Constraint-based modeling analysis of the metabolism of two <Emphasis Type="Italic">Pelobacter</Emphasis> species

Background  

Pelobacter species are commonly found in a number of subsurface environments, and are unique members of the Geobacteraceae family. They are phylogenetically intertwined with both Geobacter and Desulfuromonas species. Pelobacter species likely play important roles in the fermentative degradation of unusual organic matters and syntrophic metabolism in the natural environments, and are of interest for applications in bioremediation and microbial fuel cells.     

Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of <Emphasis Type="Italic">Pelobacter carbinolicus</Emphasis>

Background  

Pelobacter carbinolicus, a bacterium of the family Geobacteraceae, cannot reduce Fe(III) directly or produce electricity like its relatives. How P. carbinolicus evolved is an intriguing problem. The genome of P. carbinolicus contains clustered regularly interspaced short palindromic repeats (CRISPR) separated by unique spacer sequences, which recent studies have shown to produce RNA molecules that interfere with genes containing identical sequences.     

Degradation of Acetaldehyde and Its Precursors by Pelobacter carbinolicus and P. acetylenicus

Pelobacter carbinolicus and P. acetylenicus oxidize ethanol in syntrophic cooperation with methanogens. Cocultures with Methanospirillum hungatei served as model systems for the elucidation of syntrophic ethanol oxidation previously done with the lost “Methanobacillus omelianskii” coculture. During growth on ethanol, both Pelobacter species exhibited NAD⁺-dependent alcohol dehydrogenase activity. Two different acetaldehyde-oxidizing activities were found: a benzyl viologen-reducing enzyme forming acetate, and a NAD⁺-reducing enzyme forming acetyl-CoA. Both species synthesized ATP from acetyl-CoA via acetyl phosphate. Comparative 2D-PAGE of ethanol-grown P. carbinolicus revealed enhanced expression of tungsten-dependent acetaldehyde: ferredoxin oxidoreductases and formate dehydrogenase. Tungsten limitation resulted in slower growth and the expression of a molybdenum-dependent isoenzyme. Putative comproportionating hydrogenases and formate dehydrogenase were expressed constitutively and are probably involved in interspecies electron transfer. In ethanol-grown cocultures, the maximum hydrogen partial pressure was about 1,000 Pa (1 mM) while 2 mM formate was produced. The redox potentials of hydrogen and formate released during ethanol oxidation were calculated to be E_H2 = -358±12 mV and E_HCOOH = -366±19 mV, respectively. Hydrogen and formate formation and degradation further proved that both carriers contributed to interspecies electron transfer. The maximum Gibbs free energy that the Pelobacter species could exploit during growth on ethanol was −35 to −28 kJ per mol ethanol. Both species could be cultivated axenically on acetaldehyde, yielding energy from its disproportionation to ethanol and acetate. Syntrophic cocultures grown on acetoin revealed a two-phase degradation: first acetoin degradation to acetate and ethanol without involvement of the methanogenic partner, and subsequent syntrophic ethanol oxidation. Protein expression and activity patterns of both Pelobacter spp. grown with the named substrates were highly similar suggesting that both share the same steps in ethanol and acetalydehyde metabolism. The early assumption that acetaldehyde is a central intermediate in Pelobacter metabolism was now proven biochemically.     

Characterization of two strains of Pelobacter carbinolicus isolated from anaerobic digesters

In anaerobic industrial digesters treating wastewaters from food industry, the sludges showed high capacities to degrade ethanol. The main syntrophic ethanoldegrading organisms were characterized as Pelobacter carbinolicus. During acetoin degradation, butanediol isomers were shown to be transiently produced. In coculture with Methanobrevibacter arboriphilus, the strains oxidized not only primary monoalcohols but also 1,2- and 1,3-diols. Ecological importance as well as metabolic pathways of diol degradation are discussed.     

