Philippe Constant

Streptomycetes contributing to atmospheric molecular hydrogen soil uptake are widespread and encode a putative high‐affinity [NiFe]‐hydrogenase

Uptake of molecular hydrogen (H2) by soil is a biological reaction responsible for approximately 80% of the global loss of atmospheric H2. Indirect evidence obtained over the last decades suggests that free soil hydrogenases with an unusually high affinity for H2 are carrying out the reaction. This assumption has recently been challenged by the isolation of Streptomyces sp. PCB7, displaying the high-affinity H2 uptake activity previously attributed to free soil enzymes. While this finding suggests that actinobacteria could be responsible for atmospheric H2 soil uptake, the ecological importance of H2-oxidizing streptomycetes remains to be investigated. Here, we show that high-affinity H2 uptake activity is widespread among the streptomycetes. Among 14 streptomycetes strains isolated from temperate forest and agricultural soils, six exhibited a high-affinity H2 uptake activity. The gene encoding the large subunit of a putative high-affinity [NiFe]-hydrogenase (hydB-like gene sequence) was detected exclusively in the isolates exhibiting high-affinity H2 uptake. Catalysed reporter deposition-fluorescence in situ hybridization (CARD-FISH) experiments targeting hydB-like gene transcripts and H2 uptake assays performed with strain PCB7 suggested that streptomycetes spores catalysed the H2 uptake activity. Expression of the activity in term of biomass revealed that 10(6)-10(7) H2-oxidizing bacteria per gram of soil should be sufficient to explain in situ H2 uptake by soil. We propose that specialized H2-oxidizing actinobacteria are responsible for the most important sink term in the atmospheric H2 budget.

Genome Data Mining and Soil Survey for the Novel Group 5 [NiFe]-Hydrogenase To Explore the Diversity and Ecological Importance of Presumptive High-Affinity H2-Oxidizing Bacteria

ABSTRACT Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H2 possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H2-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H2 oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 106 to 108 hhyL gene copies g (dry weight)−1. Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H2-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H2 oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H2 by soil, because high-affinity H2 oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur.

Isolation of Streptomyces sp. PCB7, the first microorganism demonstrating high-affinity uptake of tropospheric H2.

Microbial-mediated soil uptake accounts for ∼80% of the global tropospheric dihydrogen (H2) sinks. Studies conducted over the last three decades provide indirect evidences that H2 soil uptake is mediated by free soil hydrogenases or by unknown microorganisms that have a high affinity for H2. The exact nature of these hypothetical free soil enzymes or of H2-consuming microorganisms remains elusive because the activity has never been observed in pure culture. Here, we present the first aerobic microorganism able to consume tropospheric H2 at ambient levels. A dynamic microcosm chamber was developed to enrich a microbial consortium with a high affinity for H2, from which selected bacterial and fungal strains were isolated and tested for H2 uptake. Strain PCB7 had a H2 consumption activity that followed a Michaelis–Menten kinetics, with an apparent Km of 11 p.p.m.v. and a H2 threshold concentration <0.100 p.p.m.v., corresponding to the high-affinity uptake of tropospheric H2 observed in soil. 16S ribosomal RNA gene sequences showed that strain PCB7 is highly related to several Streptomyces species. H2 consumption occurred during the sporulation period of the bacterium. Addition of nickel increased the activity, suggesting that the enzymes involved in H2 consumption belong to the NiFe uptake class of hydrogenases. Because this is the first microorganism showing a high-affinity uptake of tropospheric H2, we anticipate that Streptomyces sp. PCB7 will become a model organism for the understanding of the environmental factors influencing H2 soil uptake.

