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Methanoculleus marisnigri Romesser et al. 1981 is a methanogen belonging to the order Methanomicrobiales within the archaeal phylum Euryarchaeota. The type strain, JR1, was isolated from anoxic sediments of the Black Sea. M. marisnigri is of phylogenetic interest because at the time the sequencing project began only one genome had previously been sequenced from the order Methanomicrobiales. We report here the complete genome sequence of M. marisnigri type strain JR1 and its annotation. This is part of a Joint Genome Institute 2006 Community Sequencing Program to sequence genomes of diverse Archaea.
Methanoculleus marisnigri is a methanogen belonging to the order Methanomicrobiales, and strain JR1 is the type strain of this species. When it was first isolated, this organism was named Methanogenium marisnigri , but then later it was transferred to the genus Methanoculleus . The type strain was isolated from sediment of the Black Sea, while another strain was isolated from an anaerobic digestor . Other species of Methanoculleus have been isolated from different types of anaerobic digestors and marine and freshwater sediments (reviewed in ).
Methanogens have been divided into two groups known as Class I and Class II based on phylogeny . Class I includes the orders Methanococcales, Methanobacteriales, and Methanopyrales, which use H2/CO2 or formate as substrates for methanogenesis, although some can also use alcohols as electron donors. Class II includes the orders Methanosarcinales and Methanomicrobiales. Some of the Methanosarcinales are capable of using various methyl compounds as substrates for methanogenesis including acetate, methylamines, and methanol, but Methanomicrobiales are restricted to the same substrates as the Class I methanogens . Therefore Methanomicrobiales are phylogenetically closer to Methanosarcinales but physiologically more similar to Class I methanogens, making them an interesting target for genome sequencing.
In a 2006 Community Sequencing Program (CSP) project, we proposed sequencing two members of the order Methanomicrobiales: M. marisnigri and Methanocorpusculum labreanum. Previously only one genome was available from this order, that of Methanospirillum hungatei. M. marisnigri and M. labreanum are phylogenetically distant from each other and from M. hungatei (Figure 1), and they represent the three phylogenetic families within the order Methanomicrobiales. We report here the sequence and annotation of M. marisnigri type strain JR1.
Methanoculleus marisnigri JR1 was isolated from Black Sea sediment at a depth of 0.5-20 cm. The enrichment medium consisted of 30% distilled water and 70% sea water with the addition of acetate, formate, trypticase, yeast extract, vitamin solution, trace mineral solution, and volatile fatty acid solution . Cells were maintained in serum vials under an atmosphere of 80% H2 and 20% CO2 by a modification of the Hungate technique . The physiological characteristics of M. marisnigri were described as follows . The cells were irregular cocci with peritrichous flagella. The cell wall was composed of glycoprotein and lacked peptidoglycan. The optimal growth temperature was 20-25°C with growth observed between 15 and 45°C. The optimal pH for growth was 6.4 with a range of 6.0-7.5. The optimal salt concentration for growth was around 0.1 M NaCl, and growth was observed between 0.0 and 0.7 M NaCl. Neither acetate nor yeast extract was stimulatory for growth. Trypticase was required, and it could not be replaced by Casamino acids or other peptide mixtures. Coenzyme M and Coenzyme F420 were both detected in M. marisnigri. Growth was observed with H2/CO2 or formate but not with acetate or methanol. M. marisnigri was subsequently shown to grow with secondary alcohols as the electron donor for methanogenesis . The physiological and morphological features of M. marisnigri are presented in (Table 1).
Methanoculleus marisnigri was selected for sequencing based upon its phylogenetic position relative to other methanogens of the order Methanomicrobiales. It is part of a Joint Genome Institute 2006 Community Sequencing Program project that included six archaeal genomes selected for their phylogenetic diversity. A summary of the project information is shown in Table 2. The complete genome sequence was finished in February, 2007. The GenBank accession number for the project is CP000562. The genome project is listed in the Genomes OnLine Database (GOLD)  as project Gc00512. Sequencing was carried out at the Joint Genome Institute (JGI) Production Genomics Facility (PGF) in Walnut Creek, California. Quality assurance using Phred [19,20] was done by JGI-Stanford. Finishing was done by JGI-Los Alamos National Laboratory (LANL). Annotation was done by JGI-Oak Ridge National Laboratory (ORNL) and by JGI-PGF.
