Genomic and metabolomic profiling of endolithic Rhodococcus fascians strain S11 isolated from an arid serpentine environment.

Genomic and metabolomic studies of endolithic bacteria are essential for understanding their adaptations to extreme conditions of the rock environment and their contributions to mineralization and weathering processes. The endoliths of arid serpentine rocks are exposed to different environmental stresses, including desiccation and re-hydration, temperature fluctuations, oligotrophy, and high concentrations of heavy metals. Bacteria of the genus Rhodococcus commonly inhabit endolithic environments. Here, we describe genomic and metabolomic analyses of the non-pathogenic wild-type Rhodococcus fascians strain S11, isolated from weathered serpentine rock at the arid Khalilovsky massif, Russia. We found that strain S11 lacks the virulence plasmid that functions in the phytopathogenecity of some R. fascians strains. Phenotypic profiling revealed a high pH tolerance, phytase activity and siderophore production. A widely untargeted metabolome analysis performed using an Orbitrap LC-MS/MS method demonstrated the presence of chrysobactin-type siderophores in the culture medium of strain S11. The natural variation of secondary metabolites produced by strain S11 might provide a practical basis for revealing antibacterial, fungicide or insecticidal activities. Finally, plant infection and plant growth stimulation studies showed no observable effect of exposure strain S11 bacteria on the aerial and root parts of Arabidopsis thaliana plants. Based on our findings, R. fascians strain S11 might be promising tool for investigations of organo-mineral interactions, heavy metal bioremediation, and mechanisms of bacterial mediated weathering of plant-free serpentine rock to soil.

Genomic and phenotypic analysis of siderophore-producing Rhodococcus qingshengii strain S10 isolated from an arid weathered serpentine rock environment.

The success of members of the genus Rhodococcus in colonizing arid rocky environments is owed in part to desiccation tolerance and an ability to extract iron through the secretion and uptake of siderophores. Here, we report a comprehensive genomic and taxonomic analysis of Rhodococcus qingshengii strain S10 isolated from eathered serpentine rock at the arid Khalilovsky massif, Russia. Sequence comparisons of whole genomes and of selected marker genes clearly showed strain S10 to belong to the R. qingshengii species. Four prophage sequences within the R. qingshengii S10 genome were identified, one of which encodes for a putative siderophore-interacting protein. Among the ten non-ribosomal peptides synthase (NRPS) clusters identified in the strain S10 genome, two show high homology to those responsible for siderophore synthesis. Phenotypic analyses demonstrated that R. qingshengii S10 secretes siderophores and possesses adaptive features (tolerance of up to 8% NaCl and pH 9) that should enable survival in its native habitat within dry serpentine rock.

Deconstruction of plant biomass by a Cellulomonas strain isolated from an ultra-basic (lignin-stripping) spring.

Plant material falling into the ultra-basic (pH 11.5-11.9) springs within The Cedars, an actively serpentinizing site in Sonoma County, California, is subject to conditions that mimic the industrial pretreatment of lignocellulosic biomass for biofuel production. We sought to obtain hemicellulolytic/cellulolytic bacteria from The Cedars springs that are capable of withstanding the extreme alkaline conditions wherein calcium hydroxide-rich water removes lignin, making cell wall polysaccharides more accessible to microorganisms and their enzymes. We enriched for such bacteria by adding plant debris from the springs into a synthetic alkaline medium with ground tissue of the biofuel crop switchgrass (Panicum virgatum L.) as the sole source of carbon. From the enrichment culture we isolated the facultative anaerobic bacterium Cellulomonas sp. strain FA1 (NBRC 114238), which tolerates high pH and catabolizes the major plant cell wall-associated polysaccharides cellulose, pectin, and hemicellulose. Strain FA1 in monoculture colonized the plant material and degraded switchgrass at a faster rate than the community from which it was derived. Cells of strain FA1 could be acclimated through subculturing to grow at a maximal concentration of 13.4% ethanol. A strain FA1-encoded ß-1, 4-endoxylanase expressed in E. coli was active at a broad pH range, displaying near maximal activity at pH 6-9. Discovery of this bacterium illustrates the value of extreme alkaline springs in the search for microorganisms with potential for consolidated bioprocessing of plant biomass to biofuels and other valuable bio-inspired products.

