Search

The vast majority of microbes inhabiting oligotrophic shallow subsurface soil environments have not been isolated or studied under controlled laboratory conditions. In part, the challenges associated with isolating shallow subsurface microbes may persist because microbes in deeper soils are adapted to low nutrient availability or quality. Here, we use high-throughput dilution-to-extinction culturing to isolate shallow subsurface microbes from a conifer forest in Arizona, USA. We hypothesized that the concentration of heterotrophic substrates in microbiological growth medium would affect which microbial taxa were culturable from these soils. To test this, we diluted cells extracted from soil into one of two custom-designed defined growth media that differed by 100-fold in the concentration of amino acids and organic carbon. Across the two media, we isolated a total of 133 pure cultures, all of which were classified as Actinobacteria or Alphaproteobacteria The substrate availability dictated which actinobacterial phylotypes were culturable but had no significant effect on the culturability of Alphaproteobacteria We isolated cultures that were representative of the most abundant phylotype in the soil microbial community ( Bradyrhizobium spp.) and representatives of five of the top 10 most abundant Actinobacteria phylotypes, including Nocardioides spp., Mycobacterium spp., and several other phylogenetically divergent lineages. Flow cytometry of nucleic acid-stained cells showed that cultures isolated on low-substrate medium had significantly lower nucleic acid fluorescence than those isolated on high-substrate medium. These results show that dilution-to-extinction is an effective method to isolate abundant soil microbes and that the concentration of substrates in culture medium influences the culturability of specific microbial lineages. IMPORTANCE Isolating environmental microbes and studying their physiology under controlled conditions are essential aspects of understanding their ecology. Subsurface ecosystems are typically nutrient-poor environments that harbor diverse microbial communities-the majority of which are thus far uncultured. In this study, we use modified high-throughput cultivation methods to isolate subsurface soil microbes. We show that a component of whether a microbe is culturable from subsurface soils is the concentration of growth substrates in the culture medium. Our results offer new insight into technical approaches and growth medium design that can be used to access the uncultured diversity of soil microbes., (Copyright © 2020 Bartelme et al.)

Bioremediation is one of the existing techniques applied for treating oil-contaminated soil, which can be improved by the incorporation of low-cost nutritional materials. This study aimed to assess the addition of two low-cost plant residues, sugarcane bagasse (SCB) and leaf litter (LL) of the forest leguminous Mimosa caesalpiniifolia plant (sabiá), either separately or combined, to a contaminated soil from a petroleum refinery area, analyzed after 90 days of treatment. Individually, both amounts of SCB (20 and 40 g kg -1 ) favored the growth of total heterotrophic bacteria and total fungi, while LL at 20 g kg -1 better stimulated the hydrocarbon-degrading microorganism's activity in the soil. However, no TPH removal was observed under any of these conditions. Higher microbial growth was detected by the application of both plant residues in multicontaminated soil. The maximum TPH removal of 30% was achieved in amended soil with 20 g kg -1 SCB and 20 kg -1 LL. All the experimental conditions revealed changes in the microbial community structure, related to the handling of the soil, with abundance of Alphaproteobacteria. This study demonstrates the effectiveness of the plant residues SCB and LL as low-cost nutritional materials for biodegradation of hydrocarbon in real oil contaminated soil by indigenous populations.

An agar-degrading bacterium was isolated from soil collected in a vegetable cropping field. The growth of this isolate was enhanced by supplying culture supernatants of bacteria belonging to the order Rhizobiales. Phylogenetic analysis based on 16S rRNA gene sequences indicated the novel bacterium, strain KA5-B(T), belonged to the genus Steroidobacter in Gammaproteobacteria, but differed from its closest relative, Steroidobacter denitrificans FS(T), at the species level with 96.5% similarity. Strain KA5-B(T) was strictly aerobic, Gram-negative, non-motile, non-spore forming, and had a straight to slightly curved rod shape. Cytochrome oxidase and catalase activities were positive. The strain grew on media containing culture supernatants in a temperature range of 15-37°C and between pH 4.5 and 9.0, with optimal growth occurring at 30°C and pH 6.0-8.0. No growth occurred at 10 or 42°C or at NaCl concentrations more than 3% (w/v). The main cellular fatty acids were iso-C15:0, C16:1ω7c, and iso-C17:1ω9c. The main quinone was ubiquinone-8 and DNA G+C content was 62.9 mol%. In contrast, strain FS(T) was motile, did not grow on the agar plate, and its dominant cellular fatty acids were C15:0 and C17:1ω8c. Based on its phylogenetic and phenotypic properties, strain KA5-B(T) (JCM 18477(T) = KCTC 32107(T)) represents a novel species in genus Steroidobacter, for which the name Steroidobacter agariperforans sp. nov. is proposed.

