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Applied and Environmental Microbiology, November 2005, p. 7472-7482, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7472-7482.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

First Genome Data from Uncultured Upland Soil Cluster Alpha Methanotrophs Provide Further Evidence for a Close Phylogenetic Relationship to Methylocapsa acidiphila B2 and for High-Affinity Methanotrophy Involving Particulate Methane Monooxygenase

Peter Ricke,1 Michael Kube,2 Satoshi Nakagawa,3 Christoph Erkel,1 Richard Reinhardt,2 and Werner Liesack1*

Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch Strasse, D-35043 Marburg, Germany,1 Max-Planck-Institut für molekulare Genetik, Ihnestrasse 63-73, D-14195 Berlin, Germany,2 Xanagen Inc., 2-7-14 Higashinakano, Nakano-ku, Tokyo 164-0003, Japan3

Received 21 April 2005/ Accepted 11 July 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Members of upland soil cluster alpha (USC{alpha}) are assumed to be methanotrophic bacteria (MB) adapted to the trace level of atmospheric methane. So far, these MB have eluded all cultivation attempts. While the 16S rRNA phylogeny of USC{alpha} members is still not known, phylogenies constructed for the active-site polypeptide (encoded by pmoA) of particulate methane monooxygenase (pMMO) placed USC{alpha} next to the alphaproteobacterial Methylocapsa acidiphila B2. To assess whether the pmoA tree reflects the evolutionary identity of USC{alpha}, a 42-kb genomic contig of a USC{alpha} representative was obtained from acidic forest soil by screening a metagenomic fosmid library of 250,000 clones using pmoA-targeted PCR. For comparison, a 101-kb genomic contig from M. acidiphila was analyzed, including the pmo operon. The following three lines of evidence confirmed a close phylogenetic relationship between USC{alpha} and M. acidiphila: (i) tetranucleotide frequency patterns of 5-kb genomic subfragments, (ii) annotation and comparative analysis of the genomic fragments against all completely sequenced genomes available in public domain databases, and (iii) three single gene phylogenies constructed using the deduced amino acid sequences of a putative prephenate dehydratase, a staphylococcal-like nuclease, and a putative zinc metalloprotease. A comparative analysis of the pmo operons of USC{alpha} and M. acidiphila corroborated previous reports that both the pmo operon structure and the predicted secondary structure of deduced pMMO are highly conserved among all MB.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The only biological sink for atmospheric CH4 is its consumption in oxic soils (7, 51). Recently, the global terrestrial sink was estimated at 29 Tg year–1, with a wide range of uncertainty (7 to >100 Tg CH4 year–1) (51). Atmospheric CH4 is consumed in forest, agricultural, and other upland soils by aerobic methanotrophic bacteria (MB). The CH4-consuming activity of these MB is significant because the magnitude of atmospheric CH4 uptake in oxic soils is similar to the estimated excess of emissions over sinks in recent years of 37 Tg year–1 (28). Moreover, methanotrophic activity is highly susceptible to disturbance by human activities (32, 51).

Cultured MB are divided into two groups, namely, type I (further divided into types I and X) and type II. They differ in their phylogenetic affiliations (Gammaproteobacteria versus Alphaproteobacteria) and in diverse biochemical and ultrastructural characteristics (23). However, this traditional concept of methanotroph classification has become much more complex by recent descriptions of the genera Methylocella (9, 12, 17) and Methylocapsa (11). These acidophilic MB, although considered members of the type II MB, possess several unique morphological and physiological characteristics, and based on 16S rRNA phylogeny, Methylocella and Methylocapsa are more closely related to acidophilic heterotrophic bacteria of the genus Beijerinckia than to alphaproteobacterial type II MB of the Methylosinus/Methylocystis group.

Methane monooxygenase (MMO) catalyzes the first step in the metabolic pathway of MB, the conversion of CH4 to methanol. All cultivated MB possess a particulate, membrane-associated form (pMMO) of this enzyme, except for members of the genus Methylocella (9, 12, 17). pMMO is encoded by three consecutive open reading frames (pmoC, pmoA, and pmoB) in both type I and type II MB.

The identity of the microorganisms consuming atmospheric CH4 remained unclear for many years (7, 45, 49). The apparent half-saturation constant (Km) for the oxidation of atmospheric CH4 in upland soil ranges from 0.8 to 280 nM. However, the Km of cultured type I and type II MB (0.8 to 66 µM) are 1 to 3 orders of magnitude higher, and these MB are not able to survive prolonged periods using only atmospheric CH4 (7, 44, 49). It was later shown that the apparent affinity for CH4 varies with growth conditions and that type II MB of the Methylosinus/Methylocystis group might contribute to the oxidation of atmospheric CH4 in soils (15, 16). Nonetheless, the use of pmoA as a functional gene marker for the molecular characterization of methanotrophic communities revealed that, besides members of known methanotrophic genera, a novel group of MB occurs in upland soils that consume atmospheric CH4 (4, 24, 27, 33). The most recent study suggested that these novel MB (originally named forest soil cluster but now referred to as upland soil cluster alpha [USC{alpha}]) are present mainly in acidic soils with pH values of <6 (33). Activity profiles of 13C- and 14C-labeled phospholipid fatty acids provided evidence that members of USC{alpha} are highly adapted to the consumption of atmospheric CH4 and are presumably of alphaproteobacterial origin (5, 27, 33, 46).

