H2 CO2

H+

+

H2 CO2

CO2

+

+

CH4

H3C C S

O

CoA

Acetyl CoA

CO

H3C S CoM

Methyl–CoMMethanol

Acetate

Methylamines

H2O

CH2OPO32–

HC

HC

CH

HC

C

OH

OH

HO

OH

O O–

6-phosphogluconate

O

CH2OH

Hexose

CH2OPO32–

HC

C

OH

O H

Glyceraldehyde-3-phosphate

CH3

C O

O–O

Pyruvate

CH3

C

H

O

Acetaldehyde

CH3

HC

H

OH

Ethanol

c Ethanologen

b Methanogena Hydrogenogen

Pyruvatedecarboxylase

CODH/ACS

Alcoholdehydrogenase

H3C C S

O

CoA

Acetyl CoA

CO

CODH I

CODH III

H+

H+

GENOME WATCH

A more convenient truthHelena Seth-Smith

The current concern about global warming and potential future energy sources is forcing governments and researchers to think about fuel alternatives. Microorganisms have the potential to make various fuels, including ethanol, hydrogen and methane. The ability to exploit this potential is dependent on a deep understanding of the underlying biological processes. Genome sequencing and interpretation can contribute to understanding these processes, as the four genomes discussed here show.

Microorganisms live in a wide range of habi-tats, and have enormously varied metabolic repertoires. The waste products of some microorganisms can be the energy sources for others, resulting in synergistic cohabita-tion. For example, carbon monoxide is toxic to humans but can be used as a primary carbon and energy source by hydrogen-producing organisms that thrive at high temperatures in hydrothermal vents. Hydrogen that is pro-duced by hydrogenogens (FIG. 1a) can be used as a source of energy by other microorganisms, including methanogens (FIG. 1b), that in turn produce carbon monoxide as a waste product.

Carboxydothermus hydrogenoformans is a Gram positive, anaerobic bacterium that oxidizes carbon monoxide to produce car-bon dioxide, which can be fixed into bio-mass, and releases hydrogen as a waste product. Strain Z-2901 was isolated from a hot spring in Russia. Its genome is 2.4 Mb, with 2,646 predicted coding sequences (CDSs)1 (TABLE 1).

Reactions involving carbon monoxide (CO) are generally catalysed by nickel–iron carbon monoxide dehydrogenase com-plexes (CODHs). Five CDSs (cooS I–V) with homology to the catalytic subunit of anaerobic CODHs were identified in the

genome of C. hydrogenoformans. The first of these (cooS-I) is found in a CDS cluster with high identity to a characterized cluster from Rhodospirillum rubrum. These CDSs encode a membrane-bound complex that pumps protons across the membrane as CO is oxi-dized. The resulting proton gradient is used to generate ATP, the common energy cur-rency of the cell. The CDS cluster contain-ing cooS-III produces a complex (CODH/ACS acetyl-coA synthase) involved in car-bon fixation, producing acetyl-CoA. The acetyl-CoA can be used to make more ATP, or in biosynthetic pathways. The cooS-IV cluster is implicated in oxidative stress, and

the membrane-associated cooS-II cluster might be involved in NADPH generation. The cooS-V is the most divergent of the five, and its function is unknown.

Analysis of the genome revealed many unexpected homologues of sporulation-related proteins. Experimental work subse-quently identified an endospore-like state of C. hydrogenoformans, which is induced when cells are stressed. The set of sporula-tion-associated CDSs differs from those previously characterized in Bacillus or Clostridia species, so might help to define a minimal set of genes that are involved in endospore formation.

Figure 1 | Metabolic pathways of biofuel producers. Selected parts of metabolic pathways of hydrogenogens (a), methanogens (b) and ethanologens (c) are shown. Red arrows indicate steps in the pathway that are linked to proton pumping across the cell membrane.

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a b

mc

gv

C. hydrogenoformans was characterized as an obligate CO autotroph, unable to use alternative sources of carbon. Consistent with this, the genome lacks complete path-ways for sugar breakdown. However, it does contain transporters for several alternative carbon sources, and heterotrophic growth on some of these substrates has been shown, albeit at a slow rate.