Seepage of coal-bed methane (CBM) through soils is a potential source of atmospheric CH4 and also a likely source of ancient (i.e. (14) C-dead) carbon to soil microbial communities. Natural abundance (13) C and (14) C compositions of bacterial membrane phospholipid fatty acids (PLFAs) and soil gas CO2 and CH4 were used to assess the incorporation of CBM-derived carbon into methanotrophs and other members of the soil microbial community. Concentrations of type I and type II methanotroph PLFA biomarkers (16:1ω8c and 18:1ω8c, respectively) were elevated in CBM-impacted soils compared with a control site. Comparison of PLFA and 16s rDNA data suggested type I and II methanotroph populations were well estimated and overestimated by their PLFA biomarkers, respectively. The δ(13) C values of PLFAs common in type I and II methanotrophs were as negative as -67‰ and consistent with the assimilation of CBM. PLFAs more indicative of nonmethanotrophic bacteria had δ(13) C values that were intermediate indicating assimilation of both plant- and CBM-derived carbon. Δ(14) C values of select PLFAs (-351 to -936‰) indicated similar patterns of CBM assimilation by methanotrophs and nonmethanotrophs and were used to estimate that 35-91% of carbon assimilated by nonmethanotrophs was derived from CBM depending on time of sampling and soil depth., (© 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The objectives of this study were to explore the effects of long-term organic and inorganic fertilizations on the composition and abundance of nirS-type denitrifiers in black soil. Soil samples were collected from 4 treatments (i. e. no fertilizer treatment, CK; organic manure treatment, OM; chemical fertilizer treatment (NPK) and combination of organic and chemical fertilizers treatment (MNPK)) in Gongzhuling Long-term Fertilization Experiment Station. Composition and abundance of nirS-type denitrifiers were analyzed with terminal restriction fragment length polymorphism (T-RFLP) and real-time quantitative PCR (Q-PCR), respectively. Denitrification enzyme activity (DEA) and soil properties were also measured. Application of organic fertilizers (OM and MNPK) significantly increased the DEAs of black soil, with the DEAs in OM and MNPK being 5.92 and 6.03 times higher than that in CK treatment, respectively, whereas there was no significant difference between NPK and CK. OM and MNPK treatments increased the abundances of nirS-type denitrifiers by 2.73 and 3.83 times relative to that of CK treatment, respectively. The abundance of nirS-type denitrifiers in NPK treatment was not significantly different from that of CK. The T-RFLP analysis of nirS genes showed significant differences in community composition between organic and inorganic treatments, with the emergence of a 79 bp T-RF, a significant decrease in relative abundance of the 84 bp T-RF and a loss of the 99 bp T-RF in all organic treatments. Phylogenetic analysis indicated that the airS-type denitrifiers in the black soil were mainly composed of alpha, beta and gamma-Proteobacteria. The 79 bp-type denitrifiers inhabiting exclusively in organic treatments (OM and MNPK) were affiliated to Pseudomonadaceae in gamma-Proteobacteria and Burkholderiales in beta-Proteobacteria. The 84 bp-types were related to Burkholderiales and Rhodocyclales. Correlation analysis indicated that pH, concentrations of total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), nitrate (NO3(-) -N) and ammonia (NH4(+) -N) were significantly related to abundances of nirS-denitrifers (r = 0.724-0.922, P < 0.05) and the DEA (r = 0.453-0.938, P < 0.01). In addition, the DEAs were linearly and positively correlated with the abundances of nirS-type denitrifers (r = 0.85, P < 0.01). Redundancy analysis showed that except moisture, pH and concentrations of TP, TP, TOC, NH4(+) -N and NO3(-) -N were significantly correlated with the community structure of nirS-type denirifiers (r = 0.440-0.862, P < 0.01). Furthermore, the DEAs were significantly correlated with the compositions of nirS-denirifiers (r = 0.863, P < 0.01). In conclusion, the airS-type denitrifiers in the black soil are more responsive to the organic treatments than to the inorganic treatments in terms of community composition and abundance, both of which are correlated with the changes of DEAs.

