The aeroponic rhizosphere microbiome: community dynamics in early succession suggest strong selectional forces.

In the last decade there has been increased interest in the manipulation of rhizosphere microbial communities in soilless systems (hydroponics) through the addition of plant growth promoting microbes (PGPMs) to increase plant nutrition, lower plant stress response, and control pathogens. This method of crop management requires documenting patterns in communities living in plant roots throughout the growing season to inform decisions on timing of application and composition of the supplemental PGPM consortium. As a contribution to this effort, we measured changes in the bacterial community through early succession (first 26 days) in plant root biofilms growing in an indoor commercial aeroponic system where roots were sprayed with a mist of nutrient-amended water. By 12 days following seed germination, a root-associated community had established that was distinct from the source communities found circulating in the system. Successional patterns in the community over the following 2 weeks (12-26 days) included changes in abundance of bacterial groups that have been documented in published literature as able to utilize plant root exudates, release plant hormones, or augment nutrient availability. Six bacterial families/genera (Hydrogenophilaceae, Rhizobium, Legionellaceae, Methylophilus, Massilia, or Herbaspirillum) were the most abundant in each root sample, comprising 8-37% of the microbiome. Given the absence of soil-associated microbial communities in hydroponic systems, they provide an ideal design for isolating plant-microbial interactions and identifying key components possibly contributing to plant health.

Enzymatic and spectroscopic properties of a thermostable [NiFe]­hydrogenase performing H2-driven NAD+-reduction in the presence of O2.

Biocatalysts that mediate the H2-dependent reduction of NAD+ to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD+-reducing [NiFe]­hydrogenase that sustains catalytic activity at high temperatures and in the presence of O2, which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH, encoding the soluble NAD+-reducing [NiFe]­hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1T (Ht). The HtSH was recombinantly overproduced in a hydrogenase-free mutant of the well-studied, H2-oxidizing betaproteobacterium Ralstonia eutropha H16 (Re). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H2-mediated NAD+ reduction activity was observed at 80°C and pH6.5, and catalytic activity was found to be sustained at low O2 concentrations. Infrared spectroscopic analyses revealed a spectral pattern for as-isolated HtSH that is remarkably different from those of the closely related ReSH and other [NiFe]­hydrogenases. This indicates an unusual configuration of the oxidized catalytic center in HtSH. Complementary electron paramagnetic resonance spectroscopic analyses revealed spectral signatures similar to related NAD+-reducing [NiFe]­hydrogenases. This study lays the groundwork for structural and functional analyses of the HtSH as well as application of this enzyme for H2-driven cofactor recycling under oxic conditions at elevated temperatures.

Oxidative phosphorylation in a thermophilic, facultative chemoautotroph, Hydrogenophilus thermoluteolus, living prevalently in geothermal niches.

Hydrogenophilus is a thermophilic, facultative chemoautotroph, which lives prevalently in high temperature geothermal niches. Despite the environmental distribution, little is known about its oxidative phosphorylation. Here, we show that inverted membrane vesicles derived from Hydrogenophilus thermoluteolus cells autotrophically cultivated with H2 formed a proton gradient on the addition of succinate, dl-lactate, and NADH, and exhibited oxidation activity toward these three organic compounds. These indicate the capability of mixotrophic growth of this bacterium. Biochemical analysis demonstrated that the same vesicles contained an F-type ATP synthase. The F1 sector of the ATP synthase purified from H. thermoluteolus membranes exhibited optimal ATPase activity at 65°C. Transformed Escherichia coli membranes expressing H. thermoluteolus F-type ATP synthase exhibited the same temperature optimum for the ATPase. These findings shed light on H. thermoluteolus oxidative phosphorylation from the aspects of membrane bioenergetics and ATPase biochemistry, which must be fundamental and advantageous in the biogeochemical cycles occurred in the high temperature geothermal niches.

Draft genome sequence of a psychrotolerant sulfur-oxidizing bacterium, Sulfuricella denitrificans skB26, and proteomic insights into cold adaptation.