Phylogenetic trees constructed for the pmoA gene suggested that among the cultured MB, Methylocapsa acidiphila B2 is most closely related to USC{alpha} (10). In general, pmoA and 16S rRNA phylogenies show good correlation (33, 35). Despite this, the evolutionary identity of USC{alpha} is still uncertain because (i) the pmoA gene enables the inference of putative relationships only among MB and (ii) the recent detection of multiple, diverse pmoA-like genes in single genospecies of MB implies that pmoA phylogenies have to be interpreted with caution (55). Moreover, the apparent affinity for methane exhibited by M. acidiphila B2 was 1 to 2 µM, which is similar to values measured in other cultured type I and type II MB (10). Except for phospholipid fatty acid profiles and partial pmoA sequences, no additional pheno- or genotypic information is available for USC{alpha}.

The aim of our study was to gain the first insight into the genome of a USC{alpha} representative by using a metagenomic approach (13, 22). This included the extraction of high-molecular-weight (HMW) DNA from acidic forest soil, the construction of a large insert clone library in a fosmid vector, and screening of the clones by pmoA-targeted PCR. For comparison, a genomic library of M. acidiphila B2 was constructed in a bacterial artificial chromosome (BAC) vector, and genomic fragments carrying pmoA were identified. The genomic information obtained for USC{alpha} and M. acidiphila B2 should be used to clarify the phylogenetic status of USC{alpha} and to perform a detailed comparative analysis of the pmo operon.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Soil samples and bacterial strains.
The sampling site was a deciduous forest (Fagus sylvatica and Quercus robur) located near Marburg (Hesse, Germany). After the removal of rotten foliage, soil samples (100 g of Braunerde [Cambisol], pH 4.0) were taken from a soil depth of 3 to 15 cm (upper mineral horizon). The soil was stored in 20-g subsamples at –20°C until further processing. The conditions used for the growth of M. acidiphila strain B2 were adapted from the work of Heyer et al. (25). Cells were grown in liquid culture for 3 to 4 weeks at 30°C under a headspace of 10% (vol/vol) CH4, 3% CO2, and 87% air.

Extraction of HMW DNA.
Subsamples of approximately 4 g (fresh weight) of forest soil were used for DNA extraction. Each subsample was mixed with 3 ml of 120 mM sodium phosphate buffer (pH 8.0) and 250 µl of lysozyme solution (100 mg ml–1; Sigma-Aldrich, Steinheim, Germany). After incubation for 1 h at 37°C, 60 µl of proteinase K (20 mg ml–1; QIAGEN, Hilden, Germany) was added, and incubation was continued for 1 h. The soil suspension was shock frozen in liquid nitrogen and stored for 0.5 h at –80°C. After the suspension was thawed, 400 µl of a 20% (wt/vol) sodium dodecyl sulfate solution was added. The suspension was mixed gently for a few seconds and then incubated at 65°C for 45 min. The soil particles were removed by two rounds of low-speed centrifugation in an EBA 8S centrifuge (Hettich, Tuttlingen, Germany) for 5 min. The resulting supernatants were transferred to new tubes. After the second round, the supernatant was mixed with a 0.45 volume (vol/vol) of 6 M potassium acetate and incubated for 5 min at room temperature. After centrifugation in an Eppendorf microfuge for 5 min, the resulting supernatant was transferred to a new tube. To reduce the content of humic acids, the DNA extract was further purified using a FastDNASPIN kit for soil (Q-BIOgene, Heidelberg, Germany). The purification step was carried out according to the manufacturer's instructions, with the modification that four additional washing steps with 500 µl of guanidine thiocyanate solution (5.5 M in 0.1 M Tris; pH 7.5) were performed after the DNA had been loaded onto the spin column. Size fractionation of purified DNAs via pulsed-field gel electrophoresis (PFGE) in a CHEF-DR III apparatus (Bio-Rad, Madison, Wis.) and the recovery of appropriate fragment sizes by ß-agarase treatment were carried out as described previously (18, 43).

Construction of a fosmid library.
A fosmid library was constructed using a CopyControl fosmid library production kit (Epicentre Technologies, Madison, Wis.) as described by the manufacturer, with slight modifications as follows: (i) a second end-repair reaction was performed after size selection, (ii) ligation was extended overnight at 4°C, and (iii) each phage packaging reaction was extended to 4 h. The average insert size of the fosmid clones was estimated by SbfI digestion of the vector/insert constructs of 20 randomly chosen clones, followed by PFGE analysis as described by Peterson et al. (41).

