GDP-mannose pyrophosphorylase is an efficient target in Xanthomonas citri for citrus canker control.

Xanthomonas citri subsp. citri (Xcc) is a bacterium that causes citrus canker, an economically important disease that results in premature fruit drop and reduced yield of fresh fruit. In this study, we demonstrated the involvement of XanB, an enzyme with phosphomannose isomerase (PMI) and guanosine diphosphate-mannose pyrophosphorylase (GMP) activities, in Xcc pathogenicity. Additionally, we found that XanB inhibitors protect the host against Xcc infection. Besides being deficient in motility, biofilm production, and ultraviolet resistance, the xanB deletion mutant was unable to cause disease, whereas xanB complementation restored wild-type phenotypes. XanB homology modeling allowed in silico virtual screening of inhibitors from databases, three of them being suitable in terms of absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) properties, which inhibited GMP (but not PMI) activity of the Xcc recombinant XanB protein in more than 50%. Inhibitors reduced citrus canker severity up to 95%, similarly to copper-based treatment. xanB is essential for Xcc pathogenicity, and XanB inhibitors can be used for the citrus canker control. IMPORTANCE: Xcc causes citrus canker, a threat to citrus production, which has been managed with copper, being required a more sustainable alternative for the disease control. XanB was previously found on the surface of Xcc, interacting with the host and displaying PMI and GMP activities. We demonstrated by xanB deletion and complementation that GMP activity plays a critical role in Xcc pathogenicity, particularly in biofilm formation. XanB homology modeling was performed, and in silico virtual screening led to carbohydrate-derived compounds able to inhibit XanB activity and reduce disease symptoms by 95%. XanB emerges as a promising target for drug design for control of citrus canker and other economically important diseases caused by Xanthomonas sp.

Ecological indicators and biological resources for hydrocarbon rhizoremediation in a protected area.

Spillage from oil refineries, pipelines, and service stations consistently leads to soil, food and groundwater contamination. Bacterial-assisted phytoremediation is a non-invasive and sustainable solution to eliminate or decrease the concentration of xenobiotic contaminants in the environment. In the present study, a protected area interested by a fuel discharge was considered to assess a bioremediation intervention. From the spill point, a plume of contamination flowed South-West into the aquifer, eventually reaching a wetland area. Soils, groundwaters and plants belonging to the species Scirpus sylvaticus (L.) were sampled. In the majority of the soil samples, concentrations of total petroleum hydrocarbons, both C ≤ 12 and C > 12, exceeded legal limits set forth in Directive 2000/60/EC. The analysis of diatom populations, used as ecological indicators, evidenced morphology alterations and the presence of Ulnaria ulna and Ulnaria biceps species, previously detected in hydrocarbon-polluted waters. Tests for phytotoxicity and phytodegradation, carried out in soil mesocosms, planted with Zea mays and Helianthus annuus, demonstrated that both species significantly contributed to the removal of total petroleum hydrocarbons. Removal of C ≤ 12 and C > 12 petroleum hydrocarbons was in the range of 80%-82% for Z. mays and 71%-72% for H. annuus. Microbial communities inhabiting high organic carbon and vegetated soils were more active in hydrocarbon degradation than those inhabiting subsoils, as evidenced by soil slurry experiments. The abundance of functional genes encoding toluene-benzene monooxygenase (tbmD) and alkane hydroxylase (alkB), quantified in environmental samples, confirmed that the plant rhizosphere recruited a microbial community with higher biodegradation capacity. Bacterial strains isolated from the sampling site were able to grow on model hydrocarbons (hexane, hexadecane and o-, m-, p-xylene) as sole carbon and energy sources, indicating that a natural bio-attenuation process was on-going at the site. The bacterial strains isolated from rhizosphere soil, rhizoplane and endosphere showed plant growth promoting traits according to in vitro and in vivo tests on Z. mays and Oryza sativa, allowing to forecast a possible application of bacterial assisted rhizoremediation to recover the protected area.

