The Methanosarcina barkeri genome: comparative analysis with Methanosarcina acetivorans and Methanosarcina mazei reveals extensive rearrangement within methanosarcinal genomes

Abstract

We report here a comparative analysis of the genome sequence of Methanosarcina barkeri with those of Methanosarcina acetivorans and Methanosarcina mazei. The genome of M. barkeri is distinguished by having an organization that is well conserved with respect to the other Methanosarcina spp. in the region proximal to the origin of replication, with interspecies gene similarities as high as 95%. However, it is disordered and marked by increased transposase frequency and decreased gene synteny and gene density in the distal semigenome. Of the 3,680 open reading frames (ORFs) in M. barkeri, 746 had homologs with better than 80% identity to both M. acetivorans and M. mazei, while 128 nonhypothetical ORFs were unique (nonorthologous) among these species, including a complete formate dehydrogenase operon, genes required for N-acetylmuramic acid synthesis, a 14-gene gas vesicle cluster, and a bacterial-like P450-specific ferredoxin reductase cluster not previously observed or characterized for this genus. A cryptic 36-kbp plasmid sequence that contains an orc1 gene flanked by a presumptive origin of replication consisting of 38 tandem repeats of a 143-nucleotide motif was detected in M. barkeri. Three-way comparison of these genomes reveals differing mechanisms for the accrual of changes. Elongation of the relatively large M. acetivorans genome is the result of uniformly distributed multiple gene scale insertions and duplications, while the M. barkeri genome is characterized by localized inversions associated with the loss of gene content. In contrast, the short M. mazei genome most closely approximates the putative ancestral organizational state of these species.

FIG. 1.

Thin-section electron micrographs of M. barkeri Fusaro. Cells cultured in low-saline medium (A) grow as multicellular aggregates embedded in a methanochondroitin matrix (mc). Cells cultured in marine medium (B) grow as single cells without the methanochondroitin outer layer. When grown with hydrogen, gas vesicles (gv) are observed in some cells. Bar, 1.0 μm.

FIG. 2.

Venn diagram for three Methanosarcina sp. genomes, indicating the numbers of genome features with at least 80% nucleotide identity. Numbers in parentheses are the numbers of common paralog groups, and adjacent numbers are the gene counts for the contributing organisms. Values for M. barkeri are given in bold type, values for M. mazei are given in italics, and values for M. acetivorans are underlined.

FIG. 3.

Asymmetric fragmentation in M. barkeri. The top panel shows cumulative deviations from the mean in the M. barkeri genome for synteny with respect to M. acetivorans (a), M. mazei (b), or intergenic interval (c). The cumulative transposon count is superimposed (d). The bottom panel shows uniformly scaled BLASTN cross plots of the M. barkeri chromosome with those of M. mazei and M. acetivorans, with the origin regions circled.

FIG. 4.

Sequence alignment of the M. barkeri ORI A self-complementary region (1190360 to 1191059) using BLASTN. Numbers at ends of the bottom sequences are genomic locations.

FIG. 5.

Proposed mechanism for conserved repetitive sequence to provide bubbled-out repeat motifs for initiation of replication. Pairs of quasi-stable bubbles might occur in pairs at arbitrary locations on opposite strands. All motifs are essentially identical.

FIG. 6.

M. acetivorans is elongated due to distributed duplication events. Uniformly scaled, comparable 2-Mbp BLASTN cross plot comparisons reveal deviation from the dotted 45° slope of identity in M. acetivorans, with respect to M. barkeri (upper left) and M. mazei (lower right). The anticipated orthogonal relationship is observed in the M. barkeri/M. mazei comparison (upper right). The self-comparison of M. acetivorans (lower left) reveals multiple nonidentical repeat sequences characteristic of the multiple transposase genes found in all three genomes. M. barkeri comparisons show multiple strand inversions and transpositions relative to the other two genomes.