The methods for DNA isolation, genome sequencing and assembly for this genome have previously been published .
Protein-coding genes were identified using a combination of CRITICA  and Glimmer  followed by a round of manual curation using the JGI GenePRIMP pipeline . GenePRIMP points out cases where gene start sites may be incorrect based on alignment with homologous proteins. It also highlights genes that appear to be broken into two or more pieces, due to a premature stop codon or frameshift, and genes that are disrupted by transposable elements. All of these types of broken and interrupted genes are labeled as pseudogenes. Genes that may have been missed by the gene calling programs are also identified in intergenic regions. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant protein sequence database and the UniProt , TIGRFAMs , Pfam , PRIAM , KEGG , COG , and InterPro  databases. If a gene has more than one significant hit against the domain databases, then all nonoverlapping domains are recorded. Signal peptides were identified with SignalP , and transmembrane helices were determined with TMHMM . CRISPR elements were identified with the CRISPR Recognition Tool (CRT) . Paralogs are hits of a protein against another protein within the same genome with an e-value of 10-2 or lower. More details about gene annotation procedures can be found at the data processing page of the Integrated Microbial Genomes website. The tRNAScan-SE tool  was used to find tRNA genes. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform .
The genome of M. marisnigri JR1 consists of a single circular chromosome (Figure 2 and Table 3). In comparison with other methanogens, the genome size of 2.48 Mbp is larger than those of Class I methanogens, which tend to be 1.6-1.8 Mbp, but smaller than the genomes of Methanosarcina species and Methanospirillum hungatei, which range between 3.5 and 5.8 Mbp. The G+C percentage of M. marisnigri is 62.1%, the highest among sequenced methanogens. The genome contains 2,560 genes of which 2,506 are protein-coding genes and the remaining 54 are RNA genes. There were only 17 pseudogenes identified, constituting 0.68% of the total genes. In total, 1633 protein-coding genes (65.2%) were assigned a function, with the remaining annotated as hypothetical proteins. The percentage of genes with signal peptides (14.0%) is quite high compared to other methanogens, although several methanogens have similar percentages. The properties and statistics of the genome are summarized in Table 3 and genes belonging to COG functional categories are listed in Table 4.
The genome sequence of M. marisnigri JR1 shows some similarities to Class I methanogens and some to Methanosarcinales but also has some unique features. In common with Class I methanogens, M. marisnigri uses a partial reductive TCA cycle to synthesize 2-oxoglutarate, and it has the Eha membrane-bound hydrogenase. Similar to Methanosarcinales, M. marisnigri has the Ech membrane-bound hydrogenase. A unique feature of M. marisnigri and the other Methanomicrobiales is the presence of anti- and anti-anti-sigma factors, which is surprising as Archaea do not use sigma factors. These anti- and anti-anti-sigma factors must have developed a new function in the Archaea. Phylogenetic analysis of methanogenesis and cofactor biosynthesis enzymes suggests that Methanomicrobiales form a group distinct from other methanogens, and therefore methanogens can be split in to three classes .
There are also differences among the Methanomicrobiales. For instance, M. marisnigri is the only one of the three to have the F420-nonreducing hydrogenase, and it is the only one of the three to lack the 14-subunit Mbh membrane-bound hydrogenase. This has implications for the mechanism of methanogenesis: M. marisnigri may couple Coenzyme M-Coenzyme B heterodisulfide reduction to the first step of methanogenesis in the cytoplasm, similar to Class I methanogens , while the other Methanomicrobiales may couple heterodisulfide reduction to generation of a membrane ion gradient .
This work was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under Contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under Contract No. DE-AC02-06NA25396. M. L. was supported by the Department of Energy under contract DE-AC05-000R22725. M. S.-L., and W. B. W. were supported by DOE contract number DE-FG02-97ER20269.