Isolation and Characterization of Bacteria That Grow on Methane and Organic Compounds as Sole Sources of Carbon and Energy

Bacteria capable of growth on methane and a variety of complex organic substrates as sole sources of carbon and energy have been isolated. Conditions used to rigorously establish the purity of the cultures are described. One facultative methylotroph has been studied in detail. This organism has peripherally arranged pairs of intracytoplasmic membranes characteristic of obligate methylotrophs. This isolate apparently utilizes the serine pathway of formaldehyde fixation. The location of methane oxidizers in a dimictic lake indicates that these organisms prefer less than saturating levels of dissolved oxygen. Laboratory experiments confirmed the preference of these organisms for atmospheres containing less oxygen than air.     

Microbial Growth on Dichlorobiphenyls Chlorinated on Both Rings as a Sole Carbon and Energy Source

We have isolated bacterial strains capable of aerobic growth on ortho-substituted dichlorobiphenyls as sole carbon and energy sources. During growth on 2,2′-dichlorobiphenyl and 2,4′-dichlorobiphenyl strain SK-4 produced stoichiometric amounts of 2-chlorobenzoate and 4-chlorobenzoate, respectively. Chlorobenzoates were not produced when strain SK-3 was grown on 2,4′-dichlorobiphenyl.     

Analysis of Microbial Communities in a Landfill Leachate Polluted Aquifer using a New Method for Anaerobic Physiological Profiling and 16S rDNA Based Fingerprinting

Abstract Databases containing information regarding presence and activity of microbial communities will be very useful for determination of the potential for intrinsic bioremediation in landfill leachate polluted aquifers. Simple analyses such as community-level physiological profiling (CLPP) and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments yield large sets of data for inclusion into such databases. In this study we describe the development of a method for anaerobic CLPP, using commercially available Biolog plates. Incubation at the in situ temperature of the aquifer (10°C) for 28 days was optimal for obtaining a specific, reproducible physiological profile. Anaerobic incubation was essential for profiling anaerobic communities. The anaerobic cultivation-dependent CLPP method and cultivation-independent DGGE were applied to groundwater and sediment samples from the aquifer near the Coupépolder landfill in The Netherlands. A combination of computer-assisted CLPP and DGGE analysis of both groundwater and sediment samples yielded the best separating power for characterizing microbial communities in the aquifer. Communities in groundwater were significantly different from those in the corresponding sediment. Microbial communities present in subsamples from sediment cores usually were similar for the various sampling locations. Variation was observed for the heterogeneous sediment beneath the landfill. Both anaerobic CLPP and DGGE analysis clearly separated microbial communities from the polluted aquifer underneath the landfill from those in the less or not polluted aquifer downstream and upstream of the landfill. Received: 3 January 2000; Accepted: 21 March 2000; Online Publication: 28 August 2000     

The Fate of Organic Sources of Carbon in Moss‐rich Tundra Soil Microbial Communities: A Laboratory Experimental Study

Effects of glucose‐carbon supplementation on soil respiration and bacterial and protist biomass were investigated in laboratory studies of three soil samples from Alaskan tundra: spring tussock sample 1 (thin surface moss), spring tussock sample 2 (thick surface moss), and a summer tundra open field sample. Addition of 1% (w/v) glucose solution produced an immediate, pronounced two to three fold increase in respiration above basal rate, which declined over 4 h to baseline levels. Less than 1% (w/w) of glucose‐C supplement was respired during the respiratory spike, relative to the 89 μg/g added. A more substantial amount of the glucose‐C became incorporated in microbial biomass. The total difference in microbial carbon (μg/g) between the experimental treatments and controls without glucose after 1 wk was as follows: spring sample 1 (8), spring sample 2 (31), and summer sample (70). The percent (w/w) of glucose‐C incorporated was: spring sample 1 (5%), spring sample 2 (17%), and summer sample (39%), most attributed to biomass of bacteria and heterotrophic nanoflagellates. Although respiratory response to pulsed glucose‐C was minimal, the overall mean basal rate after 1 wk ranged between 4 and 6 nmol/min/g soil, indicating a significant assimilation and respiration of constituent soil organic carbon.     