To gain insight into the factors driving the structure of bacterial communities in soil, we applied real-time PCR, PCR-denaturing gradient gel electrophoreses, and phylogenetic microarray approaches targeting the 16S rRNA gene across a range of different land usages in the Netherlands. We observed that the main differences in the bacterial communities were not related to land-use type, but rather to soil factors. An exception was the bacterial community of pine forest soils (PFS), which was clearly different from all other sites. PFS had lowest bacterial abundance, lowest numbers of operational taxonomic units (OTUs), lowest soil pH, and highest C : N ratios. C : N ratio strongly influenced bacterial community structure and was the main factor separating PFS from other fields. For the sites other than PFS, phosphate was the most important factor explaining the differences in bacterial communities across fields. Firmicutes were the most dominant group in almost all fields, except in PFS and deciduous forest soils (DFS). In PFS, Alphaproteobacteria was most represented, while in DFS, Firmicutes and Gammaproteobacteria were both highly represented. Interestingly, Bacillii and Clostridium OTUs correlated with pH and phosphate, which might explain their high abundance across many of the Dutch soils. Numerous bacterial groups were highly correlated with specific soil factors, suggesting that they might be useful as indicators of soil status.

Nucleic acid-based community fingerprinting methods are valuable tools in microbial ecology, as they offer rapid and robust means to compare large series of replicates and references. To avoid the time-consuming and potentially subjective procedures of peak-based examination, we assessed the possibility to apply direct curve-based data analysis on community fingerprints produced with bacterial length heterogeneity PCR (LH-PCR). The dataset comprised 180 profiles from a 21-week rhizoremediation greenhouse experiment with three treatments and 10 sampling times. Curve-based analysis quantified the progressive effect of the plant (Galega orientalis) and the reversible effect of the contaminant (fuel oil) on bacterial succession. The major observed community shifts were assigned to changes in plant biomass and contamination level by canonical correlation analysis. A novel method to extract relative abundance data from the fingerprint curves for Shannon diversity index revealed contamination to reversibly decrease community complexity. By cloning and sequencing the fragment lengths, recognized to change in time in the averaged LH-PCR profiles, we identified Aquabacterium (Betaproteobacteria) as the putative r-strategic fuel oil degrader, and K-strategic Alphaproteobacteria growing in abundance later in succession. Curve-based community fingerprint analysis can be used for rapid data prescreening or as a robust alternative for the more heavily parameterized peak-based analysis., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