Selectivity and Activity of Benzene-1,2,4-triol and its Dimers as Antimicrobial Compounds Against Xanthomonas citri subsp. citri.

Citrus canker, caused by the bacterium Xanthomonas citri subsp. citri, is one of the main threats to citrus fruit production. Several phenolic compounds active against X. citri have been described in recent years. Benzene-1,2,4-triol is a bio-based phenolic compound that has shown high potential as a scaffold for the synthesis of new anti-X. citri compounds. However, benzene-1,2,4-triol is prone to oxidative dimerization. We evaluated the antibacterial activity of benzene-1,2,4-triol, its oxidized dimers, and analogous compounds. Benzene-1,2,4-triol has a low inhibitory concentration against X. citri (0.05 mM) and is also active against other bacterial species. Spontaneous formation of benzenetriol dimers (e. g. by contact with oxygen in aqueous solution) reduced the antimicrobial activity of benzenetriol solutions. Dimers themselves displayed lower antibacterial activity and where shown to be more stable in solution. Unlike many other phenolic compounds with anti-X. citri activity, benzene-1,2,4-triol does not act by membrane permeabilization, but seems to limit the availability of iron to cells. Benzene-1,2,4-triol is widely recognized as toxic - our results indicate that the toxicity of benzene-1,2,4-triol is largely due to spontaneously formed dimers. Stabilization of benzene-1,2,4-triol will be required to allow the safe use of this compound.

Microbial communities in paddy soils: differences in abundance and functionality between rhizosphere and pore water, the influence of different soil organic carbon, sulfate fertilization and cultivation time, and contribution to arsenic mobility and speciation.

Abiotic factors and rhizosphere microbial populations influence arsenic accumulation in rice grains. Although mineral and organic surfaces are keystones in element cycling, localization of specific microbial reactions in the root/soil/pore water system is still unclear. Here, we tested if original unplanted soil, rhizosphere soil and pore water represented distinct ecological microniches for arsenic-, sulfur- and iron-cycling microorganisms and compared the influence of relevant factors such as soil type, sulfate fertilization and cultivation time. In rice open-air-mesocosms with two paddy soils (2.0% and 4.7% organic carbon), Illumina 16S rRNA gene sequencing demonstrated minor effects of cultivation time and sulfate fertilization that decreased Archaea-driven microbial networks and incremented sulfate-reducing and sulfur-oxidizing bacteria. Different compartments, characterized by different bacterial and archaeal compositions, had the strongest effect, with higher microbial abundances, bacterial biodiversity and interconnections in the rhizosphere vs pore water. Within each compartment, a significant soil type effect was observed. Higher percentage contributions of rhizosphere dissimilatory arsenate- and iron-reducing, arsenite-oxidizing, and, surprisingly, dissimilatory sulfate-reducing bacteria, as well as pore water iron-oxidizing bacteria in the lower organic carbon soil, supported previous chemistry-based interpretations of a more active S-cycling, a higher percentage of thioarsenates and lower arsenic mobility by sorption to mixed Fe(II)Fe(III)-minerals in this soil.

Adaptation of Microbial Communities to Environmental Arsenic and Selection of Arsenite-Oxidizing Bacteria From Contaminated Groundwaters.

Arsenic mobilization in groundwater systems is driven by a variety of functionally diverse microorganisms and complex interconnections between different physicochemical factors. In order to unravel this great ecosystem complexity, groundwaters with varying background concentrations and speciation of arsenic were considered in the Po Plain (Northern Italy), one of the most populated areas in Europe affected by metalloid contamination. High-throughput Illumina 16S rRNA gene sequencing, CARD-FISH and enrichment of arsenic-transforming consortia showed that among the analyzed groundwaters, diverse microbial communities were present, both in terms of diversity and functionality. Oxidized inorganic arsenic [arsenite, As(III)] was the main driver that shaped each community. Several uncharacterized members of the genus Pseudomonas, putatively involved in metalloid transformation, were revealed in situ in the most contaminated samples. With a cultivation approach, arsenic metabolisms potentially active at the site were evidenced. In chemolithoautotrophic conditions, As(III) oxidation rate linearly correlated to As(III) concentration measured at the parental sites, suggesting that local As(III) concentration was a relevant factor that selected for As(III)-oxidizing bacterial populations. In view of the exploitation of these As(III)-oxidizing consortia in biotechnology-based arsenic bioremediation actions, these results suggest that contaminated aquifers in Northern Italy host unexplored microbial populations that provide essential ecosystem services.