PCR-based screening for pmoA.
Colonies grown on individual plates were replicated using the Lederberg technique. Colonies were washed from the replicated agar plates with 400 µl of sterile water and thoroughly mixed. One-half of the cell suspension was lysed by boiling. The cell debris was pelleted by centrifugation (10 s), and 1 µl of the supernatant was used as a template for PCR-based screening. The other half of the cell suspension was mixed with 1 volume of glycerol buffer (65% glycerol, 1.2 mM MgSO4, pH 8.0) and then shock frozen in liquid nitrogen. Storage was done at –80°C. Screening for pmoA-carrying inserts was performed by the application of two PCR assays with different target specificities. Primers A189f (GGNGACTGGGACTTCTGG) and A682b (GAASGCNGAGAAGAASGC) target a wide range of different pmoA and amoA sequence types, resulting in a 531-bp amplicon (26). Primer A189F in combination with primer Forest675R (CCYACSACATCCTTACCGAA) specifically targets pmoA sequence types of USC{alpha}, resulting in a 506-bp amplicon (35). PCRs were carried out as described in the original protocols (26, 35). In case the colony pool obtained from the replicate plate was positive for one of the two pmoA-targeted assays, the 250 to 350 colonies grown on the original master plate were picked and transferred to 96-well microtiter plates. For the identification of clones carrying pmoA, 10-µl aliquots of fosmid clone-positive Escherichia coli cells, which were grown in 12 wells (one row) of a microtiter plate, were combined. An aliquot (20 µl) of each row pool was combined to generate plate pools. Pooled cells were lysed by boiling for 15 min, and the cell debris was subsequently pelleted by centrifugation in an Eppendorf microfuge for 5 min. Microtiter plates that contained at least one test-positive fosmid clone were identified by pmoA-targeted PCR using 1 µl of the supernatant of each plate pool as the template. Row pools of test-positive plates were screened in the same way. Finally, clones of single rows that tested positive were analyzed separately. To exclude false-positive results, amplicons of the expected size were purified and sequenced using an ABI Prism 377 DNA sequencer and dye terminator chemistry as specified by the manufacturer (PE Applied Biosystems, Weiterstadt, Germany).

Construction and screening of a genomic BAC library from M. acidiphila B2.
The procedure used for the extraction of HMW DNA and construction of a BAC library followed a protocol described previously (43). Briefly, cells (approximately 1 g of fresh weight) embedded in agarose plugs were lysed in sodium dodecyl sulfate-based lysis buffer, followed by partial digestion of HMW DNA with the HindIII endonuclease (New England Biolabs, Beverly, Mass.) and size fractionation by PFGE. DNA fragments of appropriate sizes (50 to 150 kb) were recovered by ß-agarase treatment and ligated into pBeloBAC11 (Epicentre Technologies). The ligation product was desalted and transformed into E. coli ElectroMax DH10B competent cells (Invitrogen Life Technologies, Carlsbad, Calif.). The average insert size was estimated as described above for fosmid clones. Single colonies were picked and stored in 96-well microtiter plates. Starting from the microtiter plates, screening for BAC clones carrying pmo genes was performed as described above, using the primer pair A189f/A682b.

DNA sequencing and computational analysis.
The pmoA-carrying inserts of two BAC clones of M. acidiphila B2 and of two forest soil fosmid clones were characterized by a standard procedure, including shotgun cloning, sequencing, assemblage, and annotation (18, 43). Base calling of DNA sequencer traces was conducted by using the PHRED program with the quality level set to 30 (19). A threshold of 200 bp was chosen for automatic preliminary identification of putative open reading frames (ORFs) using the software package Orpheus (gene prediction; http://pedant.gsf.de/orpheus/). Subsequently, a more detailed analysis was based on high-throughput gene annotation (42) and Artemis (final manual annotation; http://www.sanger.ac.uk/Software/Artemis/). Homology searches were carried out with standard software tools, including the BLAST algorithm (3) against the nonredundant DNA database as well as against a nonredundant protein database compiled for this study from SWISSPROT, TREMBL, and PIR. Highly conserved amino acid sequence motifs were identified by comparison against the InterPro database of protein families, domains, and functional sites (http://www.ebi.ac.uk/interpro/). The locations of putative transmembrane-spanning regions of PmoCAB were computed using the programs toppred (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html) and TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) (52).

Phylogenetic trees were constructed using the ARB program package (38). For phylogenetic analysis of complete pmoCAB operons, the deduced PmoCAB and AmoCAB sequences of individual organisms were concatenated while intergenic, noncoding regions were omitted. For analyses of other protein sequences, multiple alignments of reference sequences for the respective gene type were obtained from the Pfam protein family database of alignments and HMMs (http://www.sanger.ac.uk/Software/Pfam/) or from the Clusters of Orthologous Groups (COG) database (http://www.ncbi.nlm.nih.gov/COG/). The multiple alignments were imported into ARB. The inferred protein sequences obtained for USC{alpha} and M. acidiphila B2 were aligned manually. Maximum likelihood (protML of the Molphy package [2]), TreePuzzle (54), and neighbor-joining (47) methods were applied using appropriate evolutionary models, such as PAM (8), JTT (29), and WAG (59). The resulting phylogenies were manually combined into a consensus tree. Branches for which the relative order could not be determined unambiguously by applying the different treeing methods are shown by multifurcation.

Genome signature-based phylogenetic mapping.
An analysis of tetranucleotide frequencies was performed by application of the XanaPGMap program (Xanagen). The genomic fragments of USC{alpha} and M. acidiphila B2 were subdivided into 5-kb subfragments and compared against signature maps computed by the XanaPGMap program for all completely sequenced bacterial genomes available in public domain databases at the time of analysis. The maps consisted of 5-kb genomic subfragments into which each individual bacterial genome had been subdivided. In addition, nonredundant genomic sequences available in public databases for Methylosinus spp. and Methylocystis spp. were included in the analysis.