Lack of Electricity Production by Pelobacter carbinolicus Indicates that the Capacity for Fe(III) Oxide Reduction Does Not Necessarily Confer Electron Transfer Ability to Fuel Cell Anodes

The ability of Pelobacter carbinolicus to oxidize electron donors with electron transfer to the anodes of microbial fuel cells was evaluated because microorganisms closely related to Pelobacter species are generally abundant on the anodes of microbial fuel cells harvesting electricity from aquatic sediments. P. carbinolicus could not produce current in a microbial fuel cell with electron donors which support Fe(III) oxide reduction by this organism. Current was produced using a coculture of P. carbinolicus and Geobacter sulfurreducens with ethanol as the fuel. Ethanol consumption was associated with the transitory accumulation of acetate and hydrogen. G. sulfurreducens alone could not metabolize ethanol, suggesting that P. carbinolicus grew in the fuel cell by converting ethanol to hydrogen and acetate, which G. sulfurreducens oxidized with electron transfer to the anode. Up to 83% of the electrons available in ethanol were recovered as electricity and in the metabolic intermediate acetate. Hydrogen consumption by G. sulfurreducens was important for ethanol metabolism by P. carbinolicus. Confocal microscopy and analysis of 16S rRNA genes revealed that half of the cells growing on the anode surface were P. carbinolicus, but there was a nearly equal number of planktonic cells of P. carbinolicus. In contrast, G. sulfurreducens was primarily attached to the anode. P. carbinolicus represents the first Fe(III) oxide-reducing microorganism found to be unable to produce current in a microbial fuel cell, providing the first suggestion that the mechanisms for extracellular electron transfer to Fe(III) oxides and fuel cell anodes may be different.     

General metabolism of Laribacter hongkongensis: a genome-wide analysis

Background

Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea. In this study, we performed an in-depth annotation of the genes and pathways of the general metabolism of L. hongkongensis and correlated them with its phenotypic characteristics.

Results

The L. hongkongensis genome possesses the pentose phosphate and gluconeogenesis pathways and tricarboxylic acid and glyoxylate cycles, but incomplete Embden-Meyerhof-Parnas and Entner-Doudoroff pathways, in agreement with its asaccharolytic phenotype. It contains enzymes for biosynthesis and β-oxidation of saturated fatty acids, biosynthesis of all 20 universal amino acids and selenocysteine, the latter not observed in Neisseria gonorrhoeae, Neisseria meningitidis and Chromobacterium violaceum. The genome contains a variety of dehydrogenases, enabling it to utilize different substrates as electron donors. It encodes three terminal cytochrome oxidases for respiration using oxygen as the electron acceptor under aerobic and microaerophilic conditions and four reductases for respiration with alternative electron acceptors under anaerobic conditions. The presence of complete tetrathionate reductase operon may confer survival advantage in mammalian host in association with diarrhea. The genome contains CDSs for incorporating sulfur and nitrogen by sulfate assimilation, ammonia assimilation and nitrate reduction. The existence of both glutamate dehydrogenase and glutamine synthetase/glutamate synthase pathways suggests an importance of ammonia metabolism in the living environments that it may encounter.

Conclusions

The L. hongkongensis genome possesses a variety of genes and pathways for carbohydrate, amino acid and lipid metabolism, respiratory chain and sulfur and nitrogen metabolism. These allow the bacterium to utilize various substrates for energy production and survive in different environmental niches.     

The outer membrane protein Omp35 affects the reduction of Fe(III), nitrate,and fumarate by <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> MR-1

Background  

Shewanella oneidensis MR-1 uses several electron acceptors to support anaerobic respiration including insoluble species such as iron(III) and manganese(IV) oxides, and soluble species such as nitrate, fumarate, dimethylsulfoxide and many others. MR-1 has complex branched electron transport chains that include components in the cytoplasmic membrane, periplasm, and outer membrane (OM). Previous studies have implicated a role for anaerobically upregulated OM electron transport components in the use of insoluble electron acceptors, and have suggested that other OM components may also contribute to insoluble electron acceptor use. In this study, the role for an anaerobically upregulated 35-kDa OM protein (Omp35) in the use of anaerobic electron acceptors was explored.     

