. Although all primers were highly specific according to the in silico analysis, the empirical analyses clearly exhibited a high degree of nonspecificity for many of the phyla or classes, while other primers proved to be highly specific. These findings suggest that significant care must be taken when interpreting studies whose results were obtained with target specific primers that were not adequately validated, especially where population densities or dynamics have been inferred from the data. Further, we suggest that the reliability of quantification of specific target abundance using 16S rRNA-based quantitative PCR is case specific and must be determined through rigorous empirical testing rather than solely in silico.

Extensive Phylogenetic Analysis of a Soil Bacterial Community Illustrates Extreme Taxon Evenness and the Effects of Amplicon Length, Degree of Coverage, and DNA Fractionation on Classification and Ecological Parameters

ABSTRACT To thoroughly investigate the bacterial community diversity present in a single composite sample from an agricultural soil and to examine potential biases resulting from data acquisition and analytical approaches, we examined the effects of percent G+C DNA fractionation, sequence length, and degree of coverage of bacterial diversity on several commonly used ecological parameters (species estimation, diversity indices, and evenness). We also examined variation in phylogenetic placement based on multiple commonly used approaches (ARB alignments and multiple RDP tools). The results demonstrate that this soil bacterial community is highly diverse, with 1,714 operational taxonomic units demonstrated and 3,555 estimated (based on the Chao1 richness estimation) at 97% sequence similarity using the 16S rRNA gene. The results also demonstrate a fundamental lack of dominance (i.e., a high degree of evenness), with 82% of phylotypes being encountered three times or less. The data also indicate that generally accepted cutoff values for phylum-level taxonomic classification might not be as applicable or as general as previously assumed and that such values likely vary between prokaryotic phyla or groups.

Persistence of the dominant soil phylum Acidobacteria by trace gas scavenging

Significance The mechanisms that the “dormant microbial majority” use to remain energized in nutrient-starved soil ecosystems have long remained elusive. In this work, we used an isolate of the highly abundant but poorly understood soil phylum Acidobacteria as a model for understanding microbial persistence mechanisms. When the bacterium entered a persistent state due to nutrient-exhaustion, we showed it could scavenge the trace concentrations of molecular hydrogen gas (H2) found in ambient air using a specialized high-affinity enzyme. These findings demonstrate that Acidobacteria can consume H2 and contribute to global hydrogen cycling. We propose that consumption of trace gases such as H2 provides a dependable general mechanism for dominant soil phyla to generate the maintenance energy required for long-term survival. The majority of microbial cells in global soils exist in a spectrum of dormant states. However, the metabolic processes that enable them to survive environmental challenges, such as nutrient-limitation, remain to be elucidated. In this work, we demonstrate that energy-starved cultures of Pyrinomonas methylaliphatogenes, an aerobic heterotrophic acidobacterium isolated from New Zealand volcanic soils, persist by scavenging the picomolar concentrations of H2 distributed throughout the atmosphere. Following the transition from exponential to stationary phase due to glucose limitation, the bacterium up-regulates by fourfold the expression of an eight-gene operon encoding an actinobacteria-type H2-uptake [NiFe]-hydrogenase. Whole-cells of the organism consume atmospheric H2 in a first-order kinetic process. Hydrogen oxidation occurred most rapidly under oxic conditions and was weakly associated with the cell membrane. We propose that atmospheric H2 scavenging serves as a mechanism to sustain the respiratory chain of P. methylaliphatogenes when organic electron donors are scarce. As the first observation of H2 oxidation to our knowledge in the Acidobacteria, the second most dominant soil phylum, this work identifies new sinks in the biogeochemical H2 cycle and suggests that trace gas oxidation may be a general mechanism for microbial persistence.

Atmospheric hydrogen scavenging: from enzymes to ecosystems.

ABSTRACT We have known for 40 years that soils can consume the trace amounts of molecular hydrogen (H2) found in the Earths atmosphere. This process is predicted to be the most significant term in the global hydrogen cycle. However, the organisms and enzymes responsible for this process were only recently identified. Pure culture experiments demonstrated that several species of Actinobacteria, including streptomycetes and mycobacteria, can couple the oxidation of atmospheric H2 to the reduction of ambient O2. A combination of genetic, biochemical, and phenotypic studies suggest that these organisms primarily use this fuel source to sustain electron input into the respiratory chain during energy starvation. This process is mediated by a specialized enzyme, the group 5 [NiFe]-hydrogenase, which is unusual for its high affinity, oxygen insensitivity, and thermostability. Atmospheric hydrogen scavenging is a particularly dependable mode of energy generation, given both the ubiquity of the substrate and the stress tolerance of its catalyst. This minireview summarizes the recent progress in understanding how and why certain organisms scavenge atmospheric H2. In addition, it provides insight into the wider significance of hydrogen scavenging in global H2 cycling and soil microbial ecology.