Performing practically the opposite reaction to that of C. hydrogenoformans are two methanogens whose genomes have recently been sequenced: Methanococcus maripaludis2 and Methanosarcina barkeri3. These Archaea produce methane from sev-eral substrates (FIG. 1b), and are both geneti-cally tractable, making them appealing for research. The genome of M. maripaludis is much smaller than that of M. barkeri, at 1.66 Mb as opposed to 4.84 Mb (TABLE 1). The larger genome reflects the nutritional versatility of M. barkeri, as it can grow by carbon dioxide reduction, methyl reduc-tion, acetate utilization or methylotrophic breakdown of various substrates, whereas M. maripaludis grows using carbon diox-ide, formate or acetate. Both species can fix nitrogen.

The M. maripaludis genome encodes the full pathway for carbon dioxide conversion to methane, including both selenocysteine and cysteine versions of some of the enzymes. Formate dehydrogenase, which is another selenoprotein that is present, allows this strain to grow on formate. Also encoded is one CODH/ACS cluster, which catalyses the fixation of carbon dioxide into acetyl-CoA and biomass. Six hydrogenases, grouped into three classes, are present to couple the methanogenesis/biosynthesis to the creation of ion gradients for the pro-duction of ATP. In terms of sugar metabo-lism, pathways responsible for glycolysis, gluconeogenesis and glycogen synthesis are all present, as are full amino-acid and nucleotide-biosynthesis pathways.

No clear archaeal origin of replication was identified in the M. maripaludis genome, lead-ing to the possibility that there are multiple ori-gins of replication that enable the chromosome number to increase to 3–15 copies per cell. Two possible origins of replication were identi-fied in the genome of M. barkeri, in common with other methanosarcinal genomes.

Comparing the genome of M. barkeri with the genomes of Methanosarcina acetivo-rans and Methanosarcina mazei, a substantial amount of genome rearrangement is apparent, with the most conserved gene order occurring around the origin region. The rearrangements seem to have been driven by replication-asso-ciated inversion, together with recombination between transposable elements. In addition, M. barkeri carries a plasmid that is much larger than any previously identified methanosarcinal extrachromosomal element.

All the methanogenic pathways in which M. barkeri is known to participate are repre-sented by CDSs in the genome. These include

a CODH/ACS complex that is involved in the acetate fermenting (aceticlastic) lifestyle of this organism.

Two CDSs with identity to bacterial N-acetylmuramic acid synthesis genes were found in the genome of M. barkeri. These genes have not been found in archaeal genomes before, although as no cell-wall muramic acid was detected the genes may not be expressed. Another operon that seems to have been acquired by lateral gene transfer from bacteria comprises CDSs for a P450-ferredoxin–ferredoxin reduct-ase system. These systems are commonly used for xenobiotic detoxification in aero-bic environments, so the presence of this operon in an obligate anaerobe is curious. Gas vesicles, which are possibly involved in motility in response to hydrogen gradients, have been observed in M. barkeri (FIG. 2b). A complete operon of the genes necessary for formation of gas vesicles was identified in a 33.5 Kb region flanked by transposases,

Table 1 | General features of genomes of biofuel bacteria

Species* Carboxydothermus hydrogenoformans

Methanococcus maripaludis

Methanosarcina barkeri Zymomonas mobilis

Classification Firmicute (Gram positive) Archaea Archaea Proteobacterium (Gram negative)

Class of fuel producer

Hydrogenogen Methanogen Methanogen Ethanologen

Genome size 2.40 Mb 1.66 Mb 4.84 Mb + 36Kb plasmid 2.06 Mb

CDSs 2,646 1,722 3,680 + 18 1,998

GC content 42.0% 33.10% 39.20% 46.30%

% of genome coding 91 89 70 87

*All four species are anaerobic. CDSs, predicted coding sequences.

Figure 2 | Thin-section electron micrographs of Methanosarcina 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 that lack a methanochondroitin outer layer. When grown with hydrogen, gas vesicles (gv) are observed in some cells. Scale bars represent 1.0 µm. Reproduced with permission from REF. 3 © (2006) The American Society for Microbiology.

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intermediates have been identified, and the bacterium is capable of making all but two of its required amino acids, there must be some TCA cycle activity, albeit slow. There is also evidence that Z. mobilis ZM4 is a facultative anaerobe rather than a strict anaerobe, as the genome contains CDSs for proteins that protect against oxidative stress, and there are CDSs for many enzymes involved in the aerobic electron transport chain. Growth rates of Z. mobilis ZM4 increase slightly under aerobic conditions, which might indi-cate some oxidation of NADH, despite the absence of CDSs for two cytochromes that react with oxygen.