Biological consilience of hydrogen sulfide and nitric oxide in plants: Gases of primordial earth linking plant, microbial and animal physiologies.

Hydrogen sulfide (H2S) is produced in the mammalian body through the enzymatic activities of cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). A growing number of studies have revealed that biogenic H2S produced in tissues is involved in a variety of physiological responses in mammals including vasorelaxation and neurotransmission. It is now evident that mammals utilize H2S to regulate multiple signaling systems, echoing the research history of the gaseous signaling molecules nitric oxide (NO) and carbon monoxide (CO) that had previously only been recognized for their cytotoxicity. In the human diet, meats (mammals, birds and fishes) and vegetables (plants) containing cysteine and other sulfur compounds are the major dietary sources for endogenous production of H2S. Plants are primary producers in ecosystems on the earth and they synthesize organic sulfur compounds through the activity of sulfur assimilation. Although plant H2S-producing activities have been known for a long time, our knowledge of H2S biology in plant systems has not been updated to the extent of mammalian studies. Here we review recent progress on H2S studies, highlighting plants and bacteria. Scoping the future integration of H2S, NO and O2 biology, we discuss a possible linkage between physiology, ecology and evolutional biology of gas metabolisms that may reflect the historical changes of the Earth's atmospheric composition.

Host Phenology and Leaf Effects on Susceptibility of California Bay Laurel to Phytophthora ramorum.

Spread of the plant pathogen Phytophthora ramorum, causal agent of the forest disease sudden oak death, is driven by a few competent hosts that support spore production from foliar lesions. The relationship between traits of a principal foliar host, California bay laurel (Umbellularia californica), and susceptibility to P. ramorum infection were investigated with multiple P. ramorum isolates and leaves collected from multiple trees in leaf-droplet assays. We examined whether susceptibility varies with season, leaf age, or inoculum position. Bay laurel susceptibility was highest during spring and summer and lowest in winter. Older leaves (>1 year) were more susceptible than younger ones (8 to 11 months). Susceptibility was greater at leaf tips and edges than the middle of the leaf. Leaf surfaces wiped with 70% ethanol were more susceptible to P. ramorum infection than untreated leaf surfaces. Our results indicate that seasonal changes in susceptibility of U. californica significantly influence P. ramorum infection levels. Thus, in addition to environmental variables such as temperature and moisture, variability in host plant susceptibility contributes to disease establishment of P. ramorum.

Spatiotemporal Characteristics Determining the Multifaceted Nature of Reactive Oxygen, Nitrogen, and Sulfur Species in Relation to Proton Homeostasis.

Significance: Reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS) act as signaling molecules, regulating gene expression, enzyme activity, and physiological responses. However, excessive amounts of these molecular species can lead to deleterious effects, causing cellular damage and death. This dual nature of ROS, RNS, and RSS presents an intriguing conundrum that calls for a new paradigm. Recent Advances: Recent advancements in the study of photosynthesis have offered significant insights at the molecular level and with high temporal resolution into how the photosystem II oxygen-evolving complex manages to prevent harmful ROS production during the water-splitting process. These findings suggest that a dynamic spatiotemporal arrangement of redox reactions, coupled with strict regulation of proton transfer, is crucial for minimizing unnecessary ROS formation. Critical Issues: To better understand the multifaceted nature of these reactive molecular species in biology, it is worth considering a more holistic view that combines ecological and evolutionary perspectives on ROS, RNS, and RSS. By integrating spatiotemporal perspectives into global, cellular, and biochemical events, we discuss local pH or proton availability as a critical determinant associated with the generation and action of ROS, RNS, and RSS in biological systems. Future Directions: The concept of localized proton availability will not only help explain the multifaceted nature of these ubiquitous simple molecules in diverse systems but also provide a basis for new therapeutic strategies to manage and manipulate these reactive species in neural disorders, pathogenic diseases, and antiaging efforts.

Wastewater polishing by a channelized macrophyte-dominated wetland and anaerobic digestion of the harvested phytomass.