Nucleotide sequence accession numbers.
The sequence of the 42-kb genomic fragment of the USC{alpha} representative has been deposited in the GenBank/EMBL/DDBJ databases under the accession number CT005232. The sequence of the 101-kb genomic fragment of M. acidiphila B2 was assigned the accession number CT005238. A 511-bp fragment of the gene encoding the putative prephenate dehydratase of Methylocella palustris K was assigned the accession number DQ108614.


   RESULTS AND DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Construction and screening of large insert libraries. (i) Forest soil.
Each cloning step resulted in a metagenomic library of 10,000 to 20,000 fosmid clones with an average insert size of approximately 40 kb. USC{alpha} was reported to be the predominant methanotroph population in upland soils of various geographic regions (4, 24, 27, 33). However, USC{alpha} is only a minor population of the total soil bacterial community. In the forest soil studied here, USC{alpha} was detected by real-time PCR in numbers equivalent to about 106 pmoA gene copies per gram (dry weight) of soil and comprised >90% of the detectable MB (36). Thus, a high-throughput method was established for rapid screening of a large number of fosmid clones by pmoA-targeted PCR. The screening of 250,000 fosmid clones led to the identification of two inserts carrying a pmoA gene of USC{alpha}. Shotgun-based sequencing revealed that both inserts belonged to the same or two highly related USC{alpha} genotypes, as the sequences obtained for the two inserts exhibited 100% identity over the overlapping region of approximately 30 kb. Assemblage of the two genomic fragments resulted in a 42-kb contig. It is unlikely that the screening was biased towards the identification of two USC{alpha} genomic fragments with identical pmoA sequences because the two fragments were detected using PCR assays with different pmoA target specificities (see above) (26, 35).

The nucleic acid sequence of a 495-bp stretch of the USC{alpha} pmoA gene was completely identical to that of the environmental pmoA sequence Mforest (GenBank/EMBL/DDBJ accession number AJ496664). Mforest had been obtained in a previous study from total DNA of the same forest soil that was used in this study as the starting material. The retrieval of the Mforest sequence was based on denaturing gradient gel electrophoresis of PCR-amplified pmoA genes (35). Comparative sequence analysis showed that Mforest belongs to USC{alpha} (Fig. 2 in reference 36). MForest had high identities (96.8 and 98.7%, respectively) to the forest clones Rold5 (AF148527) and RA14 (AF148521) (27, 35).



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  		FIG. 2. Schematic overview of gene arrangements in a 101-kb fragment of Methylocapsa acidiphila B2, the complete genomes of Beijerinckia japonicum and Rhodopseudomonas palustris, and a 42-kb fragment of USC{alpha}. Corresponding genomic regions are linked by grayish interconnections, except for those between USC{alpha} and M. acidiphila B2. These are highlighted in yellow. The ORF numbering corresponds to that obtained by the automatic ORF prediction program ORPHEUS (20). Color code: green, pmoCAB and, only on the USC{alpha} fragment, the associated ORF45 (OrfD [21]); blue (only on the M. acidiphila B2 fragment), genes encoding enzymes involved in H4MPT/MFR-mediated C1 metabolism (57); red, genes encoding other proteins with assigned functions; white, genes encoding hypothetical proteins; gray, genes encoding conserved hypothetical proteins.

 
(ii) M. acidiphila B2.
A genomic BAC library was constructed that consisted of 1,054 clones with an average insert size of 70 to 80 kb. Eight BAC clones were identified as carrying a pmoA gene. Restriction analysis of the clones (data not shown) resulted in the identification of two genomic fragments with a minimal overlap for shotgun-based sequencing. Finally, a 101-kb contig of nonredundant sequence information was obtained.

Genome signature-based phylogenetic mapping.
The USC{alpha} genomic fragment was analyzed with the program XanaPGMap. This program performs a highly reliable assignment of genomic fragments to phylogenetic groups based on a statistical analysis of the relative abundances of tetranucleotides in a given sequence without any regard to coding regions or sequence similarities (1, 56). Signature-based analyses have been widely discussed during recent years (14, 30, 31, 34, 48). It was shown that in most cases genomic fragments of 10 kb, and sometimes even fragments of 1 kb, retain species-specific information (1). Teeling et al. (56) reported that the discrimination of fosmid-sized genomic fragments based on tetranucleotide frequency patterns was consistent with corresponding data from 16S rRNA sequence analysis. XanaPGMap performs the assignment via an unsupervised neural network (self-organizing map [SOM]) algorithm. The algorithm is able to cluster complex, n-dimensional data (e.g., the frequencies of the analyzed tetranucleotides in different fully sequenced genomes) and to display them on two-dimensional maps. The individual squares of the resulting map thereby represent weight vectors (data points within the high-dimensional data space). The distance between two weight vectors reflects (in a nonlinear manner) the difference in genomic signatures.

