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Methanogenesis, the biological production of methane, plays a pivotal role in the global carbon cycle and contributes significantly to global warming. The majority of methane in nature is derived from acetate. Here we report the complete genome sequence of an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are the most metabolically diverse methanogens, thrive in a broad range of environments, and are unique among the Archaea in forming complex multicellular structures. This diversity is reflected in the genome of M. acetivorans.

Carbonic anhydrases catalyze the reversible hydration of CO(2) [CO(2)+H(2)Oright harpoon over left harpoon HCO(3)(-)+H(+)]. Since the discovery of this zinc (Zn) metalloenzyme in erythrocytes over 65 years ago, carbonic anhydrase has not only been found in virtually all mammalian tissues but is also abundant in plants and green unicellular algae. The enzyme is important to many eukaryotic physiological processes such as respiration, CO(2) transport and photosynthesis. Although ubiquitous in highly evolved organisms from the Eukarya domain, the enzyme has received scant attention in prokaryotes from the Bacteria and Archaea domains and has been purified from only five species since it was first identified in Neisseria sicca in 1963. Recent work has shown that carbonic anhydrase is widespread in metabolically diverse species from both the Archaea and Bacteria domains indicating that the enzyme has a more extensive and fundamental role in prokaryotic biology than previously recognized. A remarkable feature of carbonic anhydrase is the existence of three distinct classes (designated alpha, beta and gamma) that have no significant sequence identity and were invented independently. Thus, the carbonic anhydrase classes are excellent examples of convergent evolution of catalytic function. Genes encoding enzymes from all three classes have been identified in the prokaryotes with the beta and gamma classes predominating. All of the mammalian isozymes (including the 10 human isozymes) belong to the alpha class; however, only nine alpha class carbonic anhydrase genes have thus far been found in the Bacteria domain and none in the Archaea domain. The beta class is comprised of enzymes from the chloroplasts of both monocotyledonous and dicotyledonous plants as well as enzymes from phylogenetically diverse species from the Archaea and Bacteria domains. The only gamma class carbonic anhydrase that has thus far been isolated and characterized is from the methanoarchaeon Methanosarcina thermophila. Interestingly, many prokaryotes contain carbonic anhydrase genes from more than one class; some even contain genes from all three known classes. In addition, some prokaryotes contain multiple genes encoding carbonic anhydrases from the same class. The presence of multiple carbonic anhydrase genes within a species underscores the importance of this enzyme in prokaryotic physiology; however, the role(s) of this enzyme is still largely unknown. Even though most of the information known about the function(s) of carbonic anhydrase primarily relates to its role in cyanobacterial CO(2) fixation, the prokaryotic enzyme has also been shown to function in cyanate degradation and the survival of intracellular pathogens within their host. Investigations into prokaryotic carbonic anhydrase have already led to the identification of a new class (gamma) and future research will undoubtedly reveal novel functions for carbonic anhydrase in prokaryotes.

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Carbonic anhydrases catalyze the reversible hydration of CO2 and are ubiquitous in highly evolved eukaryotes. The recent identification of a third class of carbonic anhydrase (␥ class) in a methanoarchaeon and our present finding that the ␤ class also extends into thermophilic species from the Archaea domain led us to initiate a systematic search for these enzymes in metabolically and phylogenetically diverse prokaryotes. Here we show that carbonic anhydrase is widespread in the Archaea and Bacteria domains, and is an ancient enzyme. The occurrence in chemolithoautotrophic species occupying deep branches of the universal phylogenetic tree suggests a role for this enzyme in the proposed autotrophic origin of life. The presence of the ␤ and ␥ classes in metabolically diverse species spanning the Archaea and Bacteria domains demonstrates that carbonic anhydrases have a far more extensive and fundamental role in prokaryotic biology than previously recognized. Carbonic anhydrase is a zinc-containing enzyme catalyzing the reversible hydration of CO 2 [CO 2 ϩ H 2 O º HCO 3 Ϫ ϩ H ϩ ]. Since the discovery of the enzyme in bovine erythrocytes in 1933 (1), isozymes have been found in virtually all mammalian tissues and cell types, where they function in CO 2 transport and other physiological processes (2). Carbonic anhydrases are also abundant in plants and unicellular green algae, where they are essential for photosynthetic CO 2 fixation (3). Although they are ubiquitous in highly evolved organisms from the Eukarya, the extent to which carbonic anhydrases occur in the Archaea and Bacteria domains is unknown; the enzyme has been purified from only five prokaryotic species (4-8) since 1963, when it was first identified in Neisseria sicca (9).All carbonic anhydrases are divided into three distinct classes (␣, ␤, and ␥) that evolved independently and have no sequence homology (10). Carbonic anhydrases from mammals (including the 10 active human isoforms) (10, 11), together with the two periplasmic enzymes from the unicellular green alga Chlamydomonas reinhardtii (12, 13), belong to the ␣ class. The ␤ class is comprised of enzymes from the chloroplasts of both monocotyledonous and dicotyledonous plants (10). Within the Bacteria domain, the enzymes purified from Neisseria gonorrhoeae and Escherichia coli also belong to the ␣ and ␤ classes (14, 15), respectively. Recently, a gene encoding a putative ␤ type carbonic anhydrase in the methanoarchaeon Methanobacterium thermoautotrophicum was expressed in E. coli and found to encode a thermostable carbonic anhydrase (16). A carbonic anhydrase purified from the archaeon Methanosarcina thermophila is decidedly distinct from the ␣ and ␤ classes, and is the prototype of a different class, the ␥ class (17).Our detection of documented ␤ and ␥ carbonic anhydrases in the methanoarchaea prompted us to investigate the distribution of carbonic anhydrases in the Archaea and Bacteria domains. The results indicate not only that carbonic anhydrases are widely distributed in prokaryotes, but that th...