Benzenetriol-Derived Compounds against Citrus Canker.

In order to replace the huge amounts of copper salts used in citrus orchards, alternatives have been sought in the form of organic compounds of natural origin with activity against the causative agent of citrus canker, the phytopathogen Xanthomonas citri subsp. Citri. We synthesized a series of 4-alkoxy-1,2-benzene diols (alkyl-BDOs) using 1,2,4-benzenetriol (BTO) as a starting material through a three-step synthesis route and evaluated their suitability as antibacterial compounds. Our results show that alkyl ethers derived from 1,2,4-benzenetriol have bactericidal activity against X. citri, disrupting the bacterial cell membrane within 15 min. Alkyl-BDOs were also shown to remain active against the bacteria while in solution, and presented low toxicity to (human) MRC-5 cells. Therefore, we have demonstrated that 1,2,4-benzenetriol-a molecule that can be obtained from agricultural residues-is an adequate precursor for the synthesis of new compounds with activity against X. citri.

Antibacterial activity of a new monocarbonyl analog of curcumin MAC 4 is associated with divisome disruption.

Curcumin (CUR) is a symmetrical dicarbonyl compound with antibacterial activity. On the other hand, pharmacokinetic and chemical stability limitations hinder its therapeutic application. Monocarbonyl analogs of curcumin (MACs) have been shown to overcome these barriers. We synthesized and investigated the antibacterial activity of a series of unsymmetrical MACs derived from acetone against Mycobacterium tuberculosis and Gram-negative and Gram-positive species. Phenolic MACs 4, 6 and 8 showed a broad spectrum and potent activity, mainly against M. tuberculosis, Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus (MRSA), with MIC (minimum inhibitory concentration) values ranging from 0.9 to 15.6 µg/mL. The investigation regarding toxicity on human lung cells (MRC-5 and A549 lines) revealed MAC 4 was more selective than MACs 6 and 8, with SI (selectivity index) values ranging from 5.4 to 15.6. In addition, MAC 4 did not demonstrate genotoxic effects on A549 cells and it was more stable than CUR in phosphate buffer (pH 7.4) for 24 h at 37 °C. Fluorescence and phase contrast microscopies indicated that MAC 4 has the ability to disrupt the divisome of Bacillus subtilis without damaging its cytoplasmic membrane. However, biochemical investigations demonstrated that MAC 4 did not affect the GTPase activity of B. subtilis FtsZ, which is the main constituent of the bacterial divisome. These results corroborated that MAC 4 is a promising antitubercular and antibacterial agent.

Investigating the Modes of Action of the Antimicrobial Chalcones BC1 and T9A.

Xanthomonas citri subsp. citri (X. citri) is an important phytopathogen and causes Asiatic Citrus Canker (ACC). To control ACC, copper sprays are commonly used. As copper is an environmentally damaging heavy metal, new antimicrobials are needed to combat citrus canker. Here, we explored the antimicrobial activity of chalcones, specifically the methoxychalcone BC1 and the hydroxychalcone T9A, against X. citri and the model organism Bacillus subtilis. BC1 and T9A prevented growth of X. citri and B. subtilis in concentrations varying from 20 µg/mL to 40 µg/mL. BC1 and T9A decreased incorporation of radiolabeled precursors of DNA, RNA, protein, and peptidoglycan in X. citri and B. subtilis. Both compounds mildly affected respiratory activity in X. citri, but T9A strongly decreased respiratory activity in B. subtilis. In line with that finding, intracellular ATP decreased strongly in B. subtilis upon T9A treatment, whereas BC1 increased intracellular ATP. In X. citri, both compounds resulted in a decrease in intracellular ATP. Cell division seems not to be affected in X. citri, and, although in B. subtilis the formation of FtsZ-rings is affected, a FtsZ GTPase activity assay suggests that this is an indirect effect. The chalcones studied here represent a sustainable alternative to copper for the control of ACC, and further studies are ongoing to elucidate their precise modes of action.

