Detection of Methane Oxidizing Bacteria in Forest Soil by Monooxygenase PCR Amplification

Abstract Atmospheric methane oxidation by a spruce forest soil from Norway at 15°C was found to be maximal at a depth of ca 7 cm. Examination of the kinetics of this methane oxidation revealed an apparent Km of 403.1 nM and a Vmax of 2.2 nmol g^-1 dry weight soil h^-1. The low apparent Km suggested the presence of active methane oxidizing bacteria with a high affinity for methane. DNA was extracted from the 5–10 cm horizon, purified, and subjected to PCR amplification with primers directed toward the monooxygenase genes pmoA and amoA, which are essential for methane oxidation. Hybridization analysis of the clone library subsequently constructed revealed that 49% of the 76 cloned PCR fragments were putative methanotroph pmoA sequences and 16% were putative ammonium oxidizing nitrifier amoA sequences. Sequencing of 28 clones identified three major groups showing homology to pmoA from Methylococcus capsulatus, #-subdivision ammonia-oxidizers (amoA), and a new group of monooxygenase pmoA/amoA sequences.     

Effects of Arbuscular Mycorrhizas on Ammonia Oxidizing Bacteria in an Organic Farm Soil

Arbuscular mycorrhizal fungi (AMF) are potentially important in nutrient cycling in agricultural soils and particularly in soils managed for organic production; little is known, however, about the interrelationships between AMF and other members of soil microbial communities. Ammonia oxidizing bacteria (AOB) are a trophic group of bacteria having an enormous impact on nitrogen availability in soils and are expected to be influenced by the presence of AMF. In a field study, we utilized a unique genetic system comprised of a mycorrhiza defective tomato mutant (named rmc) and its mycorrhiza wild-type progenitor (named 76RMYC+). We examined the effect of AMF by comparing AOB community composition and populations in soil containing roots of the two tomato genotypes in an organically managed soil. Responses of AOB to soil N and P amendments were also studied in the same experiment. Phylogenetic analysis of cloned AOB sequences, derived from excised denaturing gradient gel electrophoresis (DGGE) bands, revealed that the organic farm soil supported a diverse yet stable AOB community, which was neither influenced by mycorrhizal colonization of roots nor by N and P addition to the soil. Real-time TaqMan polymerase chain reaction (PCR) was used to quantify AOB population sizes and showed no difference between any of the treatments. An alternative real-time PCR protocol for quantification of AOB utilizing SYBR green yielded similar results as the TaqMan real-time PCR method, although with slightly lower resolution. This alternative method is advantageous in not requiring the detailed background information about AOB community composition required for adaptation of the TaqMan system for a new soil.     

Species Diversity of Uncultured and Cultured Populations of Soil and Marine Ammonia Oxidizing Bacteria

Although molecular techniques are considered to provide a more comprehensive view of species diversity of natural microbial populations, few studies have compared diversity assessed by molecular and cultivation-based approaches using the same samples. To achieve this, the diversity of natural populations of ammonia oxidising bacteria in arable soil and marine sediments was determined by analysis of 16S rDNA sequences from enrichment cultures, prepared using standard methods for this group, and from 16S rDNA cloned from DNA extracted directly from the same environmental samples. Soil and marine samples yielded 31 and 18 enrichment cultures, respectively, which were compared with 50 and 40 environmental clones. There was no evidence for selection for particular ammonia oxidizer clusters by different procedures employed for enrichment from soil samples, although no culture was obtained in medium at acid pH. In soil enrichment cultures, Nitrosospira cluster 3 sequences were most abundant, whereas clones were distributed more evenly between Nitrosospira clusters 2, 3, and 4. In marine samples, the majority of enrichment cultures contained Nitrosomonas, whereas Nitrosospira sequences were most abundant among environmental clones. Soil enrichments contained a higher proportion of identical sequences than clones, suggesting laboratory selection for particular strains, but the converse was found in marine samples. In addition, 16% of soil enrichment culture sequences were identical to those in environmental clones, but only 1 of 40 marine enrichments was found among clones, indicating poorer culturability of marine strains represented in the clone library, under the conditions employed. The study demonstrates significant differences in species composition assessed by molecular and culture-based approaches but indicates also that, employing only a limited range of cultivation conditions, 7% of the observed sequence diversity in clones of ammonia oxidizers from these environments could be obtained in laboratory enrichment culture. Further studies and experimental approaches are required to determine which approach provides better representation of the natural community.     