The SOM-based analysis unambiguously placed 37 of 38 5-kb genomic subfragments of USC{alpha} within a map area defined by Alphaproteobacteria (Fig. 1). The only exception was a single subfragment that was placed within a map area dominated by Betaproteobacteria. This fragment contained ORF16, whose derived amino acid sequence implies a betaproteobacterial origin (also see below). The ORFs flanking ORF16 showed relatedness to alphaproteobacterial genes (Fig. 2), which is reflected by the placement of this 5-kb subfragment at the boundary between the alpha- and betaproteobacterial map areas (green arrow in Fig. 1). All 5-kb genomic subfragments analyzed for M. acidiphila were placed into the alphaproteobacterial map area, most of them in regions similar to those of USC{alpha}. The same is true for the genomic subfragments of the Methylosinus/Methylocystis group, except for a few that were placed outside of the alphaproteobacterial map area (black arrows in Fig. 1). A second analysis against a map specifically constructed for alphaproteobacterial genomes placed all 37 alphaproteobacterial-like subfragments of USC{alpha} into a map area defined by genomic subfragments of Bradyrhizobium japonicum, Rhodopseudomonas palustris, Sinorhizobium meliloti, and Agrobacterium tumefaciens (data not shown).



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  		FIG. 1. Genome signature-based phylogenetic map calculated for 5-kb subfragments of all publicly available bacterial genome sequences (only the relevant section of the total bacterial map is shown). Symbols (x) indicate the positions calculated for the 5-kb genomic subfragments of USC{alpha} (black), M. acidiphila B2 (white), and Methylosinus/Methylocystis spp. (red). Single data points may be indicative of several genomic subfragments that were mapped at the same position. The green arrow points to the position of a genomic subfragment of USC{alpha} that contains ORF16. The black arrows indicate the positions of genomic subfragments of Methylosinus/Methylocystis spp. that were placed outside of the alphaproteobacterial map area.

 
Annotation of genomic fragments.
In order to further resolve the phylogenetic status of USC{alpha} and its relationship to M. acidiphila B2, the genomic data obtained for the two taxa were thoroughly analyzed in relation to the genomic information available in public databases. A major aim was to identify gene homologs on the genomic fragments of USC{alpha} and M. acidiphila B2.

(i) USC{alpha}.
Of 71 ORFs identified on the 42-kb USC{alpha} genomic fragment by ORPHEUS (20), 47 ORFs were confirmed by manual annotation to encode proteins. Manual analysis in combination with semiautomatic annotation by the HTGA platform enabled the assignment of 27 ORFs to genes with known functions, while 20 ORFs were referred to as hypothetical or conserved hypothetical. The designation "conserved hypothetical" (5 ORFs) refers to ORFs with high similarities to predicted genes of unknown function. The coding density of the USC{alpha} genomic fragment was 85.0%, with an average ORF length of 767 bp. The overall mol% G+C content was 59.8%.

Thirty-two ORFs had at least one homologous sequence in public databases. Four of them are arranged in a gene cluster containing the pmoCAB operon (ORFs 46, 48, and 49) plus an additional gene of unknown function (ORF45) (Fig. 2). During the initial BLAST analyses, 27 of the remaining 28 ORFs showed highest similarities to genes from the Alphaproteobacteria. Fourteen of them exhibited highest similarities to genes from B. japonicum. A different subset of 14 ORFs was arranged in four gene clusters with high structural similarities to genomic regions of B. japonicum. Three of the four clusters were also found in Rhodopseudomonas palustris, which is also a member of the Bradyrhizobiaceae. The genome of B. japonicum was the only completely sequenced genome in which homologs of all four USC{alpha} gene clusters were present. However, for each of the four USC{alpha} gene clusters, at least partially conserved genomic regions could also be identified in organisms other than B. japonicum. The homologous clusters in B. japonicum could be identified easily using BLAST similarity searches. In contrast, most of the homologous gene clusters were detected in bacteria other than B. japonicum and R. palustris only by analyses of manually curated databases of orthologous genes (KEGG-KO and NCBI-COG). Since the conservation of structural genomic features is an indicator of evolutionary relationships of organisms or at least of genomic regions, the four USC{alpha} gene clusters were characterized in detail.

Cluster 1 consists of four genes that putatively encode the general secretory pathway proteins GspN, GspM, GspL, and GspK (ORFs 8, 9, 13, and 14) (Fig. 2). In the genomes of B. japonicum and four members of the gammaproteobacterial genera Xylella and Xanthomonas, these genes are part of a conserved cluster of up to 12 genes coding for secretory pathway proteins. The presence of cluster 1 in USC{alpha} and B. japonicum seems to be the exception rather than the rule for Alphaproteobacteria because these genes are missing from all other completely sequenced alphaproteobacterial genomes, including the genome of R. palustris.

Cluster 2 consists of seven genes (ORFs 26, 27, 28, 30, 31, 33, and 34) (Fig. 2). The first three ORFs encode proteins of a zinc/manganese ABC transporter system. The organization of ORFs 26, 27, and 28 is highly conserved among bacteria and is partially conserved even in some archaea. Gene homologs of ORFs 30 and 31 are located in a homologous position in some alphaproteobacterial, betaproteobacterial, and cyanobacterial genomes. An additional gene encoding acetoacetate decarboxylase (ORF33) and a paralogous gene copy of ORF31 (ORF34) complete the gene cluster. The acetoacetate decarboxylase gene (ORF33) was found in a homologous position only in the B. japonicum genome.