Hexyl gallate for the control of citrus canker caused by Xanthomonas citri subsp citri.

Brazil is the biggest producer of sweet oranges and the main exporter of concentrated orange juice in the world. Among the diseases that affect citriculture, Asiatic citrus canker, caused by the bacterial pathogen Xanthomonas citri, represents one of the most significant threats. The current Brazilian legislation regulating the control of citrus canker no longer requires the eradication of affected trees in states where the incidence of the disease is high. Instead, control involves disease control measures, including periodic preventative spraying of copper compounds. The long-term use of copper for plant disease control has raised concerns about environmental accumulation and toxicity, as well as the selective pressure it exerts leading to the emergence of copper-resistant X. citri strains. Here, we evaluated hexyl gallate (G6) as an alternative to copper compounds for citrus plant protection. G6 was able to protect citrus nursery trees against X. citri infection. Thirty days after inoculation, the trees treated with G6 developed 0.5 lesions/cm2 leaf area compared with the 2.84 lesions/cm2 observed in the untreated control trees. Also, G6 did not interfere with germination and root development of tomato, lettuce, and arugula, which is consistent with our previous data showing that G6 is safe for tissue culture cell lines. Membrane permeability tests showed that the primary target of G6 is the bacterial outer membrane. Finally, we could not isolate spontaneous X. citri mutants resistant to G6 nor induce resistance to G6 after long-term exposures to increasing concentrations of the compound, which suggests that G6 may have multiple cellular targets. This study demonstrated that G6 is a promising candidate for the development and use in citrus canker management.

Exposure to different arsenic species drives the establishment of iron- and sulfur-oxidizing bacteria on rice root iron plaques.

Iron- and sulfur-oxidizing bacteria inhabiting rice rhizoplane play a significant role on arsenic biogeochemistry in flooded rice paddies, influencing arsenic translocation to rice grains. In the present study, the selective pressure of arsenic species on these microbial populations was evaluated. Rice roots from continuously flooded plants were incubated in iron sulfide (FeS) gradient tubes and exposed to either arsenate or arsenite. The biomass developed in the visible iron-oxidation band of the enrichments was analyzed by Scanning Electron Microscopy and Energy-Dispersive Spectroscopy (SEM-EDS) and the bacterial communities were characterized by 16S rRNA gene sequencing. Different Proteobacteria communities were selected depending on exposure to arsenate and arsenite. Arsenate addition favored the versatile iron-oxidizers Dechloromonas and Azospira, associated to putative iron (hydr)oxide crystals. Arsenite exposure decreased the diversity in the enrichments, with the development of the sulfur-oxidizer Thiobacillus thioparus, likely growing on sulfide released by FeS. Whereas sulfur-oxidizers were observed in all treatments, iron-oxidizers disappeared when exposed to arsenite. These results reveal a strong impact of different inorganic arsenics on rhizospheric iron-oxidizers as well as a crucial role of sulfur-oxidizing bacteria in establishing rice rhizosphere communities under arsenic pressure.

Transcriptomic Analysis of Two Thioalkalivibrio Species Under Arsenite Stress Revealed a Potential Candidate Gene for an Alternative Arsenite Oxidation Pathway.