Oxidation and Assimilation of Atmospheric Methane by Soil Methane Oxidizers

The metabolism of atmospheric methane in a forest soil was studied by radiotracer techniques. Maximum (sup14)CH(inf4) oxidation (163.5 pmol of C cm(sup-3) h(sup-1)) and (sup14)C assimilation (50.3 pmol of C cm(sup-3) h(sup-1)) occurred at the A(inf2) horizon located 15 to 18 cm below the soil surface. At this depth, 31 to 43% of the atmospheric methane oxidized was assimilated into microbial biomass; the remaining methane was recovered as (sup14)CO(inf2). Methane-derived carbon was incorporated into all major cell macromolecules by the soil microorganisms (50% as proteins, 19% as nucleic acids and polysaccharides, and 5% as lipids). The percentage of methane assimilated (carbon conversion efficiency) remained constant at temperatures between 5 and 20(deg)C, followed by a decrease at 30(deg)C. The carbon conversion efficiency did not increase at methane concentrations between 1.7 and 1,000 ppm. In contrast, the overall methane oxidation activity increased at elevated methane concentrations, with an apparent K(infm) of 21 ppm (31 nM CH(inf4)) and a V(infmax) of 188 pmol of CH(inf4) cm(sup-3) h(sup-1). Methane oxidizers from soil depths with maximum methanotrophic activity respired approximately 1 to 3% of the assimilated methane-derived carbon per day. This apparent endogenous respiration did not change significantly in the absence of methane. Similarly, the potential for oxidation of atmospheric methane was relatively insensitive to methane starvation. Soil samples from depths above and below the zone with maximum atmospheric methane oxidation activity showed a dramatic increase in the turnover of the methane assimilated (>20 times increase). Physical disturbance such as sieving or mixing of soil samples decreased methane oxidation and assimilation by 50 to 58% but did not alter the carbon conversion efficiency. Ammonia addition (0.1 or 1.0 (mu)mol g [fresh weight](sup-1)) decreased both methane oxidation and carbon conversion efficiency. This resulted in a dramatic decrease in methane assimilation (85 to 99%). In addition, ammonia-treated soil showed up to 10 times greater turnover of the assimilated methane-derived carbon (relative to untreated soil). The results suggest a potential for microbial growth on atmospheric methane. However, growth was regulated strongly by soil parameters other than the methane concentration. The pattern observed for metabolism of atmospheric methane in soils was not consistent with the physiology of known methanotrophic bacteria.     

Molecular Existence and Diversity of Nitrite-Dependent Anaerobic Methane Oxidizing (n-Damo) Bacteria in the Lakes of Badain of the Gobi Desert

Nitrite-dependent anaerobic methane oxidation (n-damo) process, mediated by Candidatus Methylomirabilis oxyfera of the candidate phylum NC10, was discovered recently which plays an important role in coupling the global nitrogen and carbon cycles. However, the distribution and diversity of this new anaerobic methane-oxidizing microorganism have not been investigated in desert lakes yet. The present study successfully retrieved n-damo bacterial 16S rRNA and pmoA gene sequences using PCR technique from lakes in Badain Jaran Desert of China. Phylogenetic analyses showed that n-damo bacteria widely occurred in brine and freshwater lakes on the desert with high diversity, including both sediment and water samples. The results of quantitative PCR indicated that the abundance of the 16S rRNA gene in lake sediments varied from 1.12?±?0.68?×?10⁵ to 1.64?±?0.70?×?10⁵ copies g^?1 (dry weight), while that in water samples per milliliter was generally one order of magnitude lower than sediments. Correlation analyses suggested that n-damo bacterial abundance and diversity strongly depended on salinity. In lake sediments, the distribution, abundance, and diversity of n-damo bacteria were significantly associated with depth due to the concentration gradient of the NO_x^- and ammonium. This study provided new insights into both the n-damo community patterns and its interaction with ambient environmental factors in the desert lake ecosystem.     

The Ways of Plant Colonization by MethylobacteriumStrains and Properties of These Bacteria