Constructed wetlands (CW) offer a mechanism to meet increasingly stringent regulatory standards for wastewater treatment while minimizing energy inputs. Additionally, harvested wetland phytomass subjected to anaerobic digestion can serve as a source of biogas methane. To investigate CW wastewater polishing activities and potential energy yield we constructed a pair of secondary wastewater-fed channelized CW modules designed to retain easily harvestable floating aquatic vegetation and maximize exposure of water to roots and sediment. Modules that were regularly harvested averaged a nitrate removal rate of 1.1 g N m(-2) d(-1); harvesting, sedimentation and gasification were responsible for 30.5%, 8.0% and 61.5% of the N losses, respectively. Selective harvesting of a module to maintain dominance of filamentous algae had no effect on nitrate removal rate but lowered productivity by one-half. The average monthly productivity for unselectively harvested modules was 9.3 ± 1.7 g dry wt. m(-2) d(-1) (±SE). Cessation of harvesting in one module resulted in a significant increase in nitrate removal rate and decrease in phosphate removal rate. Compared to the influent, the effluent of the harvested module had significantly lower levels of estrogenic activity, as determined by a quantitative PCR-based juvenile trout bioassay, and significantly lower densities of E. coli. In mixed vertical-flow reactors anaerobic co-digestion of equal dry weight proportions of harvested aquatic vegetation, wine yeast lees and dairy manure was greatly improved when the manure was replaced with the crude glycerol by-product of biodiesel production. Remaining solids were vermicomposted for use as a soil amendment. Our results indicate that incorporation of constructed wetlands into an integrated treatment system can simultaneously enhance the economic and energetic feasibility of wastewater and organic waste treatment processes.

Sequence-based Functional Metagenomics Reveals Novel Natural Diversity of Functioning CopA in Environmental Microbiomes.

Exploring the natural diversity of functional genes/proteins from environmental DNA in high-throughput remains challenging. In this study, we developed a sequence-based functional metagenomics procedure for mining the diversity of copper resistance gene copA in global microbiomes, by combining the metagenomic assembly technology, local BLAST, evolutionary trace analysis (ETA), chemical synthesis, and conventional functional genomics. In total, 87 metagenomes were collected from a public database and subjected to copA detection, resulting in 93,899 hits. Manual curation of 1214 hits of high-confidence led to the retrieval of 517 unique CopA candidates, which were further subjected to ETA. Eventually, 175 novel copA sequences of high-quality were discovered. Phylogenetic analysis showed that almost all these putative CopA proteins are distantly related to known CopA proteins, with 55 sequences from totally unknown species. Ten novel and three known copA genes were chemically synthesized for further functional genomic tests using the Cu-sensitive Escherichia coli (ΔcopA). The growth test and Cu uptake determination showed that five novel clones had positive effects on host Cu resistance and uptake. One recombinant harboring copA-like 15 (copAL15) successfully restored Cu resistance of the host with a substantially enhanced Cu uptake. Two novel copA genes were fused with the gfp gene and expressed in E. coli for microscopic observation. Imaging results showed that they were successfully expressed and their proteins were localized to the membrane. The results here greatly expand the diversity of known CopA proteins, and the sequence-based procedure developed overcomes biases in length, screening methods, and abundance of conventional functional metagenomics.

Protozoan-induced regulation of cyclic lipopeptide biosynthesis is an effective predation defense mechanism for Pseudomonas fluorescens.

Environmental bacteria are exposed to a myriad of biotic interactions that influence their function and survival. The grazing activity of protozoan predators significantly impacts the dynamics, diversification, and evolution of bacterial communities in soil ecosystems. To evade protozoan predation, bacteria employ various defense strategies. Soil-dwelling Pseudomonas fluorescens strains SS101 and SBW25 produce the cyclic lipopeptide surfactants (CLPs) massetolide and viscosin, respectively, in a quorum-sensing-independent manner. In this study, CLP production was shown to protect these bacteria from protozoan predation as, compared to CLP-deficient mutants, strains SS101 and SBW25 exhibited resistance to grazing by Naegleria americana in vitro and superior persistence in soil in the presence of this bacterial predator. In the wheat rhizosphere, CLP-producing strains had a direct deleterious impact on the survival of N. americana. In vitro assays further showed that N. americana was three times more sensitive to viscosin than to massetolide and that exposure of strain SS101 or SBW25 to this protozoan resulted in upregulation of CLP biosynthesis genes. Enhanced expression of the massABC and viscABC genes did not require physical contact between the two organisms as gene expression levels were up to threefold higher in bacterial cells harvested 1 cm from feeding protozoans than in cells collected 4 cm from feeding protozoans. These findings document a new natural function of CLPs and highlight that bacterium-protozoan interactions can result in activation of an antipredator response in prey populations.

d-Cysteine-Induced Rapid Root Abscission in the Water Fern Azolla Pinnata: Implications for the Linkage between d-Amino Acid and Reactive Sulfur Species (RSS) in Plant Environmental Responses.

Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) have been proposed as universal signaling molecules in plant stress responses. There are a growing number of studies suggesting that hydrogen sulfide (H2S) or Reactive Sulfur Species (RSS) are also involved in plant abiotic as well as biotic stress responses. However, it is still a matter of debate as to how plants utilize those RSS in their signaling cascades. Here, we demonstrate that d-cysteine is a novel candidate for bridging our gap in understanding. In the genus of the tiny water-floating fern Azolla, a rapid root abscission occurs in response to a wide variety of environmental stimuli as well as chemical inducers. We tested five H2S chemical donors, Na2S, GYY4137, 5a, 8l, and 8o, and found that 5a showed a significant abscission activity. Root abscission also occurred with the polysulfides Na2S2, Na2S3, and Na2S4. Rapid root abscission comparable to other known chemical inducers was observed in the presence of d-cysteine, whereas l-cysteine showed no effect. We suggest that d-cysteine is a physiologically relevant substrate to induce root abscission in the water fern Azolla.

Microbial diversity and mineral composition of weathered serpentine rock of the Khalilovsky massif.

Endolithic microbial communities survive nutrient and energy deficient conditions while contributing to the weathering of their mineral substrate. This study examined the mineral composition and microbial communities of fully serpentinized weathered rock from 0.1 to 6.5 m depth at a site within the Khalilovsky massif, Orenburg Region, Southern Ural Mountains, Russia. The mineral composition includes a major content of serpentinite family (mostly consisting of lizardite and chrysotile), magnesium hydrocarbonates (hydromagnesite with lesser amounts of hydrotalcite and pyroaurite) concentrated in the upper layers, and clay minerals. We found that the deep-seated weathered serpentinites are chrysotile-type minerals, while the middle and surface serpentinites mostly consist of lizardite and chrysotile types. Microbial community analysis, based on 16S rRNA gene sequencing, showed a similar diversity of phyla throughout the depth profile. The dominant bacterial phyla were the Actinobacteria (of which unclassified genera in the orders Acidimicrobiales and Actinomycetales were most numerous), Chloroflexi (dominated by an uncultured P2-11E order) and the Proteobacteria (predominantly class Betaproteobacteria). Densities of several groups of bacteria were negatively correlated with depth. Occurrence of the orders Actinomycetales, Gaiellales, Solirubrobacterales, Rhizobiales and Burkholderiales were positively correlated with depth. Our findings show that endolithic microbial communities of the Khalilovsky massif have similar diversity to those of serpentine soils and rocks, but are substantially different from those of the aqueous environments of actively serpentinizing systems.

NO signal at the crossroads: polyamine-induced nitric oxide synthesis in plants?

Polyamines, such as spermine, spermidine and putrescine, are ubiquitous polycationic compounds that are produced by almost all living organisms, including plants, animals, fungi and bacteria. Polyamines are multifunctional and interact with polyanionic biomolecules such as DNA or protein. However, despite their potential significance, the polyamine-dependent signal transduction system has not been revealed yet. Ni Ni Tun and colleagues have recently reported a possible linkage between polyamine and nitric oxide (NO), another ubiquitous signalling molecule.

Mechanism of action and efficacy of seed meal-induced pathogen suppression differ in a brassicaceae species and time-dependent manner.

