Roots are the primary site of interaction between plants and microorganisms. To meet food demands in changing climates, improved yields and stress resistance are increasingly important, stimulating efforts to identify factors that affect plant productivity. The role of bacterial endophytes that reside inside plants remains largely unexplored, because analysis of their specific functions is impeded by difficulties in cultivating most prokaryotes. Here, we present the first metagenomic approach to analyze an endophytic bacterial community resident inside roots of rice, one of the most important staple foods. Metagenome sequences were obtained from endophyte cells extracted from roots of field-grown plants. Putative functions were deduced from protein domains or similarity analyses of protein-encoding gene fragments, and allowed insights into the capacities of endophyte cells. This allowed us to predict traits and metabolic processes important for the endophytic lifestyle, suggesting that the endorhizosphere is an exclusive microhabitat requiring numerous adaptations. Prominent features included flagella, plant-polymer-degrading enzymes, protein secretion systems, iron acquisition and storage, quorum sensing, and detoxification of reactive oxygen species. Surprisingly, endophytes might be involved in the entire nitrogen cycle, as protein domains involved in N(2)-fixation, denitrification, and nitrification were detected and selected genes expressed. Our data suggest a high potential of the endophyte community for plant-growth promotion, improvement of plant stress resistance, biocontrol against pathogens, and bioremediation, regardless of their culturability.
The potential of DNA microarray technology in high-throughput detection of bacteria and quantitative assessment of their community structures is widely acknowledged but has not been fully realised yet. A generally applicable set of techniques, based on readily available technologies and materials, was developed for the design, production and application of diagnostic microbial microarrays. A microarray targeting the particulate methane monooxygenase (pmoA) gene was developed for the detection and quantification of methanotrophs and functionally related bacteria. A microarray consisting of a set of 59 probes that covers the whole known diversity of these bacteria was validated with a representative set of extant strains and environmental clones. The potential of the pmoA microarray was tested with environmental samples. The results were in good agreement with those of clone library sequence analyses. The approach can currently detect less dominant bacteria down to 5% of the total community targeted. Initial tests assessing the quantification potential of this system with artificial PCR mixtures showed very good correlation with the expected results with standard deviations in the range of 0.4-17.2%. Quantification of environmental samples with this method requires the design of a reference mixture consisting of very close relatives of the strains within the sample and is currently limited by biases inherent in environmental DNA extraction and universal PCR amplification.
3Methane oxidation can occur in both aerobic and anaerobic environments; however, these are completely different processes involving different groups of prokaryotes. Aerobic methane oxidation is carried out by aerobic methanotrophs, and anaerobic methane oxidizers, discovered recently, thrive under anaerobic conditions and use sulfate or nitrate as electron donors for methane oxidation (11,104). This review will focus on the aerobic oxidation of methane.Aerobic methanotrophs are a unique group of methylotrophic bacteria that utilize methane as a sole carbon and energy source (52). Based on their cell morphology, ultrastructure, phylogeny, and metabolic pathways, methanotrophs can be divided into two taxonomic groups: type I and type II. Type I methanotrophs include the genera Methylobacter, Methylomicrobium, Methylomonas, Methylocaldum, Methylosphaera, Methylothermus, Methylosarcina, Methylohalobius, Methylosoma, and Methylococcus, which belong to the gamma subdivision of the Proteobacteria (Fig. 1). The type II methanotrophs Methylocystis, Methylosinus, Methylocella, and Methylocapsa are in the alpha subdivision of the Proteobacteria (52) (Fig. 1). Recently, two filamentous methane oxidizers have been described, Crenothrix polyspora (113), which has a novel pmoA, and Clonothrix fusca (125), which has a conventional pmoA. Both are gammaproteobacteria and are closely related to the type I methanotrophs. Most extant methanotrophs are cultured at 20 to 45°C and neutral pH but have also recently been isolated from extreme environments (reviewed in reference 122).The first step in the oxidation of methane to CO 2 is the conversion of methane to methanol by the enzyme methane monooxygenase. There are two forms of this enzyme: a particulate membrane bound form (pMMO) and a soluble cytoplasmic form (sMMO). The pMMO has been reported in all methanotrophs except for the genus Methylocella (121), whereas the sMMO is present only in certain methanotroph strains (94). The pMMO is a membrane bound copper and iron containing enzyme (reviewed in reference 49). The structural genes for this enzyme have been cloned and sequenced from Methylococcus capsulatus Bath (107, 114), Methylocystis sp. strain M, and Methylosinus trichosporium OB3b (45). They lie in a three-gene operon, pmoCAB, which code for three integral membrane polypeptides of approximately 23, 27, and 45 kDa, respectively. These operons are present in duplicate copies in all three organisms. These duplicate copies of pmoCAB are virtually identical and are transcribed from 70 -type promoters found upstream of the pmoC gene (45, 110).The sMMO is a cytoplasmic enzyme containing a unique di-iron site at its catalytic center. It has a broad substrate range, including trichloroethylene, alkanes, alkenes, and aromatic compounds. The biochemistry of the sMMO has been studied in detail (reviewed in reference 75). It consists of three components: a hydroxylase, which is a dimer of three subunits, (␣␥) 2 ; a regulatory protein (protein B); and a reductase (protein C). It is e...
Landfill sites are responsible for 6-12% of global methane emission. Methanotrophs play a very important role in decreasing landfill site methane emissions. We investigated the methane oxidation capacity and methanotroph diversity in lysimeters simulating landfill sites with different plant vegetations. Methane oxidation rates were 35 g methane m-2 day-1 or higher for planted lysimeters and 18 g methane m-2 day-1 or less for bare soil controls. Best methane oxidation, as displayed by gas depth profiles, was found under a vegetation of grass and alfalfa. Methanotroph communities were analysed at high throughput and resolution using a microbial diagnostic microarray targeting the particulate methane monooxygenase (pmoA) gene of methanotrophs and functionally related bacteria. Members of the genera Methylocystis and Methylocaldum were found to be the dominant members in landfill site simulating lysimeters. Soil bacterial communities in biogas free control lysimeters, which were less abundant in methanotrophs, were dominated by Methylocaldum. Type Ia methanotrophs were found only in the top layers of bare soil lysimeters with relatively high oxygen and low methane concentrations. A competetive advantage of type II methanotrophs over type Ia methanotrophs was indicated under all plant covers investigated. Analysis of average and individual results from parallel samples was used to identify general trends and variations in methanotroph community structures in relation to depth, methane supply and plant cover. The applicability of the technology for the detection of environmental perturbations was proven by an erroneous result, where an unexpected community composition detected with the microarray indicated a potential gas leakage in the lysimeter being investigated.
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