Tropospheric H2 budget and the response of its soil uptake under the changing environment

Molecular hydrogen (H(2)) is an indirect greenhouse gas present at the trace level in the atmosphere. So far, the sum of its sources and sinks is close to equilibrium, but its large-scale utilization as an alternative energy carrier would alter its atmospheric burden. The magnitude of the emissions associated with a future H(2)-based economy is difficult to predict and remains a matter of debate. Previous attempts to predict the impact that a future H(2)-based economy would exert on tropospheric chemistry were realized by considering a steady rate of microbial-mediated soil uptake, which is currently responsible of ~80% of the tropospheric H(2) losses. Although soil uptake, also known as dry deposition is the most important sink for tropospheric H(2), microorganisms involved in the activity remain elusive. Given that microbial-mediated H(2) soil uptake is influenced by several environmental factors, global change should exert a significant effect on the activity and then, assuming a steady H(2) soil uptake rate for the future may be mistaken. Here, we present an overview of tropospheric H(2) sources and sinks with an emphasis on microbial-mediated soil uptake process. Future researches are proposed to investigate the influence that global change would exert on H(2) dry deposition and to identify microorganisms involved H(2) soil uptake activity.

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.

Surveying the endomicrobiome and ectomicrobiome of bark beetles: The case of Dendroctonus simplex

Many bark beetles belonging to the Dendroctonus genus carry bacterial and fungal microbiota, forming a symbiotic complex that helps the insect to colonize the subcortical environment of the host tree. However, the biodiversity of those bacteria at the surface of the cuticle or inside the body parts of bark beetles is not well established. The aim of this study was to characterize the bacterial microbiome associated with the eastern larch beetle, Dendroctonus simplex, using bacterial 16S rRNA gene pyrosequencing. The ecto- and endomicrobiome and the subcortical galleries were investigated. Several bacterial genera were identified, among which Pseudomonas, Serratia and Yersinia are associated with the surface of the beetle cuticle, and genera belonging to Enterobacteriaceae and Gammaproteobacteria with the interior of the insect body. The index of dissimilarity indicates that the bacterial microbiome associated with each environment constitutes exclusive groups. These results suggest the presence of distinct bacterial microbiota on the surface of the cuticle and the interior of D. simplex body. Additionally, the bacterial diversity identified in the galleries is substantially different from the ectomicrobiome, which could indicate a selection by the insect. This study reports for the first time the identification of the eastern larch beetle microbiome.

Land-use influences the distribution and activity of high affinity CO-oxidizing bacteria associated to type I-coxL genotype in soil

Soil carboxydovore bacteria are the biological sink of atmospheric carbon monoxide (CO). The initial oxidation of CO is catalyzed by a CO-dehydrogenase (CODH), and the gene coxL encodes the large subunit of the enzyme. Only a few carboxydovore isolates were shown to oxidize atmospheric CO and little is known about the potential impact of global change on the ecophysiology of this functional group. The main objective of this study was to assess the impact of land-use and soil properties on coxL gene diversity and identify molecular indicators for the soil uptake of atmospheric CO. Soil samples were collected in three neighboring sites encompassing different land-use types, namely deciduous forest, larch plantation and maize field. CO uptake activity was related to total carbon and nitrogen content in soil, with the highest activity observed in deciduous forest. An extensive coxL database was assembled to optimize a PCR detection assay targeting sequences belonging to functional type I-CODH and hypothetical type II-CODH. Fully replicated coxL gene libraries unveiled a unique molecular signature in deciduous forest soil, with enrichment of type I sequences. Genetic profiles of larch and maize monocultures were not statistically different and showed higher level of coxL gene richness than deciduous forest. Soil water content and CO uptake activity explained 38% of the variation of coxL gene profiles in a canonical ordination analysis, leading to the identification of sequences belonging to the δ-Proteobacteria cluster as indicator for high affinity CO uptake activity. Enrichment of type I and δ-Proteobacteria coxL sequences in deciduous forest were confirmed by qPCR in an independent soil survey. CO uptake activity in model carboxydovore bacteria suggested that a significant fraction of detected putative high affinity CO oxidizers were active in soil. Land-use was a driving force separating coxL diversity in deciduous forest from monocultures.

Breathing air to save energy--new insights into the ecophysiological role of high-affinity [NiFe]-hydrogenase in Streptomyces avermitilis.