The pink-pigmented facultative methylotrophic bacteria (PPFMB) of the genus Methylobacteriumare indispensible inhabitants of the plant phyllosphere. Using maize Zea maysas a model, the ways of plant colonization by PPFMB and some properties of the latter that might be beneficial to plants were studied. A marked strain, Methylobacterium mesophilicumAPR-8 (pULB113), was generated to facilitate the detection of the methylotrophic bacteria inoculated into the soil or applied to the maize leaves. Colonization of maize leaves by M. mesophilicumAPR-8 (pULB113) occurred only after the bacteria were applied onto the leaf surface. In this case, the number of PPFMB cells on inoculated leaves increased with plant growth. During seed germination, no colonization of maize leaves with M. mesophilicumcells occurred immediately from the soil inoculated with the marked strain. Thus, under natural conditions, colonization of plant leaves with PPFMB seems to occur via soil particle transfer to the leaves by air. PPFMB monocultures were not antagonistic to phytopathogenic bacteria. However, mixed cultures of epiphytic bacteria containing Methylobacterium mesophilicumor M. extorquensdid exhibit an antagonistic effect against the phytopathogenic bacteria studied (Xanthomonas campestris, Pseudomonas syringae, Erwinia carotovora, Clavibacter michiganense,andAgrobacterium tumifaciens). Neither epiphytic nor soil strains of Methylobacterium extorquens, M. organophillum, M. mesophilicum, andM. fujisawaensecatalyzed ice nucleation. Hence, they cause no frost injury to plants. Thus, the results indicate that the strains of the genus Methylobacteriumcan protect plants against adverse environmental factors.     

Experimental Colonization and Alteration of Orthopyroxene by the Pleomorphic Bacteria Ramlibacter tataouinensis

The colonization of orthopyroxene crystals by a pleomorphic bacterium and the mineralogical products resulting from a prolonged interaction have been studied. We used Ramlibacter tataouinensis (strain TTB310), which is an aerobic β-Proteobacterium moving over surfaces by gliding motility and whose life cycle includes rods and spherical cysts. Analysis of cultures grown on solid media with micrometer-sized pyroxene and quartz crystals scattered over the surface revealed a taxis of the bacteria toward the crystals. Given the mineralogical non-specificity of the interaction, a mechanism of elasticotaxis is inferred. After the rods had adhered to the pyroxene surface, they differentiated into cysts leading to the formation of microcolonies that were centered on a crystal grain. This suggests an original coupling between the life cycle of R. tataouinensis and the interaction with the crystals. The alteration of orthopyroxene was studied by high-resolution transmission electron microscopy at the interface between cysts and pyroxene crystals. The pyroxene surface showed an amorphous layer that was more developed than that of abiotic control samples processed under the same conditions. Moreover, chemical analyses showed that the dissolution of pyroxene was reduced in the presence of R. tataouinensis. The origin and the significance of the amorphous layers is discussed.     

Selective Isolation of Bacillus sphaericus from Soil by Use of Acetate as the Only Major Source of Carbon

A simple chemically defined medium containing sodium acetate (37 or 74 mM) as the only major source of carbon was inoculated with soil from various locations. The bacteria which grew most rapidly at 30°C were almost entirely strains of Bacillus sphaericus and Arthrobacter species. Pasteurization of the soils made the medium selective for B. sphaericus. Several new strains were isolated, and the peptidoglycans of their cell walls were examined.     

Recovery of Soil Ammonia Oxidation After Long-Term Zinc Exposure Is Not Related to the Richness of the Bacterial Nitrifying Community

A soil sterilization–reinoculation approach was used to manipulate soil microbial diversity and to assess the effect of the diversity of the ammonia-oxidizing bacteria (AOB) on the recovery of the nitrifying community to metal stress (zinc). Gamma-irradiated soil was inoculated with 13 different combinations of up to 22 different soils collected worldwide to create varying degrees of AOB diversity. Two months after inoculation, AOB amoA DGGE based diversity (weighted richness) varied more than 10-fold among the 13 treatments, the largest value observed where the number of inocula had been largest. Subsequently, the 13 treatments were either or not amended with ZnCl₂. Initially, Zn amendment completely inhibited nitrification. After 6 months of Zn exposure, recovery of the potential nitrification activity in the Zn amended soils ranged from <10 % to >100 % of the potential nitrification activity in the corresponding non-amended soils. This recovery was neither related to DGGE-based indices of AOB diversity nor to the AOB abundance assessed 2 months after inoculation (p?>?0.05). However, recovery was significantly related (r?=?0.75) to the potential nitrification rate before Zn amendment and only weakly to the number of soil inocula used in the treatments (r?=?0.46). The lack of clear effects of AOB diversity on recovery may be related to an inherently sufficient diversity and functional redundancy of AOB communities in soil. Our data indicate that potential microbial activity can be a significant factor in recovery.     