Cluster 3 consists of two genes (ORFs 39 and 40) (Fig. 2). ORF39 was annotated as a conserved hypothetical gene. ORF40 codes for salicyl hydroxylase. The gene organization of cluster 3 is found only in alphaproteobacterial genomes, including those of B. japonicum, R. palustris, and Brucella spp.

Cluster 4 is localized directly upstream of the pmoCAB gene cluster and is composed of two genes (ORFs 54 and 56) (Fig. 2). A BLAST search of the deduced amino acid sequence encoded by ORF54 revealed significant similarities only to hypothetical proteins of B. japonicum and R. palustris. However, ORF54 could be annotated by comparison against the InterPro database as a putative member of the ‘Staphylococcus nuclease subtype’ protein family, which is widespread among bacteria, archaea, and eukaryotes. ORF56 was annotated as encoding a putative zinc metalloprotease. Besides B. japonicum and R. palustris, Pseudomonas aeruginosa was the only organism for which homologs in the same gene order could be identified. However, the similarity between ORF54 and its homolog from P. aeruginosa was very low.

Taken together, the results of comparative genomics agree well with those obtained by signature-based phylogenetic mapping and provide, based on the organisms available for analysis, strong evidence of an evolutionary relationship between USC{alpha} and members of the Bradyrhizobiaceae, particularly B. japonicum. In contrast to the overall high degree of similarity between the 42-kb contig of USC{alpha} and the genomes of Bradyrhizobiaceae, a single ORF (ORF16) was identified whose derived amino acid sequence exhibited no significant similarities to any alphaproteobacterial sequence. Instead, it showed high similarity only to a hypothetical protein of the betaproteobacterium Nitrosomonas europaea (GenBank accession number Q82s51). As mentioned above, the independent SOM-based analysis of the USC{alpha} fragment also predicted a betaproteobacterial origin for the genomic region containing ORF16. The combined interpretation of these results strongly suggests that ORF16 was acquired by horizontal gene transfer from the betaproteobacterial lineage.

(ii) M. acidiphila B2.
Of 149 ORFs identified on the 101-kb genomic fragment of M. acidiphila, 90 ORFs were confirmed by manual annotation to encode proteins. Fifty-eight ORFs could be assigned a putative function. For example, genes involved in DNA replication and recombination (encoding the chromosomal replication initiator proteins DnaA, RuvC, and RuvA) and genes encoding proteins of the ABC transporter family were identified. Thirty-two ORFs were referred to as hypothetical (24 ORFs) or conserved hypothetical (8 ORFs) genes. The coding density of the M. acidiphila B2 genomic fragment was 86.9%, with an average ORF length of 989 bp. The overall mol% G+C content was 62.0%.

Like the case for the USC{alpha} genomic fragment, BLAST analyses of the deduced amino acid sequences of predicted ORFs resulted in a large number of matches with highest similarities to genes from B. japonicum. Altogether, 48 derived protein sequences exhibited clear similarities to sequences from B. japonicum. Twenty-two genes were found to be organized into six gene clusters with similar structural organizations to those of their homologs in B. japonicum. The largest of these consists of ORFs 78, 82, and 84 to 87 (Fig. 2). ORFs 84 to 87 encode proteins of the ABC transporter family.

In addition to the aforementioned six gene clusters, we identified a gene cluster that encodes enzymes involved in tetrahydromethanopterin/methanofuran (H4MPT/MFR)-mediated C1 transfer reactions (ORFs 1 to 12) (Fig. 2), including formaldehyde-activating enzyme (Fae; ORF5), methenyl-H4MPT cyclohydrolase (Mch; ORF9), and NAD(P)-dependent methylene-H4MPT hydrogenase (MtdB; ORF11). These enzymes, which are characteristic of methanogenic and sulfate-reducing archaea, have previously been shown to be involved in formaldehyde oxidation to CO2 in the methylotrophic bacterium Methylobacterium extorquens AM1 (6, 57, 58). Thus, in correspondence to various other methylotrophic and methanotrophic bacteria, M. acidiphila seems to possess an H4MPT-dependent formaldehyde oxidation pathway. However, a comparison with the corresponding genomic region of M. extorquens suggested that the gene clusters predicted to code for enzymes involved in H4MPT/MFR-mediated C1 transfer reactions are organized differently in both organisms. Indeed, the genes encoding Fae, Mch, and MtdB are flanked by genes that can be identified in homologous positions in the genomes of M. extorquens and Methylococcus capsulatus (Bath) (57). However, in contrast to the genomes of M. extorquens and M. capsulatus (Bath), genes encoding the formyltransferase/hydrolase complex (FhcA to -D) are missing from the M. acidiphila genomic fragment.

Single gene phylogenies.
Besides the pmo operon (see below), five additional gene homologs were identified to be present on the genomic fragments of both USC{alpha} and M. acidiphila B2, including USC{alpha} ORFs 30, 42, 54, 56, and 63 (Table 1). ORFs 42, 54, and 56 contained significant phylogenetic information. The other two genes (ORFs 30 and 63) were unreliable markers for phylogenetic inference because different paralogous gene copies were identified in various alphaproteobacterial genomes.