A field experiment was conducted with cultivation of hybrid and conventional cultivars in a rice paddy from China. Rhizosphere soil was sampled and CO(2) flux was measured at tillering (S1), grain filling (S2) and ripening (S3) across the growth stages. Microbial community structure, abundance and activity were analyzed using a combination of functional (enzymes) and denaturing gradient gel electrophoresis (DGGE) and real-time PCR molecular approaches. Invertase and urease activities, total microbial biomass carbon, bacterial 16S rRNA and fungal internal transcribed spacer rRNA gene copies were found to be the highest at S2 under both cultivars, being greater under the hybrid cultivar than under the conventional cultivar across the stages. Moreover, the CO(2) flux was 11%, 16% and 25% higher under the hybrid cultivar than under the conventional cultivar at S1, S2 and S3, respectively. Principal component analyses of the PCR-DGGE profile revealed a significant difference between conventional and hybrid cultivars across growth stages. Sequencing DGGE bands of the bacterial 16S rRNA gene showed that a particular bacterial group of Alphaproteobacteria was enhanced and several distinct operational taxonomic units markedly resembled Ascomycota under the hybrid cultivar. These illustrate a significant selection of a particular group of bacteria and fungi of the hybrid cultivar. However, the potential impacts of these cultivar effects in soil C and N cycling deserve further field studies., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Changes in the abundance of bacteria and fungi and in the composition of bacterial communities during primary succession were investigated in a brown coal mine deposit area near Sokolov, the Czech Republic, using phospholipid fatty acids analysis, microarray and 16S rRNA gene sequencing. The study considered a chronosequence of sites undergoing spontaneous succession: 6-, 12-, 21- and 45-year-old and a 21-year-old site revegetated with Alnus glutinosa. During succession, organic carbon and the total nitrogen content increased while the pH and the C/N ratio decreased. Microbial biomass and bacterial diversity increased until 21 years and decreased later; bacteria dominated over fungi in the initial and late phases of succession. Bacterial community composition of the 6-year-old site with no vegetation cover largely differed from the older sites, especially by a higher content of Gammaproteobacteria, Cyanobacteria and some Alphaproteobacteria. Bacteria belonging to the genera Acidithiobacillus, Thiobacillus and related taxa, the CO(2) and N(2) fixers, dominated the community at this site. In the later phases, bacterial community development seemed to reflect more the changes in soil nutrient content and pH than vegetation with a decrease of Actinobacteria and an increase of Acidobacteria. The site revegetated with A. glutinosa resembled the 45-year-old primary succession site and exhibited an even lower pH and C/N ratio, indicating that recultivation is able to accelerate soil development., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The ability of soil bacteria to successfully compete with a range of other microbial species is crucial for their growth and survival in the nutrient-limited soil environment. In the present work, we studied the behavior and transcriptional responses of soil-inhabiting Pseudomonas fluorescens strain Pf0-1 on nutrient-poor agar to confrontation with strains of three phylogenetically different bacterial genera, that is, Bacillus, Brevundimonas and Pedobacter. Competition for nutrients was apparent as all three bacterial genera had a negative effect on the density of P. fluorescens Pf0-1; this effect was most strong during the interaction with Bacillus. Microarray-based analyses indicated strong differences in the transcriptional responses of Pf0-1 to the different competitors. There was higher similarity in the gene expression response of P. fluorescens Pf0-1 to the Gram-negative bacteria as compared with the Gram-positive strain. The Gram-negative strains did also trigger the production of an unknown broad-spectrum antibiotic in Pf0-1. More detailed analysis indicated that expression of specific Pf0-1 genes involved in signal transduction and secondary metabolite production was strongly affected by the competitors' identity, suggesting that Pf0-1 can distinguish among different competitors and fine-tune its competitive strategies. The results presented here demonstrate that P. fluorescens Pf0-1 shows a species-specific transcriptional and metabolic response to bacterial competitors and provide new leads in the identification of specific cues in bacteria-bacteria interactions and of novel competitive strategies, antimicrobial traits and genes.