Characterization of a Mutant of Chlamydomonas reinhardtii That Uses L-Methionine-S-Sulfoximine and Phosphinothricin as Nitrogen Sources for Growth

A strain of Chlamydomonas reinhardtii, named ARF-1, which grows with the glutamine synthetase (GS) inhibitor L-methionine-S-sulfoximine (MSX), has been isolated and characterized. Mutant ARF-1 is affected at a single and dominant gene, tentatively assigned to the allele msr-1-2. Neither the uptake of ammonia nor the two GS isoenzyme activities of the mutant were affected by MSX in vivo. GS activities, however, were fully abolished in vitro, thus suggesting that neither GS isoform was an altered enzyme resistant to the inhibitor. Resistance to MSX does not seem to be due to either a defect in a permease responsible for the transport of MSX or over-expression of GS activity, nor did we find an alternative enzymatic pathway for the assimilation of ammonium. Resistance was independent of the nitrogen source used and was strongly enhanced by the addition of acetate. Unlike the parental strain, mutant ARF-1 can degrade and utilize MSX as the sole nitrogen source for growth, which could account for the observed resistance. Thus, this mutant can be classified as a novel type of MSX-resistant mutant. This mutant can also use phosphinothricin, methionine sulfone, or methionine sulfoxide as the sole sources of nitrogen. This capability cosegregated in the genetic crosses and was also observed in all the diploids isolated. An MSX/[alpha]-ketoglutarate aminotransferase activity, not present in the parental strain 305, was detected in mutant ARF-1 cells. Therefore, we propose that the locus msr-1-2 either codes for this transaminase activity or its product gene is necessary to express this transaminase activity.     

Physiological Profiling of Soil Microbial Communities in a Florida Scrub-Oak Ecosystem: Spatial Distribution and Nutrient Limitations

Rapid physiological profiling of heterotrophic microbial communities enables intensive analysis of the factors affecting activity in aerobic habitats, such as soil. Previous methods for performing such profiling were severely limited due to enrichment bias and inflexibility in incubation conditions. We tested a new physiological profiling approach based on a microtiter plate oxygen sensor system (Becton Dickinson Oxygen Biosensor System (BDOBS)), which allows for testing of lower substrate addition (i.e., lower enrichment potential) and manipulation of physiochemical assay conditions, such as pH and nutrients. Soil microbial communities associated with a scrub-oak forest ecosystem on Merritt Island Wildlife Refuge in central Florida, USA, were studied in order to evaluate microbial activity in a nutrient poor soil and to provide baseline data on the site for subsequent evaluation of the effects of elevated CO₂ on ecosystem function. The spatial variation in physiological activity amongst different habitats (litter, bulk soil, and rhizosphere) was examined as a function of adaptation to local resources (i.e., water soluble extracts of roots and leaf litter) and the degree of N and P limitation. All the communities were primarily N-limited, with a secondary P limitation, which was greater in the rhizosphere and bulk soil. The litter community showed greater overall oxygen consumption when exposed to litter extracts relative to the rhizosphere or soil, suggesting acclimation toward greater use of the mixed substrates in the extract. Root extracts were readily used by communities from all the habitats with no habitat specific acclimation observed. A priming effect was detected in all habitats; addition of glucose caused a significant increase in the use of soil organic carbon. Response to added glucose was only observed with N and P addition, suggesting that C may be lost to the groundwater from these porous soils because nutrient limitation prevents C immobilization.     

Impact of Carbon and Flooding on the Metabolic Diversity of Microbial Communities in Soils

The assumption that carbon and soil water content are major determinants of microbial community structure and function is rarely questioned because of substantial evidence of the impacts of these variables on specific populations and functions. The significance of carbon and water for metabolic diversity at the microbial community level was tested on the field scale in agricultural plots varying in carbon inputs and in whether they were flooded. Surface soils in which rice straw was incorporated or burned and which were flooded or unflooded were sampled at monthly intervals three times during the flooded winter period (January to March) and again 1 month postdraining. Biomass carbon and nitrogen were not affected by treatments, active bacterial counts showed slight increases, and respiration rates were increased by carbon inputs and flooding. Biolog microplates were inoculated with soil extracts to quantify the metabolic diversity of the soil microbial community. Canonical correspondence analysis and the Monte Carlo permutation testing showed that differences in substrate utilization patterns were significantly related (P < 0.001) to carbon and flooding treatments. Biolog substrates whose metabolism was altered by the treatments were consistent across dates and tended to be positively related (utilization enhancement) to carbon inputs and negatively related to winter flooding. The importance of carbon as an environmental variable increased over time after straw treatment, whereas the importance of water became evident after flooding and decreased after drainage. The effect of long-term rice straw incorporation on substrate utilization patterns at another field site was consistent with these results despite the dissimilarities of the two soils.     