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  		TABLE 1. Putative functions of gene homologs identified on genomic fragments of USC{alpha} and M. acidiphila B2

 
The data sets used for treeing analysis of ORFs 42, 54, and 56 differed, but all three phylogenies contained, in addition to the ORFs from USC{alpha} and M. acidiphila, gene homologs of B. japonicum and R. palustris. This allowed us to resolve the phylogenetic relationships within this quartet in more detail. Although the branching patterns of all three trees showed some inconsistencies with the 16S rRNA phylogeny, the alphaproteobacterial sequences were always recovered as a monophyletic clade. USC{alpha} and M. acidiphila B2 clustered tightly together in all three trees. Likewise, B. japonicum clustered closely together with R. palustris. Also, the trees of ORFs 42 and 56 suggest a specific relationship between USC{alpha}/M. acidiphila and B. japonicum/R. palustris among the alphaproteobacterial sequences used for tree construction (Fig. 3). In these two trees, USC{alpha} is separated from M. acidiphila B2 by similar evolutionary distances to those that separate B. japonicum from R. palustris. Assuming that both genes evolve at similar rates in USC{alpha}, M. acidiphila, B. japonicum, and R. palustris, it can be hypothesized that the 16S rRNA gene sequence dissimilarity between USC{alpha} and M. acidiphila B2 is similar to that between B. japonicum and R. palustris and thus in a range that separates members of closely related genera.



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  		FIG.3. Phylogenetic dendrograms constructed for the deduced amino acid sequences of ORFs 42, 54, and 56 (Table 1). The shaded boxes indicate the highly similar branching patterns observed between the three phylogenies for USC{alpha}, M. acidiphila B2, B. japonicum, and R. palustris. {alpha} indicates the branch leading to the alphaproteobacterial clade. The numbers of amino acid positions used for tree construction were 220 (ORF42), 135 (ORF54), and 324 (ORF56). Phylogenies were constructed using a maximum frequency filter of 25% (ORFs 42 and 54) or 35% (ORF56). The dendrograms are consensus trees of phylogenies constructed using different approaches, including distance-based and position-specific algorithms (neighbor joining, Treepuzzle, and ProtML). An exception is the ORF42 gene homolog that was obtained from M. palustris K. Due to its shorter length, its deduced amino acid sequence was inserted into the tree according to maximum parsimony criteria, without allowing changes in the existing tree topology, using the appropriate function of the ARB software. Bar, 0.1 substitutions per amino acid position.

 
Finally, the PCR primers ORF42-F (5'-ATGATTCCGHATCGAGAACTC-3') and ORF42-R (5'-TRTTGAYGCCRTTGGTGGC-3') were designed to target conserved regions of ORF42. They enabled us to obtain a 511-bp fragment from Methylocella palustris K but not from Methylocystis strain SC2 or Beijerinckia indica subsp. indica (the PCR approach is not described in detail). The position of the sequenced M. palustris gene fragment in the phylogeny of ORF42 (Fig. 3) agrees well with the position of M. palustris in the 16S rRNA tree.

Comparative analysis of genes encoding pMMO. (i) Structure and phylogeny of the pmo operon.
The pmo genes of both USC{alpha} and M. acidiphila B2 are arranged as an operon in the order pmoCAB. This finding confirms the previous conclusion that the structural organization of the pmo operon is highly conserved among methanotrophs, as exemplarily shown for the gammaproteobacterial type I MB M. capsulatus (Bath) and Methylomicrobium album BG8 (50, 53) as well as the alphaproteobacterial type II MB Methylosinus trichosporium OB3b and Methylocystis sp. strain M (21). In addition to duplicate copies of the conventional pmo operon (pmo1), a novel sequence-divergent type of pmo operon (pmo2) has recently been reported to occur in many type II MB of the Methylosinus/Methylocystis group. An analysis of this paralogous copy of the pmo1 operon underscored the conserved nature of the pmoCAB gene arrangement (43).

The data set of fully sequenced pmo and amo operons enabled a phylogenetic analysis of concatenated pmoCAB and amoCAB sequences. Regardless of the phylogenetic method, parameters, and models used for tree construction, the pmoCAB sequences of USC{alpha} and M. acidiphila B2 grouped together, at both the nucleic acid (data not shown) and the deduced amino acid levels (Fig. 4). The branching pattern of the trees corresponded well with those computed in previous studies for partial PmoA sequences, with one major exception. In PmoA-based phylogenies, USC{alpha} and M. acidiphila B2 formed a common branch, together with the conventional PmoA1 sequences of the Methylosinus/Methylocystis group. The novel PmoA2 sequences formed a separate cluster (55). In contrast to the phylogeny of partial PmoA sequences, the concatenated PmoCAB sequences of USC{alpha} and M. acidiphila B2 branch clearly separately from a lineage defined, on the one hand, by PmoCAB1 of type II MB, and on the other hand, by PmoCAB2 of Methylocystis sp. strain SC2 (Fig. 4). This change in the branching pattern was due to the inclusion of PmoB/AmoB in the tree construction (data not shown).



