Stefan Radajewski

Identification of active methylotroph populations in an acidic forest soil by stable- isotope probing

Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3.5) microcosms that were exposed to (13)CH(3)OH or (13)CH(4). Distinct (13)C-labelled DNA ((13)C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the (13)C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the (13)C-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms. Enrichments targeted towards the active proteobacterial CH(3)OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the beta-subclass of the PROTEOBACTERIA: Analysis of AOB-selective 16S rDNA amplification products identified Nitrosomonas and Nitrosospira sequences in the (13)C-DNA fractions, suggesting certain AOB assimilated a significant proportion of (13)CO(2), possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the (13)C-DNA were related to the 16S rDNA sequences of members of the Acidobacterium division, the beta-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by-products of methylotrophic carbon metabolism. Results from the (13)CH(3)OH and (13)CH(4) SIP experiments thus provide a rational basis for further investigations into the ecology of methylotroph populations in situ.

Stable-isotope probing of nucleic acids: a window to the function of uncultured microorganisms

Phylogeny based on ribosomal RNA sequences alone is rarely a reliable indicator of microbial function. To circumvent this problem, nucleic acid based techniques have been developed that exploit the physical properties of stable isotopes to study microbially mediated processes within complex environmental samples. Investigations using labelled substrates, or which detect variations in the natural abundance of isotopes, have thus revealed the metabolic function of microorganisms without the need to isolate them in culture.

Identification of the Functionally Active Methanotroph Population in a Peat Soil Microcosm by Stable-Isotope Probing

ABSTRACT The active population of low-affinity methanotrophs in a peat soil microcosm was characterized by stable-isotope probing. “Heavy” 13C-labeled DNA, produced after microbial growth on 13CH4, was separated from naturally abundant 12C-DNA by cesium chloride density gradient centrifugation and used as a template for the PCR. Amplification products of 16S rRNA genes and pmoA, mxaF, and mmoX, which encode key enzymes in the CH4 oxidation pathway, were analyzed. Sequences related to extant type I and type II methanotrophs were identified, indicating that these methanotrophs were active in peat exposed to 8% (vol/vol) CH4. The 13C-DNA libraries also contained clones that were related to β-subclass Proteobacteria, suggesting that novel groups of bacteria may also be involved in CH4 cycling in this soil.

Regulation of methane oxidation in the facultative methanotroph Methylocella silvestris BL2

The molecular regulation of methane oxidation in the first fully authenticated facultative methanotroph Methylocella silvestris BL2 was assessed during growth on methane and acetate. Problems of poor growth of Methylocella spp. in small‐scale batch culture were overcome by growth in fermentor culture. The genes encoding soluble methane monooxygenase were cloned and sequenced, which revealed that the structural genes for soluble methane monooxygenase, mmoXYBZDC, were adjacent to two genes, mmoR and mmoG, encoding a σ54 transcriptional activator and a putative GroEL‐like chaperone, located downstream (3′) of mmoC. Transcriptional analysis revealed that the genes were all cotranscribed from a σ54‐dependent promoter located upstream (5′) of mmo X. The transcriptional start site was mapped. Transcriptional analysis of soluble methane monooxygenase genes and expression studies on fermentor grown cultures showed that acetate repressed transcription of sMMO in M. silvestris BL2. The possibility of the presence of a particulate, membrane‐bound methane monooxygenase enzyme in M. silvestris BL2 and the copper‐mediated regulation of soluble methane monooxygenase was investigated. Both were shown to be absent. A promoter probe vector was constructed and used to assay transcription of the promoter of the soluble methane monoxygenase genes of M. silvestris BL2 grown under various conditions and with different substrates. These data represent the first insights into the molecular physiology of a facultative methanotroph.

Effects of land-use on the activity and diversity of methane oxidizing bacteria in forest soils

Abstract Methane is an important greenhouse gas and CH 4 oxidation in soil represents a significant sink for this gas. High capacity CH 4 oxidation potentials and molecular profiles of CH 4 oxidizing bacteria in soil were compared for five land-use treatments at a fully replicated experimental site within the Gisburn Forest Experiment, to assess the effects of land-use on both the potential activity of CH 4 oxidizing bacteria and their diversity. Forestry land-use was found to have a highly significant effect on CH 4 oxidation potentials. Highest CH 4 oxidation potentials were found in soils collected under stands of oak, in grassland plots, and in one soil under Norway spruce. A negative relationship between soil water nitrate concentration and CH 4 oxidation capacity was evident across the experimental site, with the high nitrate soils under stands of alder exhibiting little or no capacity for CH 4 oxidation even at optimal temperature and water content. Molecular profiles indicated that a diverse range of bacteria with the potential to oxidize CH 4 were present in all soils, however no clear correlation with CH 4 oxidation potential was identified.

Cultivation-independent techniques for studying methanotroph ecology

Methane-oxidizing bacteria (methanotrophs) have attracted considerable attention over the past 30 years. They have the unique ability to use methane as sole carbon and energy source, they are found in a wide variety of environments and play a crucial role in the global methane cycle. Methanotrophs also show considerable potential for bioremediation processes such as degradation of ground water pollutants, and for production of bulk chemicals from cheap substrates. We review here the cultivation-independent molecular biological methods that are available for the detection and characterization of methanotrophs in the natural environment.

Degradation of organic pollutants by methane grown microbial consortia

AbstractMicrobial consortia were enriched from various environmental samples with methane as the sole carbon and energy source. Selected consortia that showed a capacity for co-oxidation of naphthalene were screened for their ability to degrade methyl-tert-butyl-ether (MTBE), phthalic acid esters (PAE), benzene, xylene and toluene (BTX). MTBE was not removed within 24 h by any of the consortia examined. One consortium enriched from activated sludge (“AAE-A2”), degraded PAE, including (butyl-benzyl)phthalate (BBP), and di-(butyl)phthalate (DBP). PAE have not previously been described as substrates for methanotrophic consortia. The apparent Km and Vmax for DBP degradation by AAE-A2 at 20 °C was 3.1 ± 1.2 mg l−1 and 8.7 ± 1.1 mg DBP (g protein × h)−1, respectively. AAE-A2 also showed fast degradation of BTX (230 ± 30 nmol benzene (mg protein × h)−1 at 20 °C). Additionally, AAE-A2 degraded benzene continuously for 2 weeks. In contrast, a pure culture of the methanotroph Methylosinustrichosporium OB3b ceased benzene degradation after only 2 days. Experiments with methane mono-oxygenase inhibitors or competitive substrates suggested that BTX degradation was carried out by methane-oxidizing bacteria in the consortium, whereas the degradation of PAE was carried out by non-methanotrophic bacteria co-existing with methanotrophs. The composition of the consortium (AAE-A2) based on polar lipid fatty acid (PLFA) profiles showed dominance of type II methanotrophs (83–92% of biomass). Phylogeny based on a 16S-rRNA gene clone library revealed that the dominating methanotrophs belonged to Methylosinus/Methylocystis spp. and that members of at least 4 different non-methanotrophic genera were present (Pseudomonas,Flavobacterium,Janthinobacterium and Rubivivax).