The genus Thioalkalivibrio includes haloalkaliphilic chemolithoautotrophic sulfur-oxidizing bacteria isolated from various soda lakes worldwide. Some of these lakes possess in addition to their extreme haloalkaline environment also other harsh conditions, to which Thioalkalivibrio needs to adapt. An example is arsenic in soda lakes in eastern California, which is found there in concentrations up to 3000 µM. Arsenic is a widespread element that can be an environmental issue, as it is highly toxic to most organisms. However, resistance mechanisms in the form of detoxification are widespread and some prokaryotes can even use arsenic as an energy source. We first screened the genomes of 76 Thioalkalivibrio strains for the presence of known arsenic oxidoreductases and found 15 putative ArxA (arsenite oxidase) and two putative ArrA (arsenate reductase). Subsequently, we studied the resistance to arsenite in detail in Thioalkalivibrio jannaschii ALM2T, and Thioalkalivibrio thiocyanoxidans ARh2T by comparative genomics and by growing them at different arsenite concentrations followed by arsenic species and transcriptomic analysis. Tv. jannaschii ALM2T, which has been isolated from Mono Lake, an arsenic-rich soda lake, could resist up to 5 mM arsenite, whereas Tv. thiocyanoxidans ARh2T, which was isolated from a Kenyan soda lake, could only grow up to 0.1 mM arsenite. Interestingly, both species oxidized arsenite to arsenate under aerobic conditions, although Tv. thiocyanoxidans ARh2T does not contain any known arsenite oxidases, and in Tv. jannaschii ALM2T, only arxB2 was clearly upregulated. However, we found the expression of a SoeABC-like gene, which we assume might have been involved in arsenite oxidation. Other arsenite stress responses for both strains were the upregulation of the vitamin B12 synthesis pathway, which can be linked to antioxidant activity, and the up- and downregulation of different DsrE/F-like genes whose roles are still unclear. Moreover, Tv. jannaschii ALM2T induced the ars gene operon and the Pst system, and Tv. thiocanoxidans ARh2T upregulated the sox and apr genes as well as different heat shock proteins. Our findings for Thioalkalivibrio confirm previously observed adaptations to arsenic, but also provide new insights into the arsenic stress response and the connection between the arsenic and the sulfur cycle.

Exploring Biodiversity and Arsenic Metabolism of Microbiota Inhabiting Arsenic-Rich Groundwaters in Northern Italy.

Arsenic contamination of groundwater aquifers is an issue of global concern. Among the affected sites, in several Italian groundwater aquifers arsenic levels above the WHO limits for drinking water are present, with consequent issues of public concern. In this study, for the first time, the role of microbial communities in metalloid cycling in groundwater samples from Northern Italy lying on Pleistocene sediments deriving from Alps mountains has been investigated combining environmental genomics and cultivation approaches. 16S rRNA gene libraries revealed a high number of yet uncultured species, which in some of the study sites accounted for more of the 50% of the total community. Sequences related to arsenic-resistant bacteria (arsenate-reducing and arsenite-oxidizing) were abundant in most of the sites, while arsenate-respiring bacteria were negligible. In some of the sites, sulfur-oxidizing bacteria of the genus Sulfuricurvum accounted for more than 50% of the microbial community, whereas iron-cycling bacteria were less represented. In some aquifers, arsenotrophy, growth coupled to autotrophic arsenite oxidation, was suggested by detection of arsenite monooxygenase (aioA) and 1,5-ribulose bisphosphate carboxylase (RuBisCO) cbbL genes of microorganisms belonging to Rhizobiales and Burkholderiales. Enrichment cultures established from sampled groundwaters in laboratory conditions with 1.5 mmol L-1 of arsenite as sole electron donor were able to oxidize up to 100% of arsenite, suggesting that this metabolism is active in groundwaters. The presence of heterotrophic arsenic resistant bacteria was confirmed by enrichment cultures in most of the sites. The overall results provided a first overview of the microorganisms inhabiting arsenic-contaminated aquifers in Northern Italy and suggested the importance of sulfur-cycling bacteria in the biogeochemistry of arsenic in these ecosystems. The presence of active arsenite-oxidizing bacteria indicates that biological oxidation of arsenite, in combination with arsenate-adsorbing materials, could be employed for metalloid removal.

Antibacterial activity of 3,3'-dihydroxycurcumin (DHC) is associated with membrane perturbation.