Molecular Profiling of Rhizosphere Microbial Communities Associated with Healthy and Diseased Black Spruce (Picea mariana) Seedlings Grown in a Nursery

Bacterial and fungal populations associated with the rhizosphere of healthy black spruce (Picea mariana) seedlings and seedlings with symptoms of root rot were characterized by cloned rRNA gene sequence analysis. Triplicate bacterial and fungal rRNA gene libraries were constructed, and 600 clones were analyzed by amplified ribosomal DNA restriction analysis and grouped into operational taxonomical units (OTUs). A total of 84 different bacterial and 31 different fungal OTUs were obtained and sequenced. Phylogenetic analyses indicated that the different OTUs belonged to a wide range of bacterial and fungal taxa. For both groups, pairwise comparisons revealed that there was greater similarity between replicate libraries from each treatment than between libraries from different treatments. Significant differences between pooled triplicate samples from libraries of genes from healthy seedlings and pooled triplicate samples from libraries of genes from diseased seedlings were also obtained for both bacteria and fungi, clearly indicating that the rhizosphere-associated bacterial and fungal communities of healthy and diseased P. mariana seedlings were different. The communities associated with healthy and diseased seedlings also showed distinct ecological parameters as indicated by the calculated diversity, dominance, and evenness indices. Among the main differences observed at the community level, there was a higher proportion of Acidobacteria, Gammaproteobacteria, and Homobasidiomycetes clones associated with healthy seedlings, while the diseased-seedling rhizosphere harbored a higher proportion of Actinobacteria, Sordariomycetes, and environmental clones. The methodological approach described in this study appears promising for targeting potential rhizosphere-competent biological control agents against root rot diseases occurring in conifer nurseries.     

A Rapid Microtiter Plate Method To Measure Carbon Dioxide Evolved from Carbon Substrate Amendments so as To Determine the Physiological Profiles of Soil Microbial Communities by Using Whole Soil

Sole-carbon-source tests (Biolog), designed to identify bacteria, have become very popular for metabolically fingerprinting soil microbial communities, despite disadvantages associated with the use of carbon source profiles that primarily select for fast-growing bacteria. In this paper we describe the use of an alternative method that combines the advantages of the Biolog community-level physiological profile (CLPP) method, in which microtiter-based detection plates are used, with the ability to measure carbon dioxide evolution from whole soil. This method facilitates measurement over short periods of time (4 to 6 h) and does not require the extraction and culturing of organisms. Deep-well microtiter plates are used as test wells into which soil is placed. The apparatus to fill the deep-well plates and interface it with a second removable detection plate is described. Two detection systems, a simple colorimetric reaction in absorbent alkali and scintillation counting with radioactive carbon sources, are described. The methods were compared to the Biolog-CLPP system by using soils under different vegetation types and soil treated with wastewater sludge. We aimed to test the hypothesis that using whole soil would have specific advantages over using extracts in that more immediate responses to substrates could be obtained that would reflect activity rather than growth. The whole-soil method was more rapid and gave earlier detection of C source use. Also, the metabolic fingerprints obtained could discriminate between sludge treatments.     

