Methanothrophic bacteria (MB) of the "upland soil cluster α" (USCα) are assumed to be responsible for methane oxidation at atmospheric methane concentrations but have as yet escaped all cultivation attempts. A metagenomic fosmide library consisting of 250.000 clones was constructed from total DNA of an acidic forest soil previously characterized as a sink for atmospheric methane. Using pmoA-targeted PCR assays, two clones were identified to carry genomic fragments of USCα representatives. Shotgun-based sequencing revealed that both inserts belonged to the same strain or two highy related USCα genotypes. Thus sequences were assembled resulting in a 42-kb contig. The next cultured relative of USCα is, based on partial pmoA phylogenies, the acidophilic M. acidiphila.
Consequently, a 100-kb fragment of the M. acidiphila genome containing the pmo operon was sequenced. Annotation and comparative analysis of both genomic fragments against publicly available completely sequenced genomes revealed highest similarities to members of the Bradyrhizobiaceae. Phylogenetic analyses of four genes identified on both fragments corroborated a close phylogenetic relation between M. acidiphila and USCα. The affiliation of USCα to the Alphaproteobacteria was also confirmed by a SOM-based analysis of di-, tri- and tetra-nucleotid patterns of the USCα genomic fragment. A detailed comparative analysis of the pmo operons of USCα and M. acidiphila showed that both the pmo operon structure and the predicted secondary structure of pMMO are highly conserved among all MB. No particularities were identified at the DNA or amino acid level that might have been interpretable as indicators for the putative capability of USCα to oxidize methane with high affinity. Together with the recently reported pmoCAB1 and pmoCAB2 of Methylocystis strain SC2, this study doubled the number of completely analysed pMMO operons. It enabled a phylogenetic analysis of concatenated pmoCAB sequences rather than only of partial pmoA, thereby roughly sextupling the amount of underlying genetic information.
Introduction
Aerobic methanotrophic Bacteria (MB) are able to use methane (CH4) as sole source of carbon and energy. Based on phylogenetic, physiological, morphological, and biochemical characteristics, methanotrophs have been divided into three phylogenetically and physiologically distinct groups, type X, type I and type II MB, forming coherent clusters within the Gammaproteobacteria (type X and type I) and Alphaproteobacteria (type II) (Hanson and Hanson, 1996). The gammaproteobacterial type I MB comprises the genera Methylomonas, Methylocaldum, Methylomicrobium, Methylobacter, Methylosarcina, and Methylosphaera. While the type X MB Methylococcus also belong to the Gammaproteobacteria the alphaproteobacterial type II MB consists of the genera Methylocystis and Methylosinus. With the advent of novel MB isolates and environmental studies, this classification has been challenged. Novel isolates were obtained, that could not be assigned into the type-system. For example, the recently described acidophilic MB that are represented by two genera, Methylocella and Methylocapsa, possess several unique morphological and physiological characteristics, and, based on 16S rRNA phylogeny, Methylocella and Methylocapsa are evolutionarily more closely related to acidophilic heterotrophic bacteria of the genus Beijerinckia than to the Methylosinus/Methylocystis group.
The first step in CH4 oxidation, the conversion of methane to methanol, is carried out by a methane monooxygenase (MMO). This enzyme exists in two forms, a particulate, membrane-associated form (pMMO) and a soluble form (sMMO). The two forms of the enzyme differ in structure, in kinetic properties, and in the range of substrates which are utilized. Only a restricted number of MB species possess sMMO, while almost all MB possess pMMO. The only MB lacking pMMO are members of the genus Methylocella. In MB that harbor both forms of MMO, sMMO is synthesized under copper-deficient conditions, while in the presence of even a minuscule amount of available Cu(II) (0.85 to 1.0 µmol/g [dry weight] of cells) only pMMO is synthesized.
The pMMO is composed of two subunits, a hydroxylase and a NADH-oxidoreduktase.
The hydroxylase subunit has been demonstrated to be encoded by three consecutive open reading frames (pmoC, pmoA, and pmoB) in both type X MB and type II MB. The pmo operon is homologous to the amoCAB operon that encodes ammonium monooxygenase (AMO), the key enzyme of autotrophic nitrifiers. AMO catalyzes not only the oxidation of ammonia but also that of CH4, albeit at a much lower activity.
PmoA and AmoA are presumed to contain the active sites of the respective enzymes because they have been shown to be specifically labeled by [14C] acetylene, a suicide substrate for MMO and AMO. pmoA has been used in numerous studies on the diversity and distribution of MOB, as the phylogeny of pmoA sequences shows a strong congruity with that of 16S rDNA sequences (Murrell et al., 1998). It is an ideal marker for environmental studies because it enables the functional detection of novel, as-yet uncultured MB.
The only biological sink for CH4 is oxidation in soil. Atmospheric CH4 is consumed in forest, agricultural, and other upland soils. CH4 consumption in these soils is caused by MB which are characterized by a high apparent affinity for methane of 10–200 nM (reviewed by (Dunfield et al., 1999). In forest soils that are sinks for atmospheric CH4, novel pmoA sequence types (referred to as "upland soil cluster α" or "USCα") have been described frequently. More recently, an additional novel pmoA lineage was detected in upland soils.
Since it grouped distantly related to type I MB in pmoA-based phylogenies, it was designated "upland soil cluster γ" (USCγ). USCγ was detected in soils with pH values greater than 6.0, whereas pmoA of USCα has been retrieved mainly from acidic soils. No pure cultures are known for these putative atmospheric methane oxidizers.
Phylogenies constructed for partial pmoA sequences group USCα sequences within pmoA sequences of alphaproteobacterial origin. The inferred amino acid sequences of USCα exhibited closest relatedness to PmoA of Methylocapsa acidiphila. In phylogenetic trees USCα and M. acidiphila partial PmoA sequences formed a common branch, clustering distinct from PmoA sequences of conventional type II MB of the Methylosinus/Methylocystis group (Dedysh et al., 2001). Thus, based on pmoA phylogenies M. acidiphila has been regarded as the closest cultivated relative of USCα. However, the apparent affinity for methane exhibited by Methylocapsa acidophila B2 was 1–2 µM, which is similar to values measured in other methanotrophic bacteria.
Since 16S rDNA-based phylogenetic information is still unavailable for USCα, its true evolutionary origin remained to be elucidated. We used a metagenomic approach to obtain a first insight into the genome of USCα representatives. Besides final elucidation of the evolutionary origin, the retrieval of USCα genomic fragments also aimed at assessing whether the pMMO of USCα representatives exhibits any unusual feature(s) that correspond(s) to the assumed capability of USCα to consume atmospheric methane. The pmo operon sequence and flanking genomic regions of M. acidiphila were analysed for