ABSTRACT The effect of seed meals derived from Brassica juncea, B. napus, or Sinapis alba on suppression of soilborne pathogens inciting replant disease of apple was evaluated in greenhouse trials. Regardless of plant source, seed meal amendment significantly improved apple growth in all orchard soils; however, relative differences in pathogen suppression were observed. All seed meals suppressed root infection by native Rhizoctonia spp. and an introduced isolate of Rhizoctonia solani AG-5, though B. juncea seed meal often generated a lower level of disease control relative to other seed meal types. When introduction of the pathogen was delayed until 4 to 8 weeks post seed meal amendment, disease suppression was associated with proliferation of resident Streptomyces spp. and not qualitative or quantitative attributes of seed meal glucosinolate content. Using the same experimental system, when soils were pasteurized prior to pathogen infestation, control of R. solani was eliminated regardless of seed meal type. In the case of B. juncea seed meal amendment, the mechanism of R. solani suppression varied in a temporal manner, which initially was associated with the generation of allylisothiocyanate and was not affected by soil pasteurization. Among those tested, only B. juncea seed meal did not stimulate orchard soil populations of Pythium spp. and infection of apple roots by these oomycetes. Although application of B. napus seed meal alone consistently induced an increase in Pythium spp. populations, no significant increase in Pythium spp. populations was observed in response to a composite B. juncea and B. napus seed meal amendment. Suppression of soil populations and root infestation by Pratylenchus spp. was dependent upon seed meal type, with only B. juncea providing sustained nematode control. Collectively, these studies suggest that use of a composite B. juncea and B. napus seed meal mixture can provide superior control of the pathogen complex inciting apple replant disease relative to either seed meal used alone.

Comparative Metagenomic Analysis of Electrogenic Microbial Communities in Differentially Inoculated Swine Wastewater-Fed Microbial Fuel Cells.

Bioelectrochemical systems such as microbial fuel cells (MFCs) are promising new technologies for efficient removal of organic compounds from industrial wastewaters, including that generated from swine farming. We inoculated two pairs of laboratory-scale MFCs with sludge granules from a beer wastewater-treating anaerobic digester (IGBS) or from sludge taken from the bottom of a tank receiving swine wastewater (SS). The SS-inoculated MFC outperformed the IGBS-inoculated MFC with regard to COD and VFA removal and electricity production. Using a metagenomic approach, we describe the microbial diversity of the MFC planktonic and anodic communities derived from the different inocula. Proteobacteria (mostly Deltaproteobacteria) became the predominant phylum in both MFC anodic communities with amplification of the electrogenic genus Geobacter being the most pronounced. Eight dominant and three minor species of Geobacter were found in both MFC anodic communities. The anodic communities of the SS-inoculated MFCs had a higher proportion of Clostridium and Bacteroides relative to those of the IGBS-inoculated MFCs, which were enriched with Pelobacter. The archaeal populations of the SS- and IGBS-inoculated MFCs were dominated by Methanosarcina barkeri and Methanothermobacter thermautotrophicus, respectively. Our results show a long-term influence of inoculum type on the performance and microbial community composition of swine wastewater-treating MFCs.

An Overview of Methods in Plant Nitric Oxide (NO) Research: Why Do We Always Need to Use Multiple Methods?

The free radical nitric oxide (NO) is a universal signaling molecule among living organisms. To investigate versatile functions of NO in plants it is essential to analyze biologically produced NO with an appropriate method. Owing to the uniqueness of NO, plant researchers may encounter difficulties in applying methods that have been developed for mammalian study. Based on our experience, we present here a practical guide to NO measurement fitted to plant biology.

Genome Sequence of the Electrogenic Petroleum-Degrading Thalassospira sp. Strain HJ.

We present the draft genome of the petroleum-degrading Thalassospira sp. strain HJ, isolated from tidal marine sediment. Knowledge of this genomic information will inform studies on electrogenesis and means to degrade environmental organic contaminants, including compounds found in petroleum.

Bimodal effect of hydrogen peroxide and oxidative events in nitrite-induced rapid root abscission by the water fern Azolla pinnata.

