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TRENDS in Microbiology

Vol.12 No.1 January 2004

time a case in which different transport mechanisms are genetically linked to a conserved carbohydrate metabolic cluster. Even transporters of the unusual class of binding protein-dependent secondary transporters were found linked to this cluster. It was suggested that the different classes of transporters have evolved from secondary transporters by association with binding proteins, ATPases and phosphorylating enzymes, thereby gaining higher substrate afnities and translocation power [12]. It is possible that in this cluster we can see the subtlety by which evolution can take place at the genome level.
Acknowledgements
We thank Wil Konings for helpful discussion. T.H.P. was supported by the Netherlands Organization for Scientic Research (NWO) grant no. 805 19 046 P.

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References
nchez, J.C. et al. (1994) Activation of a cryptic gene encoding a 1 Sa kinase for L-xylulose opens a new pathway for the utilization of L-lyxose by Escherichia coli. J. Biol. Chem. 269, 29665 29669 2 Bada, J. et al. (1998) A rare 920-kilobase chromosomal inversion mediated by IS1 transposition causes constitutive expression of the yiaK S operon for carbohydrate utilization in Escherichia coli. J. Biol. Chem. 273, 8376 8381 n 3 Iba ez, E. et al. (2000) Regulation of expression of the yiaKLMNOPQRS operon for carbohydrate utilization in Escherichia coli: involvement of the main transcriptional factors. J. Bacteriol. 182, 4617 4624 n 4 Iba ez, E. et al. (2000) Role of the yiaR and yiaS genes of Escherichia coli in metabolism of endogenously formed L-xylulose. J. Bacteriol. 182, 4625 4627 5 Reizer, J. et al. (1997) Is the ribulose monophosphate pathway widely distributed in bacteria? Microbiology 143, 2519 2520 6 Plantinga, T.H. et al. (2003) Functional characterization of the

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Escherichia coli K-12 yiaMNO transport protein genes. Mol. Mem. Biol. 10.1080/09687680310001607369 (www.tandf.co.uk) Yew, W.S. and Gerlt, J.A. (2002) Utilization of L-ascorbate by Escherichia coli K-12: assignments of functions to products of the yjf-sga and yia-sgb operons. J. Bacteriol. 184, 302 306 Yasueda, H. et al. (1999) Bacillus subtillis yckG and yckF encode two key enzymes of the ribulose monophosphate pathway used by methylotrophs, and yckH is required for their expression. J. Bacteriol. 181, 7154 7160 Zhang, Z. et al. (2003) The ascorbate transporter of Escherichia coli. J. Bacteriol. 185, 2243 2250 Postma, P.W. et al. (1996) Phosphoenolpyruvate:carbohydrate phosphotransferase systems. In Escherichia coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F.C. et al., eds), pp. 1149 1174, ASM Press Driessen, A.J.M. et al. (1997) A new family of prokaryotic transport proteins: binding protein-dependent secondary transporters. Mol. Microbiol. 24, 879 883 Driessen, A.J.M. et al. (2000) Diversity of transport mechanisms: common structural principles. Trends Biochem. Sci. 25, 397 401 Rabus, R. et al. (1999) TRAP transporters: an ancient family of extracytoplasmic solute-receptor-dependent secondary active transporters. Microbiology 145, 3431 3445 Kelly, D.J. and Thomas, G.H. (2001) The tri-partite ATP-independent periplasmic (TRAP) transporters of bacteria and archaea. FEMS Microbiol. Rev. 25, 405 424 Poolman, B. and Konings, W.N. (1993) Secondary solute transport in bacteria. Biochim. Biophys. Acta 1183, 5 39 Higgins, C.F. (2001) ABC transporters: physiology, structure and mechanism - an overview. Res. Microbiol. 152, 205 210 Campos, E. et al. (2002) The gene yjfQ encodes the repressor of the yjfR-X regulon (ula) which is involved in L-ascorbate metabolism in Escherichia coli. J. Bacteriol. 184, 6065 6068 Tamames, J. (2001) Evolution of gene order conservation in prokaryotes. Genome Biol. RESEARCH0020 (www.genomebiology.com)