Cultivation of syntrophic anaerobic bacteria in membrane-separated culture devices

A dialysis culture device was used for growth of syntrophic fatty acid-oxidizing and ethanol-oxidizing anaerobic bacteria. A pure culture of the fatty acid oxidizer Clostridium bryantii was grown inside dialysis tubing which was surrounded by a pure culture of Desulfovibrio vulgaris. The same apparatus was used for the syntrophic cultivation of Pelobacter acetylenicus and Acetobacterium woodii with ethanol as substrate. In both cases, substrate degradation and product formation were about half as fast as with the homogeneously mixed control cultures. In the compartment of the hydrogen producer, the concentration of free hydrogen during syntrophic ethanol degradation was about 10 times as high as in that of the hydrogen utilizer, whereas the homogeneously mixed culture exhibited an intermediate hydrogen partial pressure.     

Environmental controls and the influence of tree species on temporal variation in soil respiration in subtropical China

Background and aims

The knowledge of individual tree species impacts on soil respiration based on rigorous experimental designs is limited, but is crucial to help guide selection of species for reforestation and carbon (C) management purposes.

Methods

We assessed monthly soil respiration and its components, litterfall input, fine root production and mortality under 19-year-old native coniferous Cunninghamia lanceolata and broadleaved Mytilaria laosensis plantations in sub-tropical China.

Results

Total soil respiration from October 2011 to March 2013 was significantly lower under the C. lanceolata than the M. laosensis plantation. The difference in respiration rates derived from fine roots and the litter layer explained much of the variation of total soil respiration between the two tree species. We used an exponential equation and base temperature (10 °C) to normalize soil respiration rate and its components (R₁₀) and determined the correlation between R₁₀ and soil moisture. Although soil moisture had a positive relationship with R₁₀ derived from roots or litter under both C. lanceolata and M. laosensis forests, these positive correlations were masked by negative relationships between soil moisture and R₁₀ derived from root-free soil, which resulted in a neutral correlation between total R₁₀ and soil moisture under C. lanceolata forests. Monthly litterfall input was associated with variation in concurrent total soil respiration rate under the M. laosensis plantation and respiration rate lagging 3 months behind under the C. lanceolata plantation, which may suggest that litterfall input from M. laosensis can more rapidly produce C substrates for microbial respiration than litterfall from C. lanceolata.

Conclusions

This study highlighted that tree species-induced variation in the quality and quantity of fine roots and litterfall can impact not only the soil respiration rate but also the seasonal variation model of forest soil respiration.     

Comparative Metagenomics of Anode-Associated Microbiomes Developed in Rice Paddy-Field Microbial Fuel Cells

In sediment-type microbial fuel cells (sMFCs) operating in rice paddy fields, rice-root exudates are converted to electricity by anode-associated rhizosphere microbes. Previous studies have shown that members of the family Geobacteraceae are enriched on the anodes of rhizosphere sMFCs. To deepen our understanding of rhizosphere microbes involved in electricity generation in sMFCs, here, we conducted comparative analyses of anode-associated microbiomes in three MFC systems: a rice paddy-field sMFC, and acetate- and glucose-fed MFCs in which pieces of graphite felt that had functioned as anodes in rice paddy-field sMFC were used as rhizosphere microbe-bearing anodes. After electric outputs became stable, microbiomes associated with the anodes of these MFC systems were analyzed by pyrotag sequencing of 16S rRNA gene amplicons and Illumina shotgun metagenomics. Pyrotag sequencing showed that Geobacteraceae bacteria were associated with the anodes of all three systems, but the dominant Geobacter species in each MFC were different. Specifically, species closely related to G. metallireducens comprised 90% of the anode Geobacteraceae in the acetate-fed MFC, but were only relatively minor components of the rhizosphere sMFC and glucose-fed MFC, whereas species closely related to G. psychrophilus were abundantly detected. This trend was confirmed by the phylogenetic assignments of predicted genes in shotgun metagenome sequences of the anode microbiomes. Our findings suggest that G. psychrophilus and its related species preferentially grow on the anodes of rhizosphere sMFCs and generate electricity through syntrophic interactions with organisms that excrete electron donors.     