The Streptomyces avermitilis genome encodes a putative high‐affinity [NiFe]‐hydrogenase conferring the ability to oxidize tropospheric H2 in mature spores. Here, we used a combination of transcriptomic and mutagenesis approaches to shed light on the potential ecophysiological role of the enzyme. First, S. avermitilis was either exposed to low or hydrogenase‐saturating levels of H2 to investigate the impact of H2 on spore transcriptome. In total, 1293 genes were differentially expressed, with 1127 and 166 showing lower and higher expression under elevated H2 concentration, respectively. High H2 exposure lowered the expression of the Sec protein secretion pathway and ATP‐binding cassette‐transporters, with increased expression of genes encoding proteins directing carbon metabolism toward sugar anabolism and lower expression of NADH dehydrogenase in the respiratory chain. Overall, the expression of relA responsible for the synthesis of the pleiotropic alarmone ppGpp decreased upon elevated H2 exposure, which likely explained the reduced expression of antibiotic synthesis and stress response genes. Finally, deletion of hhySL genes resulted in a loss of H2 uptake activity and a dramatic loss of viability in spores. We propose that H2 is restricted to support the seed bank of Streptomyces under a unique survival–mixotrophic energy mode and discuss important ecological implications of this finding.

H2-saturation of high affinity H2-oxidizing bacteria alters the ecological niche of soil microorganisms unevenly among taxonomic groups

Soil microbial communities are continuously exposed to H2 diffusing into the soil from the atmosphere. N2-fixing nodules represent a peculiar microniche in soil where H2 can reach concentrations up to 20,000 fold higher than in the global atmosphere (0.530 ppmv). In this study, we investigated the impact of H2 exposure on soil bacterial community structure using dynamic microcosm chambers simulating soil H2 exposure from the atmosphere and N2-fixing nodules. Biphasic kinetic parameters governing H2 oxidation activity in soil changed drastically upon elevated H2 exposure, corresponding to a slight but significant decay of high affinity H2-oxidizing bacteria population, accompanied by an enrichment or activation of microorganisms displaying low-affinity for H2. In contrast to previous studies that unveiled limited response by a few species, the relative abundance of 958 bacterial ribotypes distributed among various taxonomic groups, rather than a few distinct taxa, was influenced by H2 exposure. Furthermore, correlation networks showed important alterations of ribotype covariation in response to H2 exposure, suggesting that H2 affects microbe-microbe interactions in soil. Taken together, our results demonstrate that H2-rich environments exert a direct influence on soil H2-oxidizing bacteria in addition to indirect effects on other members of the bacterial communities.

The tale of a neglected energy source: Elevated hydrogen exposure affects both microbial diversity and function in soil.

ABSTRACT The enrichment of H2-oxidizing bacteria (HOB) by H2 generated by nitrogen-fixing nodules has been shown to have a fertilization effect on several different crops. The benefit of HOB is attributed to their production of plant growth-promoting factors, yet their interactions with other members of soil microbial communities have received little attention. Here we report that the energy potential of H2, when supplied to soil, alters ecological niche partitioning of bacteria and fungi, with multifaceted consequences for both generalist and specialist microbial functions. We used dynamic microcosms to expose soil to the typical atmospheric H2 mixing ratio (0.5 ppmv) permeating soils, as well as mixing ratios comparable to those found at the soil-nodule interface (10,000 ppmv). Elevated H2 exposure exerted direct effects on two HOB subpopulations distinguished by their affinity for H2 while enhancing community level carbon substrate utilization potential and lowering CH4 uptake activity in soil. We found that H2 triggered changes in the abundance of microorganisms that were reproducible yet inconsistent across soils at the taxonomic level and even among HOB. Overall, H2 exposure altered microbial process rates at an intensity that depends upon soil abiotic and biotic features. We argue that further examination of direct and indirect effects of H2 on soil microbial communities will lead to a better understanding of the H2 fertilization effect and soil biogeochemical processes. IMPORTANCE An innovative dynamic microcosm chamber system was used to demonstrate that H2 diffusing in soil triggers changes in the distribution of HOB and non-HOB. Although the response was uneven at the taxonomic level, an unexpected coordinated response of microbial functions was observed, including abatement of CH4 oxidation activity and stimulation of carbon turnover. Our work suggests that elevated H2 rewires soil biogeochemical structure through a combination of direct effects on the growth and persistence of HOB and indirect effects on a variety of microbial processes involving HOB and non-HOB.