Interactive Effects of Nitrogen and Phosphorus on Soil Microbial Communities in a Tropical Forest

Elevated nitrogen (N) deposition in humid tropical regions may exacerbate phosphorus (P) deficiency in forests on highly weathered soils. However, it is not clear how P availability affects soil microbes and soil carbon (C), or how P processes interact with N deposition in tropical forests. We examined the effects of N and P additions on soil microbes and soil C pools in a N-saturated old-growth tropical forest in southern China to test the hypotheses that (1) N and P addition will have opposing effects on soil microbial biomass and activity, (2) N and P addition will alter the composition of the microbial community, (3) the addition of N and P will have interactive effects on soil microbes and (4) addition-mediated changes in microbial communities would feed back on soil C pools. Phospholipid fatty acid (PLFA) analysis was used to quantify the soil microbial community following four treatments: Control, N addition (15 g N m⁻² yr⁻¹), P addition (15 g P m⁻² yr⁻¹), and N&P addition (15 g N m⁻² yr⁻¹ plus 15 g P m⁻² yr⁻¹). These were applied from 2007 to 2011. Whereas additions of P increased soil microbial biomass, additions of N reduced soil microbial biomass. These effects, however, were transient, disappearing over longer periods. Moreover, N additions significantly increased relative abundance of fungal PLFAs and P additions significantly increased relative abundance of arbuscular mycorrhizal (AM) fungi PLFAs. Nitrogen addition had a negative effect on light fraction C, but no effect on heavy fraction C and total soil C. In contrast, P addition significantly decreased both light fraction C and total soil C. However, there were no interactions between N addition and P addition on soil microbes. Our results suggest that these nutrients are not co-limiting, and that P rather than N is limiting in this tropical forest.     

A Comparative Study of the Microbial Communities Between the Mineral Surface and the Bioleaching Solution Using the Microarray Method

In order to explore the bioleaching mechanism and improve the bioleaching efficiency,the micro-bial community in the bioleaching solution was compared with that on the surface of minerals based on the microarray analysis.Meanwhile,the elements composition in the bioleaching solution was analyzed using the ICP-AES method.Results showed that there was a high concentration of S and Cu in the leaching solution which up to 2 380 mg/L and 1 378 mg/L,respectively,after continuously bioleaching of copper-ore concen-trate for 30 days by a mixed culture associated with 12 species of bioleaching microorganisms.Based on the data of microarray,the total of cell number in the surface of minerals was far higher than that in the bi-oleaching solution.Furthermore,the dominant communities on the surface of minerals,such as Acidithiobacillus ferrooxidans,Acidithiobacillus thiooxidans and Acidithiobacillus caldus,were similar to that in the bioleaching solution.However,the relative level of some bacteria,such as Sulfobacillus aci-dophilus and Sulfobacillus thermosulfidooxidans,showed great discrepancy with lower presence in the bi-oleaching solution with respect to the mineral surface.     

Modeling the Kinetics of Contaminant Biodegradation by a Mixed Microbial Consortium Based on Carbon Mass Balance

The fate of contaminant carbon was monitored during aerobic biodegradation in the presence of a mixed indigenous microbial consortium in order to calibrate a microbial-growth-based biokinetic model. The methodology simultaneously monitored mineralization, substrate depletion and microbial population evolution in biomass extract spiked with¹⁴C-labeled hexadecane. Hexadecane depletion and hexadecane-degrader population were monitored using sacrificed microcosms by centrifuging the extract so that the supernatant and the residue contained residual hexadecane and microbial population, respectively. This methodology allowed verification of the carbon mass balance (average¹⁴C-carbon recovery of 90.33 ± 1.62% for biotic microcosms) and calibration of a biokinetic model. Four biokinetic parameters and three yield coefficients were identified (Haldane kinetic parameters:μ_S = 1.3639 d^-1, K_s = 0.4295 mg-C, KI = 6.6457 mg-C; decay kinetic parameter:μ_d = 1.3.10² d^-1; substrate/biomass, carbon dioxide/ biomass during growth and carbon dioxide/biomass during decay yield coefficients: Y_s = 1.5948 mg-C/mg-C, Y_P ^g = 0.4554 mg-C/mg-C, Y_P ^d = 1.3263 mg-C/mg-C) and compared with the literature data. The methodology can facilitate the identification of biodegradation models by decoupling the intrinsic ability of microorganisms to degrade contaminant from restrictions imposed by limiting conditions.     