Identification and characterization of insect-specific proteins by genome data analysis

Background

Previous studies have sought to identify a link between the distribution of variable genes amongst isolates of Campylobacter jejuni and particular host preferences. The genomic sequence data available currently was obtained using only isolates from human or chicken hosts. In order to identify variable genes present in isolates from alternative host species, five subtractions between C. jejuni isolates from different sources (rabbit, cattle, wild bird) were carried out, designed to assess genomic variability within and between common multilocus sequence type (MLST) clonal complexes (ST-21, ST-42, ST-45 and ST-61).

Results

The vast majority (97%) of the 195 subtracted sequences identified had a best BLASTX match with a Campylobacter protein. However, there was considerable variation within and between the four clonal complexes included in the subtractions. The distributions of eight variable sequences, including four with putative roles in the use of alternative terminal electron acceptors, amongst a panel of C. jejuni isolates representing diverse sources and STs, were determined.

Conclusion

There was a clear correlation between clonal complex and the distribution of the metabolic genes. In contrast, there was no evidence to support the hypothesis that the distribution of such genes may be related to host preference. The other variable genes studied were also generally distributed according to MLST type. Thus, we found little evidence for widespread horizontal gene transfer between clonal complexes involving these genes.     

A native Glomus intraradices strain from a Mediterranean saline area exhibits salt tolerance and enhanced symbiotic efficiency with maize plants under salt stress conditions

Aims

Arbuscular mycorrhizal (AM) fungi have been shown to occur naturally in saline environments and it has been suggested that differences in fungal behaviour and efficiency can be due to the origin and adaptation of the AM fungus. These findings invite to look out for AM fungal species isolated in saline environments and compare their salt-tolerance mechanisms with those of species living in non-saline areas.

Methods

A fungal strain of G. intraradices (Gi CdG) isolated from a region with serious problems of salinity and affected by desertification, has been compared with a collection strain of the same species, used as a model fungus. An in vitro experiment tested the ability of both AM fungi to grow under increasing salinity and an in vivo experiment compared their symbiotic efficiency with maize plants grown under salt stress conditions.

Results

The isolate Gi CdG developed better under saline conditions and induced considerably the expression of GintBIP, Gint14-3-3 and GintAQP1 genes, while it showed a lower induction of GintSOD1 gene than the collection G. intraradices strain. The isolate Gi CdG also stimulated the growth of maize plants under two levels of salinity more than the collection strain. The higher symbiotic efficiency of Gi CdG was corroborated by the enhanced efficiency of photosystem II and stomatal conductance and the lower electrolyte leakage exhibited by maize plants under the different conditions assayed.

Conclusions

The higher tolerance to salinity and symbiotic efficiency exhibited by strain Gi CdG as compared to the collection G. intraradices strain may be due to a fungal adaptation to saline environments. Such adaptation may be related to the significant up-regulation of genes encoding chaperones or genes encoding aquaporins. The present study remarks that AM fungi isolated from areas affected by salinity can be a powerful tool to enhance the tolerance of crops to saline stress conditions.     

Influence of interactions between litter decomposition and rhizosphere activity on soil respiration and on the temperature sensitivity in a subtropical montane forest in SW China

Aims

The aims were to identify the effects of interactions between litter decomposition and rhizosphere activity on soil respiration and on the temperature sensitivity of soil respiration in a subtropical forest in SW China.

Methods

Four treatments were established: control (CK), litter removal (NL), trenching (NR) and trenching together with litter removal (NRNL). Soil CO₂ efflux, soil temperature, and soil water content were measured once a month over two years. Soil respiration was divided into four components: the decomposition of basic soil organic matter (SOM), litter respiration, root respiration, and the interaction effect between litter decomposition and rhizosphere activity. A two-factor regression equation was used to correct the value of soil CO₂ efflux.