In the genus Azolla rapid abscission of roots from floating fronds occurs within minutes in response to a variety of stresses, including exposure to nitrite. We found that hydrogen peroxide, though itself not an inducer of root abscission, modulates nitrite-induced root abscission by Azolla pinnata in a dose-dependent manner, with 2 mM H2O2 significantly diminishing the responsiveness to 2 mM NaNO2, and 10 mM H2O2 slightly enhancing it. Hypoxia, which has been found in other plants to result in autogenic production of H2O2, dramatically stimulated root abscission of A. pinnata in response to nitrite, especially for plants previously cultivated in medium containing 5 mM KNO3 compared to plants cultivated under N2-fixing conditions without combined nitrogen. Plants, including Azolla, produce the small signaling molecule nitric oxide (NO) from nitrite using nitrate reductase. We found Azolla plants to display dose-dependent root abscission in response to the NO donor spermine NONOate. Treatment of plants with the thiol-modifying agents S-methyl methanethiosulfonate or glutathione inhibited the nitrite-induced root abscission response. Synchrotron radiation-based Fourier transform infrared spectromicroscopy revealed higher levels of carbonylation in the abscission zone of dropped roots, indicative of reaction products of polysaccharides with potent free radical oxidants. We hypothesize that metabolic products of nitrite and NO react with H2O2 in the apoplast leading to free-radical-mediated cleavage of structural polysaccharides and consequent rapid root abscission.

Taxonomic and functional metagenomic analysis of anodic communities in two pilot-scale microbial fuel cells treating different industrial wastewaters.

The combined processes of microbial biodegradation accompanied by extracellular electron transfer make microbial fuel cells (MFCs) a promising new technology for cost-effective and sustainable wastewater treatment. Although a number of microbial species that build biofilms on the anode surfaces of operating MFCs have been identified, studies on the metagenomics of entire electrogenic communities are limited. Here we present the results of whole-genome metagenomic analysis of electrochemically active robust anodic microbial communities, and their anaerobic digester (AD) sludge inocula, from two pilot-scale MFC bioreactors fed with different distillery wastewaters operated under ambient conditions in distinct climatic zones. Taxonomic analysis showed that Proteobacteria, Bacteroidetes and Firmicutes were abundant in AD sludge from distinct climatic zones, and constituted the dominant core of the MFC microbiomes. Functional analysis revealed species involved in degradation of organic compounds commonly present in food industry wastewaters. Also, accumulation of methanogenic Archaea was observed in the electrogenic biofilms, suggesting a possibility for simultaneous electricity and biogas recovery from one integrated wastewater treatment system. Finally, we found a range of species within the anode communities possessing the capacity for extracellular electron transfer, both via direct contact and electron shuttles, and show differential distribution of bacterial groups on the carbon cloth and activated carbon granules of the anode surface. Overall, this study provides insights into structural shifts that occur in the transition from an AD sludge to an MFC microbial community and the metabolic potential of electrochemically active microbial populations with wastewater-treating MFCs.

Genome Sequence of the Alkaline-Tolerant Cellulomonas sp. Strain FA1.

We present the genome of the cellulose-degrading Cellulomonas sp. strain FA1 isolated from an actively serpentinizing highly alkaline spring. Knowledge of this genome will enable studies into the molecular basis of plant material degradation in alkaline environments and inform the development of lignocellulose bioprocessing procedures for biofuel production.

Synergistic effect of deoxyanthocyanins from symbiotic fern Azolla spp. on hrmA gene induction in the cyanobacterium Nostoc punctiforme.

The hrmA gene of the N2-fixing cyanobacterium Nostoc punctiforme functions in repressing the formation of transitory motile filaments, termed hormogonia, by plant-associated vegetative filaments. Here, we report that anthocyanins can contribute to induction of hrmA expression. Aqueous extract from fronds of the fern Azolla pinnata, a host of symbiotic Nostoc spp., was found to be a potent inducer of hrmA-luxAB in N. punctiforme strain UCD 328. The hrmA-luxAB inducing activities of A. pinnata, as well as Azolla filiculoides, were positively correlated with levels of frond deoxyanthocyanins. Analyses of the deoxyanthocyanins in frond extracts revealed, in order of predominance, an acetylated glycoside derivative of luteolinidin (m/z 475) and of apigeninidin (m/z 459) and minor amounts of a second luteolinidin derivative. At up to 150 microM, a purified preparation of deoxyanthocyanins only weakly induced hrmA-luxAB on its own, but mixtures with hrmA-luxAB inducers (A. filiculoides extract or the flavonoid naringin) synergistically doubled to tripled their inducing activities. These results suggest that appropriately localized deoxyanthocyanins could function in plant-mediated mechanisms for repressing Nostoc spp. hormogonium formation.