2-Methyl-4-chlorophenoxyacetic acid (MCPA) is a widely used phenoxyalkanoic acid herbicide and subject to aerobic microbial degradation. Earthworms stimulate both growth and activity of MCPA-degrading bacteria in soil. Thus, active MCPA degraders in soil and drilosphere (i.e. burrow walls, gut content and cast) were assessed by 16S rRNA stable isotope probing in soil columns under experimental conditions designed to minimize laboratory incubation biases. Agriculturally relevant concentrations of [(13) C]MCPA (20 µg g(dw) (-1)) were degraded in soil within 23 and 27 days in the presence and absence of earthworms respectively. Total 16S rRNA analysis revealed 73 operational taxonomic units indicative of active Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes, Gemmatimonadetes, Planctomycetes, Proteobacteria and Verrucomicrobia in soil and drilosphere derived material. Seven operational taxonomic units indicative of Alpha-, Beta-, Gammaproteobacteria and Firmicutes consumed MCPA-[(13) C]. Dominant consumers of MCPA-[(13) C] were Alphaproteobacteria (Sphingomonadaceae and Bradyrhizobiaceae) in soil and drilosphere. Beta- (Comamonadaceae) and Gammaproteobacteria (Xanthomonadaceae) were also important MCPA-[(13) C] consumers in burrow walls only, indicating that earthworms favour betaproteobacterial MCPA degraders. In oxic microcosms with bulk soil, burrow walls and cast, 20 and 300-400 µg g(dw) (-1) [(13) C]MCPA were consumed within 24 h and 20 days respectively. Gut contents did not facilitate the degradation of [(13) C]MCPA. Sphingomonadaceae dominated MCPA-[(13) C] consumers in bulk soil and burrow wall microcosms, while Beta- and Gammaproteobacteria (Burkholderiacea, Comamonadaceae, Oxalobacteraceae and Xanthomonadaceae) dominated MCPA-[(13) C] consumers in microcosms of cast, indicating that the latter taxa are prone to respond to MCPA in cast. The collective data indicated that Alphaproteobacteria are major MCPA degraders in soil and drilosphere., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The bacterial diversity present in sediments of a well-preserved mangrove in Ilha do Cardoso, located in the extreme south of São Paulo State coastline, Brazil, was assessed using culture-independent molecular approaches (denaturing gradient gel electrophoresis (DGGE) and analysis of 166 sequences from a clone library). The data revealed a bacterial community dominated by Alphaproteobacteria (40.36% of clones), Gammaproteobacteria (19.28% of clones) and Acidobacteria (27.71% of clones), while minor components of the assemblage were affiliated to Betaproteobacteria, Deltaproteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. The clustering and redundancy analysis (RDA) based on DGGE were used to determine factors that modulate the diversity of bacterial communities in mangroves, such as depth, seasonal fluctuations, and locations over a transect area from the sea to the land. Profiles of specific DGGE gels showed that both dominant ('universal' Bacteria and Alphaproteobacteria) and low-density bacterial communities (Betaproteobacteria and Actinobacteria) are responsive to shifts in environmental factors. The location within the mangrove was determinant for all fractions of the community studied, whereas season was significant for Bacteria, Alphaproteobacteria, and Betaproteobacteria and sample depth determined the diversity of Alphaproteobacteria and Actinobacteria.

Shifts in the activity and diversity of microbes involved in aliphatic and aromatic hydrocarbon degradation in contaminated soil were investigated. Subsurface soil was collected from a gas station that had been abandoned since 1995 owing to ground subsidence. The total petroleum hydrocarbon content of the sample was approximately 2,100 mg/kg, and that of the soil below a gas pump was over 23,000 mg/kg. Enrichment cultures were grown in mineral medium that contained hexadecane (H) or naphthalene (N) at a concentration of 200 mg/l. In the Henrichment culture, a real-time PCR assay revealed that the 16S rRNA gene copy number increased from 1.2x105 to 8.6x106 with no lag phase, representing an approximately 70-fold increase. In the N-enrichment culture, the 16S rRNA copy number increased about 13-fold after 48 h, from 6.3x104 to 8.3x105. Microbial communities in the enrichment cultures were studied by denaturing gradient gel electrophoresis and by analysis of 16S rRNA gene libraries. Before the addition of hydrocarbons, the gas station soil contained primarily Alpha- and Gammaproteobacteria. During growth in the H-enrichment culture, the contribution of Bacteriodetes to the microbial community increased significantly. On the other hand, during N-enrichment, the Betaproteobacteria population increased conspicuously. These results suggest that specific phylotypes of bacteria were associated with the degradation of each hydrocarbon.

The aim of this work is to describe the diversity and phylogeny of rhizobial bacteria associated to nodules of Ononis tridentata L. in different geographical regions of Spain. Twenty-two bacterial isolates were characterized using several molecular techniques (16S amplified ribosomal deoxyribonucleic acid restriction analysis, fingerprinting, and sequencing) and phylogenies were inferred from their 16S and nodC gene sequences. Phylogenetically, the isolates grouped with the genera Rhizobium, Mesorhizobium, Phylobacterium, and Bosea. The nodC gene, essential for nodulation, was detected for the first time in isolates close to the genera Bosea and Phyllobacterium. The bacteria isolated showed a high diversity at the genus, species, and strain level regardless of the geographical origin of the host plant. This is the first report describing bacteria associated to nodules of O. tridentata. This shrub legume is highly prized for the revegetation of gypsum soils in semiarid Mediterranean areas. Our molecular description of bacteria associated to this legume improves the current understanding of the ecology of this plant species. Our findings have implications for formulating suitable bacterial inocula to recover gypsum ecosystems.