Growth of Bacteria on 3-Nitropropionic Acid as a Sole Source of Carbon,Nitrogen, and Energy

3-Nitropropionic acid (3NPA) is a widespread nitroaliphatic toxin found in a variety of legumes and fungi. Several enzymes have been reported that can transform the compound, but none led to the mineralization of 3NPA. We report here the isolation of bacteria that grow on 3NPA and its anion, propionate-3-nitronate (P3N), as the sole source of carbon, nitrogen, and energy. Experiments with resting cells, cell extracts, and purified enzymes indicate that the pathway involves conversion of 3NPA to P3N, which upon denitration yields malonic semialdehyde, nitrate, nitrite, and traces of H₂O₂. Malonic semialdehyde is decarboxylated to acetyl coenzyme A. The gene that encodes the enzyme responsible for the denitration of P3N was cloned and expressed, and the enzyme was purified. Stoichiometry of the reaction indicates that the enzyme is a monooxygenase. The gene sequence is related to a large group of genes annotated as 2-nitropropane dioxygenases, but the P3N monooxygenase and closely related enzymes form a cluster within COG2070 that differs from previously characterized 2-nitropropane dioxygenases by their substrate specificities and reaction products. The results suggest that the P3N monooxygenases enable bacteria to exploit 3NPA in natural habitats as a growth substrate.Large-scale release of synthetic nitroaromatic compounds to the biosphere followed the invention of nitrobenzene around 1830. In less than 200 years, microorganisms adapted to the presence of nitroaromatic compounds in the environment by developing catalytic pathways to exploit them as growth substrates. Such rapid development suggests that the pathways did not develop de novo but evolved from preexisting degradation pathways such as might be found in microorganisms that degrade naturally occurring compounds.3-Nitropropionic acid (3NPA) is a widespread naturally occurring nitroaliphatic compound. It is a principal toxic component of Astragalus locoweeds and has been found in hundreds of species of legumes (20, 39) and a variety of fungi (6). The compound causes irreversible inhibition of succinate dehydrogenase, which makes it deadly to eukaryotes (1). Plants that make 3NPA also contain an enzyme, 3NPA oxidase (NPAO) (19, 20), which converts the compound to malonic semialdehyde (MSA) to protect the plant against the toxic effects of the compound (20). Given its widespread occurrence, we hypothesized that there must be bacteria in soil that degrade 3NPA and play a major role in determining the flux of the compound.Although bacteria that degrade 3NPA have previously been sought, the focus has been on organisms that ingest 3NPA-containing plant matter. Rumen microorganisms reduce 3NPA to β-alanine (4), and in the grasshopper gut, 3NPA is bound to glycine to form inert conjugates which are then eliminated (24). The plant enzyme NPAO converts 3NPA and O₂ to MSA, nitrate, nitrite, and hydrogen peroxide (19). It is similar to propionate-3-nitronate (P3N) oxidase (P3NO; EC 1.7.3.5) from Penicillium atrovenetum that converts the P3N form of the compound to MSA (36). The enzymes mentioned above are “orphan enzymes” (28), which means that the gene(s) has not been identified. None of the previously studied microorganisms can use 3NPA as a growth substrate, and the physiological roles of the enzymes have not been established.MSA appears to be a central intermediate in the eukaryotic transformation of 3NPA and its analogs. However, the transformations involve distinctly different types of reactions and metabolites released. P3N and 3NPA release nitrate and nitrite in a 2:1 ratio when attacked by the fungal or plant oxidases. When 3NPA is reduced to β-alanine by rumen microorganisms, β-alanine is further metabolized (4), possibly by deamination to MSA (18).We report here the isolation from soil of aerobic bacteria that grow on 3NPA as the sole source of carbon, nitrogen, and energy. The genes that encode the initial enzymes of the degradation pathway were cloned, and recombinant proteins were purified and partially characterized to allow determination of the initial steps in the catabolic pathway.(Preliminary reports of this work have been presented previously at the 106th General Meeting of the American Society for Microbiology [32a] and the 108th General Meeting of the American Society for Microbiology [32b].)     