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  		FIG. 4. Phylogenetic dendrogram constructed for the deduced amino acid sequences of concatenated pmoCAB/amoCAB sequences. The tree was derived by maximum likelihood analysis but was also confirmed by TreePuzzle and neighbor-joining methods. Bar, 0.1 substitutions per amino acid position.

 
(ii) Features of the inferred pMMO.
pMMO is composed of three subunits, encoded by pmoB ({alpha}, ~45 kDa), pmoA (ß, ~26 kDa), and pmoC ({gamma}, ~23 kDa) (39), with each containing predicted membrane-spanning helices (43). A comparison of the predicted secondary structures with those reported previously for the derived Pmo proteins of type I and type II MB (21, 39, 43) revealed a high degree of similarity (data not shown). For both USC{alpha} and M. acidiphila B2, the same numbers of membrane-spanning helices were predicted for the derived PmoCAB subunits (numbers are given in parentheses) PmoA (6), PmoB (3), and PmoC (6).

(iii) Identification of a conserved ORF downstream of pmoB.
An interesting finding is ORF45 on the USC{alpha} genomic fragment. It exhibits high sequence similarities to gene homologs present in the downstream region of amoB in autotrophic nitrifiers, and it is located in a homologous position, directly downstream of pmoB (Fig. 2). Genes homologous to ORF45 have been previously reported for Nitrosomonas europaea, Nitrosospira sp. strain NpAV, and Nitrosococcus oceani (ORF4) (40). In N. europaea, an additional gene homolog has been identified directly downstream of ORF4 (ORF NE2060), which we refer to as ORF5.

In Methylosinus trichosporium OB3b, a gene homolog of ORF45 is located directly downstream of pmoB (ORFD). Besides M. trichosporium OB3b, no other type II MB has yet been reported to possess a gene homolog of ORFD. Also, no gene with significant similarity to ORF45 could be identified on the 101-kb genomic fragment of M. acidiphila B2. However, BLAST similarity searches identified a single homologous copy of this gene (MCA2130) on the complete genome of M. capsulatus (Bath). In contrast to ORFs 45 and D, this gene homolog is located separately from the duplicate copies of the pmo operon.

Based on both similarity values and phylogenetic analyses, the amino acid sequence deduced from ORF45 exhibited the highest similarities to those of ORF4 and ORF5 from N. europaea, but especially to that of ORF5 (57% similarity, 40% identity). Both upstream and downstream of ORF45, factor-independent terminators could be identified on the USC{alpha} genomic fragment. In addition, a Shine-Dalgarno sequence was detected upstream of ORF45. However, no putative promoter region could be identified. The evolutionarily conserved nature of both the deduced amino acid sequence and the location immediately downstream of pmoCAB and amoCAB suggests a functional significance of ORF45/ORFD (MB) and ORF4/ORF5 (nitrifiers), respectively.

Final remarks.
The collective results of both signature-based phylogenetic mapping and comparative genomic analysis confirmed the alphaproteobacterial origin of USC{alpha}. The identification of gene homologs on the genomic fragments of USC{alpha} and M. acidiphila B2 enabled the reconstruction of three independent gene phylogenies. Their branching patterns provided proof of a close phylogenetic relationship between USC{alpha} and M. acidiphila B2.

Very recently, the expression of pmoA genes from USC{alpha} methanotrophs in acidic forest soil with ambient methane mixing ratios was demonstrated (36). This finding, in combination with our detection of a complete pmoCAB operon in the genome of USC{alpha} methanotrophs, provides strong evidence that high-affinity methanotrophy involves the known type of pMMO. The putative presence of pMMO in USC{alpha} methanotrophs might have been expectable since a pmoA-based diversity survey of MB in upland soils led to the first detection of USC{alpha} (27). However, the highly conserved nature of the secondary structure predicted for the pMMO of USC{alpha} and the small number of substitutions among 25 amino acid residues that had been identified in the pMMO of M. capsulatus (Bath) as possibly being involved in metal binding and/or the formation of a catalytic center (37) appear surprising. The only peculiarity among the 25 residues was the replacement of aspartate by asparagine at position 45 of PmoC. Thus, the molecular basis for high-affinity methane oxidation and the functional role of USC{alpha} ORF45 will have to be elucidated in future studies.


   ACKNOWLEDGMENTS
 
This research was conducted as part of the Network Göttingen "Genome research on bacteria" (GenoMik) financed by the German Federal Ministry of Education and Research (contract 031U213A).

We are very grateful to Sabrina Patzak, Julia Diehl, and Sonja Fleissner for their technical support. We especially thank Kazunari Kawase and Tokio Kozuki (Xanagen Inc.) for their contribution to the XanaPGMap analyses. We thank Svetlana N. Dedysh (S. N. Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow) for providing Methylocapsa acidiphila strain B2.


   FOOTNOTES
 
* Corresponding author. Mailing address: Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Str., D-35043 Marburg, Germany. Phone: 49-6421-178720. Fax: 49-6421-178809. E-mail: liesack{at}staff.uni-marburg.de. Back


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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
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Applied and Environmental Microbiology, November 2005, p. 7472-7482, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.7472-7482.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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J. Bacteriol. Microbiol. Mol. Biol. Rev. Eukaryot. Cell All ASM Journals