หน้าหลัก

NA; Lane 2, digested with ApaI; Lane 3, digested with MluI; Lane 4, concatenated NA; Lane 2, digested with ApaI; Lane 3, digested with MluI; Lane 4, concatenated lambda marker (fragment sizes, from bottom in Kb, are 48.5, 97, 145.5, 194, 242.5, 291, 339.5, 388 and 436.5). Panel B: Maps of ApaI and MluI restriction endonuclease cleavage sites within the AbM4 genome.A GAMOLA/ARTEMIS [31,32] software suite was used to manage genome annotation. Proteinencoding open reading frames (ORFs) were identified using the ORF-prediction program Glimmer [33] and BLASTX [34,35]. A manual inspection was performed to verify or, if necessary, redefine the start and stop codons of each ORF. Assignment of protein function to ORFs was performed manually using results from the following sources; BLASTP [34] to both a non-redundant protein database provided by the National Centre for Biotechnology Information (NCBI) [36] and clusters of orthologous groups (COG) database [37]. HMMER [38] was used to identify protein motifs to both the PFAM [39] and TIGRFAM [40] libraries. TMHMM [41,42] was used to predict transmembrane sequences, and SignalP, versionGenome annotation4.1 [43] was used for the prediction of signal peptides. Ribosomal RNA genes were detected on the basis of BLASTN searches to a custom GAMOLA ribosomal database. Transfer RNA genes were identified using tRNAscan-SE [44]. Miscellaneouscoding RNAs were identified using the Rfam database [45] utilizing the INFERNAL software package [46]. The AbM4 genome sequence was prepared for NCBI submission using Sequin [47]. The adenine residue of the start codon of the Cdc6-1 replication initiation protein (Abm4_0001) gene was chosen as the first base for the AbM4 genome. The nucleotide sequence of the Methanobrevibacter sp. AbM4 chromosome has been deposited in Genbank under accession number CP004050.Standards in Genomic SciencesThe genome of Methanobrevibacter sp. AbM4 consists of a single 1,998,189 bp circular chromosome with an average G+C content of 29 . A total of 1,730 genes were predicted, 1,671 of which were proteincoding genes. A putative function was assigned to 1,258 of the protein-coding genes, while the remaining protein coding genes were annotated as hypothetical proteins. The properties and statistics of the genome are summarized in Tables 3 and 4, and are compared with genomes of other sequenced gut methanogens for the order Methanobacteriales in Table 5. Comparative analysis of the orfeomes of the rumen methanogen genome sequences, AbM4, Methanobrevibacter ruminantium M1 and the draft genome sequence of Methanobrevibacter sp. JHI [48], reveal that their gene content is largely comparable, particularly AbM4 and JHI (Figure 4). This suggests that the central metabolism and the methanogenesis pathway of these strains are similar. Methanobrevibacter sp. AbM4 is a hydrogenotrophic methanogen and the genes involved in the methanogenesis pathway, and associated functions are shown in Figure 5. The presence or absence of these genes is indicated within complete genomes of gut methanogens of the order Methanobacteriales. The methane formation pathway in AbM4 is very similar to that of M1 and each of the 7 enzymatic steps expected for the reduction of CO2 (or formate) through to methane using H2 is present. AbM4 and M1 are distinguished from the human gut methanogens, Methanobrevibacter smithii PS [49] and Methanosphaera stadtmanae MCB-3 [50], by the absence of the methanol:cobalamin methyltransferase genes (mtaABC) which mediate methanol utilization in these organisms. Hydrogenotroph.