Except for several conspicuous cases, very little is known about sulfur oxidizers living in natural freshwater environments. Sulfuricella denitrificans skB26 is a psychrotolerant sulfur oxidizer recently isolated from a freshwater lake as a representative of a new genus in the class Betaproteobacteria. In this study, an approximately 3.2-Mb draft genome sequence of strain skB26 was obtained. In the draft genome, consisting of 23 contigs, a single rRNA operon, 43 tRNA genes, and 3,133 coding sequences were identified. The identified genes include those required for sulfur oxidation, denitrification, and carbon fixation. Comparative proteomic analysis was conducted to assess cold adaptation mechanisms of this organism. From cells grown at 22°C and 5°C, proteins were extracted for analysis by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry. In the cells cultured at 5°C, relative abundances of ribosomal proteins, cold shock proteins, and DEAD/DEAH box RNA helicases were increased in comparison to those at 22°C. These results suggest that maintenance of proper translation is critical for growth under low-temperature conditions, similar to the case for other cold-adapted prokaryotes.

Heterologous synthesis of cytochrome c' by Escherichia coli is not dependent on the System I cytochrome c biogenesis machinery.

Hydrogenophilus thermoluteolus cytochrome c' (PHCP) has typical spectral properties previously observed for other cytochromes c', which comprise Ambler's class II cytochromes c. The PHCP protein sequence (135 amino acids) deduced from the cloned gene is the most homologous (55% identity) to that of cytochrome c' from Allochromatium vinosum (AVCP). These findings indicate that PHCP forms a four-helix bundle structure, similar to AVCP. Strikingly, PHCP with a covalently bound heme was heterologously synthesized in the periplasm of Escherichia coli strains deficient in the DsbD protein, a component of the System I cytochrome c biogenesis machinery. The heterologous synthesis of PHCP by aerobically growing E. coli also occurred without a plasmid carrying the genes for Ccm proteins, other components of the System I machinery. Unlike Ambler's class I general cytochromes c, the synthesis of PHCP is not dependent on the System I machinery and exhibits similarity to that of E. coli periplasmic cytochrome b(562), a 106-residue four-helix bundle.

Hydrogenophilus islandicus sp. nov., a thermophilic hydrogen-oxidizing bacterium isolated from an Icelandic hot spring.

A novel chemolithotrophic bacterium, strain 16C(T), was isolated from a hot spring in Graendalur, south-west Iceland. Cells of this organism were Gram-negative, rod-shaped and motile. The isolate was aerobic and capable of chemolithotrophic growth on hydrogen and carbon dioxide, heterotrophic growth on butyrate and several other organic compounds, and mixotrophic growth on butyrate, hydrogen and carbon dioxide. Heterotrophic growth was generally enhanced in the presence of yeast extract. Autotrophic growth on hydrogen was observed at pH values between 6.0 and 10.0 and temperatures between 35 and 60 °C; optimum growth conditions were pH 7.0 and 55 °C. The DNA G+C content was 63.9 mol%. 16S rRNA gene sequence analysis showed that strain 16C(T) was a member of a distinct species belonging to the class Betaproteobacteria and was most closely related to Hydrogenophilus thermoluteolus NBRC 14978(T) and Hydrogenophilus hirschii DSM 11420(T). The major cellular fatty acids were straight-chain C(16 : 0) (44.98 %) and C(18 : 1)ω7c (17.93 %), as well as cyclic C(17 : 0) (13.90 %) and C(19 : 0)ω8c (4.67 %) fatty acids. Based on its physiological and molecular properties, it is concluded that strain 16C(T) represents a novel species within the genus Hydrogenophilus, for which the name Hydrogenophilus islandicus is proposed; the type strain is 16C(T) (=DSM 21442(T)=JCM 16106(T)).

Ferrous iron oxidation and rusticyanin in halotolerant, acidophilic 'Thiobacillus prosperus'.

The halotolerant acidophile 'Thiobacillus prosperus' was shown to require chloride for growth. With ferrous iron as substrate, growth occurred at a rate similar to that of the well-studied acidophile Acidithiobacillus ferrooxidans. Previously, the salt (NaCl) requirement of 'T. prosperus' was not clear and its growth on ferrous iron was described as poor. A subtractive hybridization of cDNAs from ferrous-iron-grown and sulfur-grown 'T. prosperus' strain V6 led to identification of a cluster of genes similar to the rus operon reported to encode ferrous iron oxidation in A. ferrooxidans. However, the 'T. prosperus' gene cluster did not contain a homologue of cyc1, which is thought to encode a key cytochrome c in the pathway of electron transport from ferrous iron in A. ferrooxidans. Rusticyanin, another key protein in ferrous iron oxidation by A. ferrooxidans, was present in 'T. prosperus' at similar concentrations in cells grown on either ferrous iron or sulfur.

Thiosulfate oxidation by a moderately thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus.