A Gram-type negative or variable, aerobic, rod-shaped, nitrogen-fixing bacterium, designated strain CC4(T), was isolated on nutrient agar from a soil sample collected at a regional agricultural research field station located in Kaohsiung County, Taiwan. 16S rRNA gene sequence analysis demonstrated that this isolate is unique, showing <92.5 % similarity with respect to species of the genera Xanthobacter (maximum of 92.2 % similarity), Prosthecomicrobium (92.0 %), Devosia (91.9 %), Kaistia (91.9 %) and Methylocystis (91.9 %). The organism utilized acetic acid, formic acid, beta-hydroxybutyric acid, dl-lactic acid, succinic acid, bromosuccinic acid, l-alaninamide, l-alanine, l-alanyl glycine, l-glutamic acid and l-proline as substrates, but not methanol or methylamine. Chemotaxonomic data revealed that strain CC4(T) contains ubiquinone Q-10 as the major respiratory quinone. The characteristic diamino acid of the peptidoglycan was meso-diaminopimelic acid. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, an unknown lipid and an unknown aminolipid. The fatty acid profile differed from those of members of the genera Xanthobacter, Prosthecomicrobium, Devosia and Kaistia. The predominant fatty acids were C(18 : 1)omega7c, C(19 : 0) cyclo and C(16 : 0). On the basis of 16S rRNA gene sequence analysis in combination with chemotaxonomic and physiological data, strain CC4(T) represents a novel genus and species, for which we propose the name Pseudoxanthobacter soli gen. nov., sp. nov. The type strain of Pseudoxanthobacter soli is CC4(T) (=DSM 19599(T) =CIP 109513(T)).

For extremely ionizing radiation-resistant bacteria, survival has been attributed to protection of proteins from oxidative damage during irradiation, with the result that repair systems survive and function with far greater efficiency during recovery than in sensitive bacteria. Here we examined the relationship between survival of dry-climate soil bacteria and the level of cellular protein oxidation induced by desiccation. Bacteria were isolated from surface soils of the shrub-steppe of the US Department of Energy's Hanford Site in Washington State. A total of 63 isolates were used for phylogenetic analysis. The majority of isolates were closely related to members of the genus Deinococcus, with Chelatococcus, Methylobacterium and Bosea also among the genera identified. Desiccation-resistant isolates accumulated high intracellular manganese and low iron concentrations compared to sensitive bacteria. In vivo, proteins of desiccation-resistant bacteria were protected from oxidative modifications that introduce carbonyl groups in sensitive bacteria during drying. We present the case that survival of bacteria that inhabit dry-climate soils are highly dependent on mechanisms, which limit protein oxidation during dehydration.

2,6-Dichlorobenzamide (BAM), a persistent metabolite from the herbicide 2,6-dichlorobenzonitrile (dichlobenil), is the pesticide residue most frequently detected in Danish groundwater. A BAM-mineralizing bacterial community was enriched from dichlobenil-treated soil sampled from the courtyard of a former plant nursery. A BAM-mineralizing bacterium (designated strain MSH1) was cultivated and identified by 16S rRNA gene sequencing and fatty acid analysis as being closely related to members of the genus Aminobacter, including the only cultured BAM degrader, Aminobacter sp. strain ASI1. Strain MSH1 mineralized 15 to 64% of the added [ring-U-(14)C]BAM to (14)CO(2) with BAM at initial concentrations in the range of 7.9 nM to 263.1 muM provided as the sole carbon, nitrogen, and energy source. A quantitative enzyme-linked immunoassay analysis with antibodies against BAM revealed residue concentrations of 0.35 to 18.05 nM BAM following incubation for 10 days, corresponding to a BAM depletion of 95.6 to 99.9%. In contrast to the Aminobacter sp. strain ASI1, strain MSH1 also mineralized the herbicide itself along with several metabolites, including ortho-chlorobenzonitrile, ortho-chlorobenzoic acid, and benzonitrile, making it the first known dichlobenil-mineralizing bacterium. Aminobacter type strains not previously exposed to dichlobenil or BAM were capable of degrading nonchlorinated structural analogs. Combined, these results suggest that closely related Aminobacter strains may have a selective advantage in BAM-contaminated environments, since they are able to use this metabolite or structurally related compounds as a carbon and nitrogen source.