H2 metabolism is widespread and diverse among human colonic microbes

ABSTRACT Microbial molecular hydrogen (H2) cycling is central to metabolic homeostasis and microbial composition in the human gastrointestinal tract. Molecular H2 is produced as an endproduct of carbohydrate fermentation and is reoxidised primarily by sulfate-reduction, acetogenesis, and methanogenesis. However, the enzymatic basis for these processes is incompletely understood and the hydrogenases responsible have not been investigated. In this work, we surveyed the genomic and metagenomic distribution of hydrogenase-encoding genes in the human colon to infer dominant mechanisms of H2 cycling. The data demonstrate that 70% of gastrointestinal microbial species listed in the Human Microbiome Project encode the genetic capacity to metabolise H2. A wide variety of anaerobically-adapted hydrogenases were present, with [FeFe]-hydrogenases predominant. We subsequently analyzed the hydrogenase gene content of stools from 20 healthy human subjects. The hydrogenase gene content of all samples was overwhelmingly dominated by fermentative and electron-bifurcating [FeFe]-hydrogenases emerging from the Bacteroidetes and Firmicutes. This study supports that H2 metabolism in the human gut is driven by fermentative H2 production and interspecies H2 transfer. However, it suggests that electron-bifurcation rather than respiration is the dominant mechanism of H2 reoxidation in the human colon, generating reduced ferredoxin to sustain carbon-fixation (e.g. acetogenesis) and respiration (via the Rnf complex). This work provides the first comprehensive bioinformatic insight into the mechanisms of H2 metabolism in the human colon.

Functional Response of a Near-Surface Soil Microbial Community to a Simulated Underground CO2 Storage Leak

Understanding the impacts of leaks from geologic carbon sequestration, also known as carbon capture and storage, is key to developing effective strategies for carbon dioxide (CO2) emissions management and mitigation of potential negative effects. Here, we provide the first report on the potential effects of leaks from carbon capture and storage sites on microbial functional groups in surface and near-surface soils. Using a simulated subsurface CO2 storage leak scenario, we demonstrate how CO2 flow upward through the soil column altered both the abundance (DNA) and activity (mRNA) of microbial functional groups mediating carbon and nitrogen transformations. These microbial responses were found to be seasonally dependent and correlated to shifts in atmospheric conditions. While both DNA and mRNA levels were affected by elevated CO2, they did not react equally, suggesting two separate mechanisms for soil microbial community response to high CO2 levels. The results did not always agree with previous studies on elevated atmospheric (rather than subsurface) CO2 using FACE (Free-Air CO2 Enrichment) systems, suggesting that microbial community response to CO2 seepage from the subsurface might differ from its response to atmospheric CO2 increases.

Use of Microcalorimetry To Determine the Costs and Benefits to Pseudomonas putida Strain KT2440 of Harboring Cadmium Efflux Genes

ABSTRACT 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.

Phylogenetic and functional potential links pH and N2O emissions in pasture soils

Denitrification is mediated by microbial, and physicochemical, processes leading to nitrogen loss via N2O and N2 emissions. Soil pH regulates the reduction of N2O to N2, however, it can also affect microbial community composition and functional potential. Here we simultaneously test the link between pH, community composition, and the N2O emission ratio (N2O/(NO + N2O + N2)) in 13 temperate pasture soils. Physicochemical analysis, gas kinetics, 16S rRNA amplicon sequencing, metagenomic and quantitative PCR (of denitrifier genes: nirS, nirK, nosZI and nosZII) analysis were carried out to characterize each soil. We found strong evidence linking pH to both N2O emission ratio and community changes. Soil pH was negatively associated with N2O emission ratio, while being positively associated with both community diversity and total denitrification gene (nir & nos) abundance. Abundance of nosZII was positively linked to pH, and negatively linked to N2O emissions. Our results confirm that pH imposes a general selective pressure on the entire community and that this results in changes in emission potential. Our data also support the general model that with increased microbial diversity efficiency increases, demonstrated in this study with lowered N2O emission ratio through more efficient conversion of N2O to N2.