The moderately thermophilic Betaproteobacterium, Hydrogenophilus thermoluteolus, not only oxidizes hydrogen, the principal electron donor for growth, but also sulfur compounds including thiosulfate, a process enabled by sox genes. A periplasmic extract of H. thermoluteolus showed significant thiosulfate oxidation activity. Ten genes apparently involved in thiosulfate oxidation (soxEFCDYZAXBH) were found on a 9.7-kb DNA fragment of the H. thermoluteolus chromosome. The proteins SoxAX, which represent c-type cytochromes, were co-purified from the cells of H. thermoluteolus; they enhanced the thiosulfate oxidation activity of the periplasmic extract when added to the latter.

Presence of Hydrogenophilus thermoluteolus DNA in accretion ice in the subglacial Lake Vostok, Antarctica, assessed using rrs, cbb and hox.

The 3561 m Vostok ice core sample originating from the subglacial Lake Vostok accretion (frozen lake water) ice with sediment inclusions was thoroughly studied by various means to confirm the presence of the thermophile bacterium Hydrogenophilus thermoluteolus reported earlier in the 3607 m accretion ice sample. PCR and molecular-phylogenetic analyses performed in two independent laboratories were made using different 16S rRNA gene (rrs) targeted primers. As a result, rrs-targeted PCR permitted to recover several very closely related clones with a small genetic distance to Hydrogenophilus thermoluteolus (< 1%). In addition, RubisCO (cbbL or rbcL) and NiFe-Hydrogenase (hoxV or hupL) targeted PCR have also allowed to recover sequences highly related to Hydrogenophilus thermoluteolus. All these results point to the presence of thermophilic chemoautotrophic microorganisms in Lake Vostok accretion ice. They presumably originate from deep faults in the bedrock cavity containing the lake in which episodes of seismotectonic activity would release debris along with microbial cells.

Community and cultivation analysis of arsenite oxidizing biofilms at Hot Creek.

At Hot Creek in California, geothermally derived arsenite is rapidly oxidized to arsenate. This process is mediated by microorganisms colonizing the surfaces of submerged aquatic macrophytes in the creek. Here we describe a multifaceted approach to characterizing this biofilm community and its activity. Molecular techniques were used to describe the community as a function of 16S-rRNA gene diversity. Cultivation-based strategies were used to enumerate and isolate three novel arsenite oxidizers, strains YED1-18, YED6-4 and YED6-21. All three strains are beta-Proteobacteria, of the genus Hydrogenophaga. Because these strains were isolated from the highest (i.e. million-fold) dilutions of disrupted biofilm suspensions, they represent the most numerically significant arsenite oxidizers recovered from this community. One clone (Hot Creek Clone 44) obtained from an inventory of the 16S rDNA sequence diversity present in the biofilm was found to be 99.6% identical to the 16S rDNA sequence of the isolate YED6-21. On the basis of most probable number (MPN) analyses, arsenite-oxidizing bacteria were found to account for 6-56% of the cultivated members of the community. Using MPN values, we could estimate an upper bound on the value of V(max) for the community of 1 x 10(-9)micromole arsenite min(-1) cell(-1). This estimate represents the first normalization of arsenite oxidation rates to MPN cell densities for a microbial community in a field incubation experiment.

Tepidiphilus margaritifer gen. nov., sp. nov., isolated from a thermophilic aerobic digester.

A moderately thermophilic bacterium is described, strain N2-214(T), that was isolated from an enrichment culture, growing on caprolactone, obtained from a sample from a water-treatment sludge aerobic digester operating at temperatures around 60 degrees C. The organism was aerobic, Gram-negative, oxidase- and catalase-positive, with a polar flagellum, and capable of growth at temperatures as high as 61 degrees C. The major fatty acids of strain N2-214(T) were C(16 : 0), C(18 : 1) and cyclo-C(19 : 0). The phylogenetic relationships of the strain, derived from 16S rRNA gene sequence comparisons, demonstrated it to be a member of the beta-subclass of the PROTEOBACTERIA: The highest 16S rDNA sequence similarity of isolate N2-214(T) was to Azoarcus buckelii (91.9 %), Thauera aromatica (92 %) and Hydrogenophilus thermoluteolus (92.7 %). On the basis of phylogenetic analyses and physiological and chemotaxonomic characteristics, it is proposed that isolate N2-214(T) (=DSM 15129(T)=LMG 21637(T)) represents a new genus and species, Tepidiphilus margaritifer gen. nov., sp. nov.