A time series phospholipid fatty acid (PLFA) 13C-labeling study was undertaken to determine methanotrophic taxon, calculate methanotrophic biomass, and assess carbon recycling in an upland brown earth soil from Bronydd Mawr (Wales, United Kingdom). Laboratory incubations of soils were performed at ambient CH4 concentrations using synthetic air containing 2 parts per million of volume of 13CH4. Flowthrough chambers maintained a stable CH4 concentration throughout the 11-week incubation. Soils were analyzed at weekly intervals by gas chromatography (GC), GC-mass spectrometry, and GC-combustion-isotope ratio mass spectrometry to identify and quantify individual PLFAs and trace the incorporation of 13C label into the microbial biomass. Incorporation of the 13C label was seen throughout the experiment, with the rate of incorporation decreasing after 9 weeks. The delta13C values of individual PLFAs showed that 13C label was incorporated into different components to various extents and at various rates, reflecting the diversity of PLFA sources. Quantitative assessments of 13C-labeled PLFAs showed that the methanotrophic population was of constant structure throughout the experiment. The dominant 13C-labeled PLFA was 18:1omega7c, with 16:1omega5 present at lower abundance, suggesting the presence of novel type II methanotrophs. The biomass of methane-oxidizing bacteria at optimum labeling was estimated to be about 7.2 x 10(6) cells g(-1) of soil (dry weight). While recycling of 13C label from the methanotrophic biomass must occur, it is a slower process than initial 13CH4 incorporation, with only about 5 to 10% of 13C-labeled PLFAs reflecting this process. Thus, 13C-labeled PLFA distributions determined at any time point during 13CH4 incubation can be used for chemotaxonomic assessments, although extended incubations are required to achieve optimum 13C labeling for methanotrophic biomass determinations.

Described genera of methanotrophic bacteria are present in most upland soils, but it is not known whether these are sufficiently oligotrophic to oxidize methane at its trace atmospheric mixing ratio of 1.75 ppmv. Members of the genera Methylocystis, Methylosinus, Methylocaldum and Methylobacter were isolated from different upland soils and compared with type strains for growth and activity under low methane mixing ratios. The specific affinity (a0s) varied by about one order of magnitude among different methanotrophs. It was highest in some Methylocystis spp., suggesting that these were the most oligotrophic. In direct tests, the threshold mixing ratio of methane required by most methanotrophs for growth ranged from 100 to greater than 1000 ppmv. However, two Methylocystis strains grew at only 10-100 ppmv of methane and one oxidized atmospheric methane for >3 months with little or no decline in the absolute rate. The results show that some cultivated methanotrophic bacteria are much more oligotrophic than others, and may contribute to atmospheric methane oxidation in soils. However, it is likely that these need additional energy sources for long-term survival, and that uncultivated groups of methanotrophic bacteria are primarily responsible for the process in soils possessing high methane oxidation rates.

This study is the first demonstration that a diverse facultatively methylotrophic microbiota exists in some Antarctic locations. PCR amplification of genes diagnostic for methylotrophs was carried out with bacterial DNA isolated from 14 soil and sediment samples from ten locations on Signy Island, South Orkney Islands, Antarctica. Genes encoding the mxaF of methanol dehydrogenase, the fdxA for Afipia ferredoxin, the msmA of methanesulfonate monooxygenase, and the 16S rRNA gene of Methylobacterium were detected in all samples tested. The mxaF gene sequences corresponded to those of Hyphomicrobium, Methylobacterium, and Methylomonas. Over 30 pure cultures of methylotrophs were isolated on methanesulfonate, dimethylsulfone, or dimethylsulfide from ten Signy Island lakes. Some were identified from 16S rRNA gene sequences (and morphology) as Hyphomicrobium species, strains of Afipia felis, and a methylotrophic Flavobacterium strain. Antarctic environments thus contain diverse methylotrophic bacteria, growing on various C1-substrates, including C1-sulfur compounds.