Results

We found a significant effect of the interaction between litter decomposition and rhizosphere activity (R _INT) on total soil respiration, and R _INT exhibited significant seasonal variation, accounting for 26 and 31 % of total soil respiration in the dry and rainy seasons, respectively. However, we found no significant interaction effect on the temperature sensitivity of soil respiration. The temperature sensitivity was significantly increased by trenching compared with the control, but was unchanged by litter removal.

Conclusions

Though the interaction between litter decomposition and rhizosphere activity had no effects on temperature sensitivity, it had a significant positive effect on soil respiration. Our results not only showed strong influence of rhizosphere activity on temperature sensitivity, but provided a viable way to identify the contribution of SOM to soil respiration, which could help researchers gain insights on the carbon cycle.     

Seagrass (Zostera marina) Colonization Promotes the Accumulation of Diazotrophic Bacteria and Alters the Relative Abundances of Specific Bacterial Lineages Involved in Benthic Carbon and Sulfur Cycling

Seagrass colonization changes the chemistry and biogeochemical cycles mediated by microbes in coastal sediments. In this study, we molecularly characterized the diazotrophic assemblages and entire bacterial community in surface sediments of a Zostera marina-colonized coastal lagoon in northern China. Higher nitrogenase gene (nifH) copy numbers were detected in the sediments from the vegetated region than in the sediments from the unvegetated region nearby. The nifH phylotypes detected were mostly affiliated with the Geobacteraceae, Desulfobulbus, Desulfocapsa, and Pseudomonas. Redundancy analysis based on terminal restriction fragment length polymorphism analysis showed that the distribution of nifH genotypes was mostly shaped by the ratio of total organic carbon to total organic nitrogen, the concentration of cadmium in the sediments, and the pH of the overlying water. High-throughput sequencing and phylogenetic analyses of bacterial 16S rRNA genes also indicated the presence of Geobacteraceae and Desulfobulbaceae phylotypes in these samples. A comparison of these results with those of previous studies suggests the prevalence and predominance of iron(III)-reducing Geobacteraceae and sulfate-reducing Desulfobulbaceae diazotrophs in coastal sedimentary environments. Although the entire bacterial community structure was not significantly different between these two niches, Desulfococcus (Deltaproteobacteria) and Anaerolineae (Chloroflexi) presented with much higher proportions in the vegetated sediments, and Flavobacteriaceae (Bacteroidetes) occurred more frequently in the bare sediments. These data suggest that the high bioavailability of organic matter (indicated by relatively lower carbon-to-nitrogen ratios) and the less-reducing anaerobic condition in vegetated sediments may favor Desulfococcus and Anaerolineae lineages, which are potentially important populations in benthic carbon and sulfur cycling in the highly productive seagrass ecosystem.     

Metabolic strategies of marine subseafloor Chloroflexi inferred from genome reconstructions

Uncultured members of the Chloroflexi phylum are highly enriched in numerous subseafloor environments. Their metabolic potential was evaluated by reconstructing 31 Chloroflexi genomes from six different subseafloor habitats. The near ubiquitous presence of enzymes of the Wood–Ljungdahl pathway, electron bifurcation, and ferredoxin-dependent transport-coupled phosphorylation indicated anaerobic acetogenesis was central to their catabolism. Most of the genomes simultaneously contained multiple degradation pathways for complex carbohydrates, detrital protein, aromatic compounds, and hydrogen, indicating the coupling of oxidation of chemically diverse organic substrates to ubiquitous CO₂ reduction. Such pathway combinations may confer a fitness advantage in subseafloor environments by enabling these Chloroflexi to act as primary fermenters and acetogens in one microorganism without the need for syntrophic H₂ consumption. While evidence for catabolic oxygen respiration was limited to two phylogenetic clusters, the presence of genes encoding putative reductive dehalogenases throughout the phylum expanded the phylogenetic boundary for potential organohalide respiration past the Dehalococcoidia class.