Z. mobilis ZM4 can tolerate concentra-tions of ethanol up to 16%. This is a useful property for bioethanol production. Several CDSs for stress-responsive chaperones and alternative sigma factors were identified that might have a role in alcohol tolerance. It would be advantageous to produce etha-nol from cheaper substrates than hexose sugars, such as pentoses, which are break-down products of plant-derived lignocel-lulose. Although Z. mobilis ZM4 does not normally metabolize pentoses, this strain has been genetically manipulated to enable it to do so. The genetic tractability of this strain is an obvious advantage for its use in industrial applications.

Although it is unlikely that any of these microbial technologies will produce enough energy to sustain society, they might sup-plement our energy production and pro-vide energy sources locally where required. Certainly, continuing research is required for the potential of these microorganisms to be realized.Helena Seth-Smith is at the Sanger Institute, Wellcome

Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. e-mail: microbes@sanger.ac.uk

doi:10.1038/nrmicro1644

1. Wu, M. et al. Life in hot carbon monoxide: the complete genome sequence of Carboxydothermus hydrogenoformans Z-2901. PLoS Genet. 1, e65 (2005).

2. Hendrickson, E. L. et al. Complete genome sequence of the genetically tractable hydrogenotrophic methanogen Methanococcus maripaludis. J. Bacteriol. 186, 6956–6969 (2004).

3. Maeder, D.L. et al. The Methanosarcina barkeri genome: comparative analysis with Methanosarcina acetivorans and Methanosarcina mazei reveals extensive rearrangement within methanosarcinal genomes. J. Bacteriol. 188, 7922–7931 (2006).

4. Seo, J.-S. et al. The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4. Nature Biotechnol. 23, 63–68 (2005).

DATABASESThe following terms in this article are linked online to:Entrez Genome Project: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genomeprjCarboxydothermus hydrogenoformans | Methanococcus maripaludis | Methanosarcina acetivorans | Methanosarcina barkeri | Methanosarcina mazei | Rhodospirillum rubrum | Zymomonas mobilisAccess to this links box is available online.

which might indicate horizontal acquisition of these genes.

In contrast to the efficient recycling of substrates and products in the examples above, Zymomonas mobilis ZM4 is a dramat-ically inefficient energy producer. Thanks to this property, however, it is an efficient producer of ethanol, which has great poten-tial as a fuel of the future. In aerobic organ-isms, one molecule of glucose can produce up to 30 molecules of ATP. However, owing to its anaerobic lifestyle and the absence of a number of enzymes that are involved in important metabolic processes, Z. mobi-lis ZM4 can only produce one molecule of ATP per molecule of glucose, with the pro-duction of two molecules of ethanol. As a result, to maintain growth, Z. mobilis ZM4 takes up sugars at a high rate and metabo-lizes them rapidly, producing large amounts of ethanol (FIG. 1c).

Z. mobilis ZM4 was isolated from sugar cane juice, and was found to be a better ethanol producer than the type strain, ZM1. This feature, in addition to its ability to be genetically manipulated, led to the sequenc-ing of the Z. mobilis ZM4 genome, the

results of which have provided insights into many of its characteristics. At 2.06 Mb, the genome is relatively small, containing 1,998 predicted CDSs4 (TABLE 1).

There are two pathways by which glucose can be catabolized anaerobically to release energy. In Z. mobilis ZM4, one of these pathways is non-functional, owing to the absence of a crucial enzyme: 6-phosphof-ructokinase. Therefore glucose can only be catabolized by the Entner-Doudoroff pathway, giving low energy yields. The product of this pathway is the ubiquitous pyruvate, which is converted to ethanol in a two-step process using pyruvate decar-boxylase (uncommon in bacteria) and an alcohol dehydrogenase. Both of these com-ponents have been identified in the genome of Z. mobilis ZM4, and microarray studies have shown them to be expressed at high levels.

The tricarboxylic acid (TCA) cycle is an important pathway in aerobic respiration, but is also crucial for the biosynthesis of amino acids and nucleotides. Z. mobilis ZM4 lacks CDSs for two of the enzymes that are involved in the TCA cycle, but as pathway

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