The Influence of Different Land Uses on the Structure of Archaeal Communities in Amazonian Anthrosols Based on 16S rRNA and amoA Genes

Soil from the Amazonian region is usually regarded as unsuitable for agriculture because of its low organic matter content and low pH; however, this region also contains extremely rich soil, the Terra Preta Anthrosol. A diverse archaeal community usually inhabits acidic soils, such as those found in the Amazon. Therefore, we hypothesized that this community should be sensitive to changes in the environment. Here, the archaeal community composition of Terra Preta and adjacent soil was examined in four different sites in the Brazilian Amazon under different anthropic activities. The canonical correspondence analysis of terminal restriction fragment length polymorphisms has shown that the archaeal community structure was mostly influenced by soil attributes that differentiate the Terra Preta from the adjacent soil (i.e., pH, sulfur, and organic matter). Archaeal 16S rRNA gene clone libraries indicated that the two most abundant genera in both soils were Candidatus nitrosphaera and Canditatus nitrosocaldus. An ammonia monoxygenase gene (amoA) clone library analysis indicated that, within each site, there was no significant difference between the clone libraries of Terra Preta and adjacent soils. However, these clone libraries indicated there were significant differences between sites. Quantitative PCR has shown that Terra Preta soils subjected to agriculture displayed a higher number of amoA gene copy numbers than in adjacent soils. On the other hand, soils that were not subjected to agriculture did not display significant differences on amoA gene copy numbers between Terra Preta and adjacent soils. Taken together, our findings indicate that the overall archaeal community structure in these Amazonian soils is determined by the soil type and the current land use.     

一种新颖简便的荧光实时 RT-PCR相对定量方法的建立

为建立一种新颖、简便的荧光实时 RT-PCR 相对定量方法,根据实时定量标准曲线,推导出相对定量基因表达的公式 . 公式显示相对表达指数只与 CT 值和标准曲线的斜率相关 . 构建标准曲线的标准品需要通过克隆和体外转录获得,实验过程繁琐 . 当人为成比例增减标准品各个稀释度的具体拷贝数时,标准曲线的斜率并不改变,说明标准曲线斜率与标准品的具体拷贝数无关 . 因此,新的相对定量方法可以用任何一个待测样品的总 RNA ( 或 cDNA) ,经系列稀释后作为标准品,来构建相对定量标准曲线,获得斜率 . 与绝对定量法比较,新方法获得了基本相同的斜率和非常一致的定量结果 ( 差异小于4%) ,而传统的 2 -ΔΔCT法却表现出较大的定量误差 . 这些结果表明,新的相对定量方法是一种简便、准确和高效的定量基因表达的方法 .     

MDA技术在转基因检测中的研究与应用

PCR技术是转基因植物及其产品检测的主要方法,但是因为其对模板纯度和质量有较高要求,对于低质量模板难以获得稳定的检测结果。本研究利用MDA技术可将低至10 copies的微量DNA样品扩增至可用PCR方法稳定检出;进一步利用MDA技术对转基因玉米有证标准物质Bt11粉末的单颗粒痕量样品释出DNA进行扩增,结合PCR扩增检测,玉米粉末颗粒中内源参照基因的检出率达到70%以上,转基因特异性序列检出与内源参照基因检出的比值,与标准物质标称值基本相符,为建立基于MDA技术转基因植物及其产品粉末颗粒等痕量样品检测方法奠定了基础,同时也为加工产品中复合性状转基因作物的检测提供了潜在的解决方案。     

MiCA: A Web-Based Tool for the Analysis of Microbial Communities Based on Terminal-Restriction Fragment Length Polymorphisms of 16S and 18S rRNA Genes

A web-based resource, Microbial Community Analysis (MiCA), has been developed to facilitate studies on microbial community ecology that use analyses of terminal-restriction fragment length polymorphisms (T-RFLP) of 16S and 18S rRNA genes. MiCA provides an intuitive web interface to access two specialized programs and a specially formatted database of 16S ribosomal RNA sequences. The first program performs virtual polymerase chain reaction (PCR) amplification of rRNA genes and restriction of the amplicons using primer sequences and restriction enzymes chosen by the user. This program, in silico PCR and Restriction (ISPaR), uses a binary encoding of DNA sequences to rapidly scan large numbers of sequences in databases searching for primer annealing and restriction sites while permitting the user to specify the number of mismatches in primer sequences. ISPaR supports multiple digests with up to three enzymes. The number of base pairs between the 5′ and 3′ primers and the proximal restriction sites can be reported, printed, or exported in various formats. The second program, APLAUS, infers a plausible community structure(s) based on T-RFLP data supplied by a user. APLAUS estimates the relative abundances of populations and reports a listing of phylotypes that are consistent with the empirical data. MiCA is accessible at .