Curcumin is a plant diphenylheptanoid and has been investigated for its antibacterial activity. However, the therapeutic uses of this compound are limited due to its chemical instability. In this work, we evaluated the antimicrobial activity of diphenylheptanoids derived from curcumin against Gram-positive and Gram-negative bacteria, and also against Mycobacterium tuberculosis in terms of MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) values. 3,3'-Dihydroxycurcumin (DHC) displayed activity against Enterococcus faecalis, Staphylococcus aureus and M. tuberculosis, demonstrating MIC values of 78 and 156 µg/mL. In addition, DHC was more stable than curcumin in acetate buffer (pH 5.0) and phosphate buffer (pH 7.4) for 24 h at 37 °C. We proposed that membrane and the cell division protein FtsZ could be the targets for DHC due to that fact that curcumin exhibits this mode of antibacterial action. Fluorescence microscopy of Bacillus subtilis stained with SYTO9 and propidium iodide fluorophores indicated that DHC has the ability to perturb the bacterial membrane. On the other hand, DHC showed a weak inhibition of the GTPase activity of B. subtilis FtsZ. Toxicity assay using human cells indicated that DHC has moderate capacity to reduce viability of liver cells (HepG2 line) and lung cells (MRC-5 and A549 lines) when compared with doxorubicin. Alkaline comet assay indicated that DHC was not able to induce DNA damage in A549 cell line. These results indicated that DHC is promising compound with antibacterial and antitubercular activities.

A simplified curcumin targets the membrane of Bacillus subtilis.

Curcumin is the main constituent of turmeric, a seasoning popularized around the world with Indian cuisine. Among the benefits attributed to curcumin are anti-inflammatory, antimicrobial, antitumoral, and chemopreventive effects. Besides, curcumin inhibits the growth of the gram-positive bacterium Bacillus subtilis. The anti-B. subtilis action happens by interference with the division protein FtsZ, an ancestral tubulin widespread in Bacteria. FtsZ forms protofilaments in a GTP-dependent manner, with the concomitant recruitment of essential factors to operate cell division. By stimulating the GTPase activity of FtsZ, curcumin destabilizes its function. Recently, curcumin was shown to promote membrane permeabilization in B. subtilis. Here, we used molecular simplification to dissect the functionalities of curcumin. A simplified form, in which a monocarbonyl group substituted the ß-diketone moiety, showed antibacterial action against gram-positive and gram-negative bacteria of clinical interest. The simplified curcumin also disrupted the divisional septum of B. subtilis; however, subsequent biochemical analysis did not support a direct action on FtsZ. Our results suggest that the simplified curcumin exerted its function mainly through membrane permeabilization, with disruption of the membrane potential necessary for FtsZ intra-cellular localization. Finally, we show here experimental evidence for the requirement of the ß-diketone group of curcumin for its interaction with FtsZ.

Bioelectrochemical Nitrogen fixation (e-BNF): Electro-stimulation of enriched biofilm communities drives autotrophic nitrogen and carbon fixation.

A new approach to microbial electrosynthesis is proposed, aimed at producing whole biomass from N2 and inorganic carbon, by electrostimulation of complex microbial communities. On a carbon-based conductor under constant polarization (-0.7 V vs SHE), an electroactive biofilm was enriched with autotrophic nitrogen fixing microorganims and led to biomass synthesis at higher amounts (up to 18 fold), as compared to controls kept at open circuit (OC). After 110 days, the electron transfer had increased by 30-fold, as compared to abiotic conditions. Metagenomics evidenced Nif genes associated with autotrophs (both Archaea and Bacteria) only in polarized biofilms, but not in OC. With this first proof of concept experiment, we propose to call this promising field 'bioelectrochemical nitrogen fixation' (e-BNF): a possible way to 'power' biological nitrogen fixation, organic carbon storage and soil fertility against desertification, and possibly a new tool to study the development of early prokaryotic life in extreme environments.