A gram negative chemolithotrophic bacterium (RPI) with facultative mode of nutrition was isolated from the soil. Enzymological studies confirmed presence of Thiosulphate oxidase, sulphite oxidase and Rhodanese, all of which play role in sulfur metabolism pathway. A set of degenerate oligonucleotide primer pairs was used for thermal amplification of a major part of the coding region of the Cytochrome c gene locus of this bacterium. Nucleotide and translated amino acid sequence revealed the gene to be a diheme Cytochrome c, different from the monoheme Cytochrome c observed in Chloribium limicola, a photosynthetic green sulfur bacterium. Significant homology at the nucleotide level could be detected only with Pseudoaminobacter salicylatoxidans. On the contrary, significant homology at the amino acid level was observed with Bradyrhizobium japonicum, Silicobacter pomeroyi apart from P. salicylatoxidans. This is possibly because of codon degeneracy observed within the diverse members of chemolithotrophs. Greater homology at amino acid level with P. salicylatoxidans and B. japonicum compared to that with P. denitrificans hint at possibly grouping of RP1 with the Rhizobium-Agrobacterium sub group of alpha Proteobacteria.

Forty-six Mesorhizobium strains associated with the leguminous plants Leucaena leucocephala and Sesbania herbacea in an uncultivated Mexican field were characterized using a polyphasic approach. The strains were identified as Mesorhizobium plurifarium based upon the close relationships with the reference strains for this species in PCR-based restriction fragment length polymorphism analyses, sequencing of 16S rRNA genes, multilocus enzyme electrophoresis, and DNA-DNA hybridization. Although the strains isolated from both plants formed the same group in multilocus enzyme electrophoresis and cross-nodulations were observed in the laboratory, different electrophoretic types were obtained from the two plants grown in natural soils, indicating the existence of a preferable association between the plants and the rhizobia. The M. plurifarium strains from Mexico and the reference strains from Africa and Brazil formed different phenotypic clusters in a numerical taxonomy. The Mexican strains did not grow at 37 degrees C and were sensitive to salty-alkaline conditions, while the reference strains from Africa and Brazil grew at 42 degrees C and were more resistant to salty-alkaline conditions. These results demonstrate that both the plants and environmental factors affected the evolution of rhizobia and that the Mexican strains had adapted to the neutral soils and the cool climate where they were isolated.

Aims: To determine the biodiversity of rhizobial strains nodulating Cicer arietinum L. in representative soils from various areas of Morocco., Methods and Results: Symbiotic traits, utilization of 49 carbohydrate sources, resistance to antibiotics and heavy metals, tolerance to salinity, to extreme temperatures and pH were studied as phenotypic markers. In addition, restriction fragment length polymorphism (RFLP) of PCR-amplified 16S rDNAs were compared with those of reference strains. Numerical analysis of the phenotypic characteristics showed that the 48 strains studied fell into three distinct groups. RFLP analysis of 16S rRNA genes revealed an additional heterogeneity and four ribotypes were identified., Conclusions: Chickpea rhizobia isolated from Moroccan soils are both phenotypically and genetically diverse. Most of these rhizobia belong to the Mesorhizobium genus. However, some strains originating from a particular soil appeared to have 16S rRNA genes similar to Sinorhizobium as well as very distinct auxanographic characteristics compared with Mesorhizo- bium isolates., Significance and Impact of the Study: A well characterized collection of chickpea-nodulating rhizobia in representative soils of Morocco has been generated, which can be used to develop efficient inoculants for this crop. This is the first report evidencing that chickpeas may be nodulated by bacteria from the Sinorhizobium genus.

Carboxydotrophic activity in forest soils was enriched by incubation in a flowthrough system with elevated concentrations of headspace CO (40 to 400 ppm). CO uptake increased substantially over time, while the apparent K(m) ((app)K(m)) for uptake remained similar to that of unenriched soils (<10 to 20 ppm). Carboxydotrophic activity was transferred to and further enriched in sterile sand and forest soil. The (app)K(m)s for secondary and tertiary enrichments remained similar to values for unenriched soils. CO uptake by enriched soil and freshly collected forest soil was inhibited at headspace CO concentrations greater than about 1%. A novel isolate, COX1, obtained from the enrichments was inhibited similarly. However, in contrast to extant carboxydotrophs, COX1 consumed CO with an (app)K(m) of about 15 ppm, a value comparable to that of fresh soils. Phylogenetic analysis based on approximately 1,200 bp of its 16S rRNA gene sequence suggested that the isolate is an alpha-proteobacterium most closely related to the genera Pseudaminobacter, Aminobacter, and Chelatobacter (98.1 to 98.3% sequence identity).