Relative Brain and Brain Part Sizes Provide Only Limited Evidence that Machiavellian Behaviour in Cleaner Wrasse Is Cognitively Demanding

It is currently widely accepted that the complexity of a species’ social life is a major determinant of its brain complexity, as predicted by the social brain hypothesis. However, it remains a challenge to explain what social complexity exactly is and what the best corresponding measures of brain anatomy are. Absolute and relative size of the brain and of the neocortex have often been used as a proxy to predict cognitive performance. Here, we apply the logic of the social brain hypothesis to marine cleaning mutualism involving the genus Labroides. These wrasses remove ectoparasites from ‘client’ reef fish. Conflict occurs as wrasse prefer client mucus over ectoparasites, where mucus feeding constitutes cheating. As a result of this conflict, cleaner wrasse show remarkable Machiavellian-like behaviour. Using own data as well as available data from the literature, we investigated whether the general brain anatomy of Labroides provides any indication that their Machiavellian behaviour is associated with a more complex brain. Neither data set provided evidence for an increased encephalisation index compared to other wrasse species. Published data on relative sizes of brain parts in 25 species of the order Perciformes suggests that only the diencephalon is relatively enlarged in Labroides dimidiatus. This part contains various nuclei of the social decision making network. In conclusion, gross brain anatomy yields little evidence for the hypothesis that strategic behaviour in cleaning selects for larger brains, while future research should focus on more detailed aspects like the sizes of specific nuclei as well as their cryoarchitectonic structure and connectivity.     

Relationship between Rapid, Firm Adhesion and Long-Term Colonization of Roots by Bacteria

For rhizobacteria to exert physiological effects on plant growth, the bacteria must first effectively colonize the root surface. To examine the relationship between long-term colonization of root systems and adherence to roots in the short term, a binding assay was developed. Adherence was determined by incubating roots of intact radish seedlings with bacteria, washing and homogenizing the roots, and dilution plating the resulting homogenate. Irreversible binding of bacteria was rapid, reaching half-maximum by 5 min. All of the rhizosphere bacteria tested showed similar, concentration-dependent binding (ranging from 10⁴ to 10⁸ CFU/ml), as well as long-term colonization of radish roots under sterile conditions. Escherichia coli, which is not a root colonizer, showed about 10-fold less binding, but still demonstrated concentration-dependent binding and rapid kinetics of adherence at high concentrations (10⁶ to 10⁸ CFU/ml). The bacteria tested were very different with respect to source or habitat and plant response, yet they showed similar concentration-dependent binding. There was no correlation between the relative hydrophobicities of the cell surfaces of strains and the adherence of the strains to roots. Binding of Pseudomonas fluorescens E6-22 was promoted by divalent cations (Ca²⁺ and Mg²⁺) at concentrations of 5 to 10 mM, whereas monovalent cations (Na⁺ and K⁺) had little effect; electrostatic phenomena may partially explain adherence in the short term, an important prelude to long-term colonization of root surfaces.     

Revegetation following Soil Disturbance and Invasion in a Californian Meadow: a 10-year History of Recovery

Disturbance is necessary for the regeneration of many native plant species, but can also facilitate biological invasions. As a result, disturbance can play complex roles in vulnerable habitats such as remnant Californian perennial grasslands. To investigate these conflicts, plots in a northern Californian coastal grassland were experimentally disturbed in the winter of 1990–1991; these plots differed in the area and intensity (depth) of the soil disturbance applied. When these plots were revisited after 10 growing seasons, patterns of revegetation differed significantly from those observed early in recolonization (0–3 years). At the earlier samplings, exotic annual grasses rapidly increased in most disturbance types. After 10 years, these exotic annuals had retreated from the depth experiment, which had recovered to a vegetation dominated by native perennials in all but the most severely disturbed plots. In contrast, although differences between control and disturbed plots also disappeared in the area experiment, the average abundance of aliens did not decline substantially relative to 1993 levels, especially in larger disturbances. Nonetheless, populations of aliens remained small compared to the peak populations in the depth experiment, probably reflecting wetter soils at the site used for the area experiment. These results differ from those of other recent studies of soil disturbance in coastal Californian ecosystems, which indicate disturbance may result in the permanent replacement of native perennial vegetation by dense populations of exotic annual grasses. This difference may reflect the high resilience of northern coastal grasslands as well as the scale of disturbances considered by different studies.     

Quantitative Aspects of Growth of the Methane Oxidizing Bacterium Methylococcus capsulatus on Methane in Shake Flask and Continuous Chemostat Culture

S ummary : Experiments were directed towards the production of high biomass concentrations in cultures of Methylococcus capsulatus. In shake flasks the effects of ammonium ion, phosphate ion and various trace metals on growth were studied. In the chemostat the effects on growth of methane limitation, oxygen limitation, aeration, dilution rate, pH value and temperature were studied and carbon balances were made in steady state conditions. Growth in the fermenter was stimulated by the use of Amberlite CG–120 ion exchange resin in the medium. The probability that Amberlite removed a growth, inhibitor is discussed.     