Design of Antibacterial Agents: Alkyl Dihydroxybenzoates against Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (Xcc) causes citrus canker, affecting sweet orange-producing areas around the world. The current chemical treatment available for this disease is based on cupric compounds. For this reason, the objective of this study was to design antibacterial agents. In order to do this, we analyzed the anti-Xcc activity of 36 alkyl dihydroxybenzoates and we found 14 active compounds. Among them, three esters with the lowest minimum inhibitory concentration values were selected; compounds 4 (52 µM), 16 (80 µM) and 28 (88 µM). Our study demonstrated that alkyl dihydroxybenzoates cause a delay in the exponential phase. The permeability capacity of alkyl dihydroxybenzoates in a quarter of MIC was compared to nisin (positive control). Compound 28 was the most effective (93.8), compared to compound 16 (41.3) and compound 4 (13.9) by percentage values. Finally, all three compounds showed inhibition of FtsZ GTPase activity, and promoted changes in protofilaments, leading to depolymerization, which prevents bacterial cell division. In conclusion, heptyl dihydroxybenzoates (compounds 4, 16 and 28) are promising anti-Xcc agents which may serve as an alternative for the control of citrus canker.

Rice Paddy Nitrospirae Carry and Express Genes Related to Sulfate Respiration: Proposal of the New Genus "Candidatus Sulfobium".

Nitrospirae spp. distantly related to thermophilic, sulfate-reducing Thermodesulfovibrio species are regularly observed in environmental surveys of anoxic marine and freshwater habitats. Here we present a metaproteogenomic analysis of Nitrospirae bacterium Nbg-4 as a representative of this clade. Its genome was assembled from replicated metagenomes of rice paddy soil that was used to grow rice in the presence and absence of gypsum (CaSO4·2H2O). Nbg-4 encoded the full pathway of dissimilatory sulfate reduction and showed expression of this pathway in gypsum-amended anoxic bulk soil as revealed by parallel metaproteomics. In addition, Nbg-4 encoded the full pathway of dissimilatory nitrate reduction to ammonia (DNRA), with expression of its first step being detected in bulk soil without gypsum amendment. The relative abundances of Nbg-4 were similar under both treatments, indicating that Nbg-4 maintained stable populations while shifting its energy metabolism. Whether Nbg-4 is a strict sulfate reducer or can couple sulfur oxidation to DNRA by operating the pathway of dissimilatory sulfate reduction in reverse could not be resolved. Further genome reconstruction revealed the potential to utilize butyrate, formate, H2, or acetate as an electron donor; the Wood-Ljungdahl pathway was expressed under both treatments. Comparison to publicly available Nitrospirae genome bins revealed the pathway for dissimilatory sulfate reduction also in related Nitrospirae recovered from groundwater. Subsequent phylogenomics showed that such microorganisms form a novel genus within the Nitrospirae, with Nbg-4 as a representative species. Based on the widespread occurrence of this novel genus, we propose for Nbg-4 the name "Candidatus Sulfobium mesophilum," gen. nov., sp. nov.IMPORTANCE Rice paddies are indispensable for the food supply but are a major source of the greenhouse gas methane. If it were not counterbalanced by cryptic sulfur cycling, methane emission from rice paddy fields would be even higher. However, the microorganisms involved in this sulfur cycling are little understood. By using an environmental systems biology approach with Italian rice paddy soil, we could retrieve the population genome of a novel member of the phylum Nitrospirae This microorganism encoded the full pathway of dissimilatory sulfate reduction and expressed it in anoxic paddy soil under sulfate-enriched conditions. Phylogenomics and comparison to the results of environmental surveys showed that such microorganisms are actually widespread in freshwater and marine environments. At the same time, they represent an undiscovered genus within the little-explored phylum Nitrospirae Our results will be important for the design of enrichment strategies and postgenomic studies to further understanding of the contribution of these novel Nitrospirae spp. to the global sulfur cycle.

A study of microbial communities on terracotta separator and on biocathode of air breathing microbial fuel cells.