Unexpected Errors in Gas Chromatographic Analysis of Methane Production by Thermophilic Bacteria

Unexpected errors in methane measurement by gas chromatography occurred when samples at thermophilic temperatures were analyzed. With a standard curve prepared at room temperature (25°C), stoppered bottles incubated and sampled at 37 to 85°C showed more methane upon analysis than bottles incubated at 25°C: values at 50, 63, and 85°C were 109, 126, and 125%, respectively, of the 25°C value. All variation between 4 and 50°C can be explained by the temperature difference between culture bottle and sampling syringe, and the variation of methane concentration can be predicted by the gas law. Between 50 and 63°C, there was a more dramatic rise than predicted by theory. These variations are important to consider if thermophilic methane production is to be measured accurately. Methods to avoid errors are discussed.     

Higher Abundance of Ammonia Oxidizing Archaea than Ammonia Oxidizing Bacteria and Their Communities in Tibetan Alpine Meadow Soils under Long-term Nitrogen Fertilization

Increasing usage of nitrogen fertilizer for food production has resulted in severely environmental problems of nutrients enrichment. This study aimed to examine the response of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) to a long-term nitrogen fertilization in Tibetan alpine meadow. The abundance and composition of both AOB and AOA were assessed using quantitative real-time PCR, cloning and sequencing techniques based on amoA gene under different fertilization gradient (0, 30, 60, 90, and 120 g m^?2 year^?1). Our results showed that, abundances of AOA amoA genes (ranging from 1.48 × 10⁹ to 2.00 × 10⁹ copies per gram of dry soil) were significantly higher than those of AOB amoA genes (1.25 × 10⁷ to 2.62 × 10⁸ copies per gram of dry soil) under fertilization scenario. The abundance of AOB amoA genes increased with increasing nitrogen fertilization, whereas fertilization had little effect on AOA abundance. Sequences of clone libraries of the different treatments revealed that AOB communities were dominated by representatives of Cluster 4, constituting 48.94–64.44% in each clone library. Sequences of Clusters 9, 1 and 2 were prevalent in soils under higher fertilization. All archaeal amoA sequences recovered were affiliated with the soil/sediment clade and marine sediment clade, and no significant difference was observed on the community structure among different fertilization treatments. Variations in the AOB community structure and abundance were linked to ammonium-N and soil pH induced by different fertilization treatments. These results showed that the abundance and structure of the AOB community respond to the fertilization gradient, not AOA.     

Metabolism of Di- and Mono-n-Butyl Phthalate by Soil Bacteria

Samples for mycological analysis were collected from surfaces in the Skylab spacecraft before launch and during flight for each manned mission. Fungal contamination levels were low during the first two flights; however, the species recovered were different for each mission. On the third mission, widespread contamination of the Skylab spacecraft with Aspergillus and Pencillium spp. was detected. This contamination was traced to several contaminated space suit undergarments.     

A Burkholderia Strain Living Inside the Arbuscular Mycorrhizal Fungus Gigaspora margarita Possesses the vacB Gene,Which Is Involved in Host Cell Colonization by Bacteria

The arbuscular mycorrhizal (AM) fungus Gigaspora margarita harbors a resident population of endosymbiontic Burkholderia in its cytoplasm. Nothing is known about the acquisition of such bacteria and about the molecular bases which allow colonization of the fungus. We wondered whether the intracellular Burkholderia strain possesses genetic determinants involved in colonization of a eukaryotic cell. Using degenerated oligonucleotide primers for vacB, a gene involved in host cell colonization by pathogenic bacteria, an 842 bp DNA fragment was cloned, sequenced, and identified as a part of the vacB gene in Burkholderia sp. The insert was used as a probe to screen a fungal library that, because of the presence of intracellular Burkholderia cells, was also representative of the bacterial genome. The complete nucleotide sequence of vacB and flanking genes was determined. The bacterial origin of this genomic region was established by PCR, using specific vacB primers on DNA from Gigasporaceae that did or did not contain cytoplasmic Burkholderia, as well as on DNA from other bacteria, including free-living Burkholderia. We hypothesize that the vacB gene is part of a new genetic region acquired by a rhizospheric Burkholderia strain, which became able to establish a symbiotic interaction with the AM fungus G. margarita.