Recently, terracotta has attracted interest as low-cost and biocompatible material to build separators in microbial fuel cells (MFCs). However, the influence of a non-conductive material like terracotta on electroactive microbiological communities remains substantially unexplored. This study aims at describing the microbial pools developed from two different seed inocula (bovine and swine sewage) in terracotta-based air-breathing MFC. A statistical approach on microbiological data confirmed different community enrichment in the MFCs, depending mainly on the inoculum. Terracotta separators impeded the growth of electroactive communities in contact with cathodes (biocathodes), while a thick biofilm was observed on the surface (anolyte-side) of the terracotta separator. Terracotta-free MFCs, set as control experiments, showed a well-developed biocathode, Biocathode-MFCs resulted in 4 to 6-fold higher power densities. All biofilms were analyzed by high-throughput Illumina sequencing applied to 16S rRNA gene. The results showed more abundant (3- to 5-fold) electroactive genera (mainly Geobacter, Pseudomonas, Desulfuromonas and Clostridia MBA03) in terracotta-free biocathodes. Nevertheless, terracotta separators induced only slight changes in anodic microbial communities.

Influence of water management on the active root-associated microbiota involved in arsenic, iron, and sulfur cycles in rice paddies.

In recent years, the role of microorganisms inhabiting rice rhizosphere in promoting arsenic contamination has emerged. However, little is known concerning the species and metabolic properties involved in this phenomenon. In this study, the influence of water management on the rhizosphere microbiota in relation to arsenic dissolution in soil solution was tested. Rice plants were cultivated in macrocosms under different water regimes: continuous flooding, continuous flooding with a 2-week period drainage before flowering, and dry soil watered every 10 days. The active bacterial communities in rhizosphere soil and in rhizoplane were characterized by 16S rRNA pyrosequencing. An in-depth analysis of microbial taxa with direct or indirect effects on arsenic speciation was performed and related contribution was evaluated. Continuous flooding promoted high diversity in the rhizosphere, with the plant strongly determining species richness and evenness. On the contrary, under watering the communities were uniform, with little differences between rhizosphere soil and rhizoplane. Arsenic-releasing and arsenite-methylating bacteria were selected by continuous flooding, where they represented 8% of the total. On the contrary, bacteria decreasing arsenic solubility were more abundant under watering, with relative abundance of 10%. These values reflected arsenic concentrations in soil solution: 135 µg L-1 and negligible in continuous flooding and under watering, respectively. When short-term drainage was applied before flowering, intermediate conditions were achieved. This evidence strongly indicates an active role of the rhizosphere microbiota in driving arsenic biogeochemistry in rice paddies, influenced by water management, explaining amounts and speciation of arsenic often found in rice grains.

Influences of dissolved oxygen concentration on biocathodic microbial communities in microbial fuel cells.

Dissolved oxygen (DO) at cathodic interface is a critical factor influencing microbial fuel cells (MFC) performance. In this work, three MFCs were operated with cathode under different DO conditions: i) air-breathing (A-MFC); ii) water-submerged (W-MFC) and iii) assisted by photosynthetic microorganisms (P-MFC). A plateau of maximum current was reached at 1.06±0.03mA, 1.48±0.06mA and 1.66±0.04mA, increasing respectively for W-MFC, P-MFC and A-MFC. Electrochemical and microbiological tools (Illumina sequencing, confocal microscopy and biofilm cryosectioning) were used to explore anodic and cathodic biofilm in each MFC type. In all cases, biocathodes improved oxygen reduction reaction (ORR) as compared to abiotic condition and A-MFC was the best performing system. Photosynthetic cultures in the cathodic chamber supplied high DO level, up to 16mgO2L-1, which sustained aerobic microbial community in P-MFC biocathode. Halomonas, Pseudomonas and other microaerophilic genera reached >50% of the total OTUs. The presence of sulfur reducing bacteria (Desulfuromonas) and purple non-sulfur bacteria in A-MFC biocathode suggested that the recirculation of sulfur compounds could shuttle electrons to sustain the reduction of oxygen as final electron acceptor. The low DO concentration limited the cathode in W-MFC. A model of two different possible microbial mechanisms is proposed which can drive predominantly cathodic ORR.