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| Microbial Genomics

All things great and small

Claire M. Fraser
The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, USA

The report in 2002 of a novel archaeal species, the rst representative of an unknown phylum, reminded us of the tremendous microbial diversity that remains to be discovered on this planet [1]. Nanoarchaeum equitans (the tiny archaea that rides the re ball) is a small spherical cell that grows attached to the surface of its archaeal host, Igniococcus spp. These organisms were isolated from a hot submarine vent in the Kolbeinsey ridge, north of Iceland, and can be grown in the laboratory in anaerobic cultures at 90 8C in the presence of S, H2 and CO2. It was by microscopic examination of the Igniococcus cells that their rider, N. equitans, was discovered and by which it was subsequently demonstrated that N. equitans is an obligate symbiont of Igniococcusspp. that cannot be grown alone in culture. Equally surprising was that universal primers designed to amplify ssRNA genes from bacterial and
Corresponding author: Claire M. Fraser (cmfraser@tigr.org).
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archaeal species failed to yield a product from N. equitans by the polymerase chain reaction (PCR). Subsequent comparison of the ssRNA gene sequence from N. equitans with those of other archeal species identied it as the rst member of a novel phylum, the Nanoarchaeota, which represents the most deeply branching archaeal lineage to date. Estimation of genome size by pulse eld gel electrophoresis suggested that N. equitans represents a minimal organism with a genome of , 500 kb. The much anticipated genomic sequence of N. equitans was published recently [2]. Its genome size of 490 885 bp makes this the smallest prokaryotic genome deciphered to date. Despite its small genome size, this archaeon has one of the highest gene densities reported to date, with , 95% of the genome representing predicted coding sequences and stable RNAs. Most of the genes involved in information processing (DNA replication, transcription and translation) show most similarity to their counterparts in

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Vol.12 No.1 January 2004

the Euryarchaeota. In contrast to the genomes described for other minimal microbial species that appear to be undergoing reductive evolution, N. equitans contains few pseudogenes. It is not at all surprising, given its small genome size, that N. equitans lacks essentially all known metabolic pathways and contains only a minimal number of putative transporters. It does, however, contain several proteases and peptidases that might allow it to degrade proteins that are derived from either its environment or host. Perhaps more surprising is what is present in this small genome. N. equitans contains an impressive repertoire of DNA repair enzymes, a feature not usually seen in other minimal genomes. It is probable that this full set of DNA repair genes is necessary to repair the DNA damage that occurs at its high growth temperature, although it is tempting to speculate that there might be other reasons for this capability. Another unusual feature of the N. equitans genome is the presence of a large number of split noncontiguous genes that are fused in other archaeal genomes. The splits, for the most part, occur between functional domains in the translated proteins. It has been postulated that multidomain proteins have evolved from the fusion of less complex domains [3]. The nding of several split genes, together with the placement of N. equitans in the most deeply branching archaeal lineage is consistent with its early divergence in the evolution of the Archaea. Whether other members of the phylum Nanoarchaeota exist, waiting to be discovered, remains to be determined. If they can be found, it is probable that additional genome sequence data from these smallest of microbes would reveal much about the evolution of microbial life. At the other end of the microbial spectrum is the marine planctomycete, Pirellula spp. strain 1, which has the largest circular genome of 7.145 Mbp that has been sequenced to date [4]. The only larger genomes to be found are those from Streptomyces coelicolour [5] and Streptomyces avermitilis [6], but these are linear chromsomes. Pirellula spp. are ubiquitously distributed in terrestrial and marine environments, and are important because of the roles that they play in global carbon and nitrogen cycles. They are members of the Planctomycetes, which might represent the deepest branching bacterial lineage [7]. However, this phylogenetic placement is still somewhat controversial. Planctomycetes share several unusual features including a budding mode of reproduction similar to that observed for Caulobacter crescentus, a cell wall that lacks peptidoglycan, and a membrane surrounding the chromosome. Therefore, there is much to be learned about this relatively unknown bacterial group. Given the paucity of molecular studies on Planctomycetes, it was not unexpected to nd that more than half of the predicted coding sequences do not display a signicant similarity to sequences in the available databases. What was surprising was that less than 25% of the predicted coding sequences in the Pirellula spp. genome represent gene paralogs, because in many larger prokaryotic

genomes ,30 50% of the predicted coding sequences appear to have arisen through gene duplication events. It was notable that almost 20% of the potential proteins for which a signcant BLAST hit was obtained were most similar to proteins in the Archaea or Eukaryotes, and the remaining 80% that were most similar to bacterial proteins were widely distributed across the phylogenetic tree. Phylogenetic analysis of a limited set of genes suggested a distant relationship to Chlamydia species, which is consistent with the fact that both lack a peptidoglycan cell wall. Genome analysis has revealed several contradictions for an environmental organism with a genome size of 7.145 Mbp. Fewer transporters and predicted regulatory genes were identied than might be expected, given that the number of genes in these functional categories tends to correlate with genome size. The presence of a large number of predicted coding sequences for nitrate transport and nitrate or nitrite reduction are an exception, and this observation is consistent with the fact that Pirellula must survive in a nitrogen-limited marine environment. Another feature that sets Pirellula apart from other microbes is the large number of sulfatases encoded in the genome at least two orders of magnitude greater than seen in any other completely sequenced microbial genome to date. Because inorganic sulfur is not limited in marine environments, this raises questions regarding the roles that the large numbers of sulfatases play in the biology of Pirellula. It has been suggested that these might be important in the degradation of sulfated glycopolymers that are found in abundance in marine snow and to which Pirellula can attach using a polar stalk. It will be of great interest to compare the genome of Pirellula spp. to that of Gemmata obscuriglobus, a freshwater Planctomycete, for which genome sequencing is underway. As the analysis of the smallest and largest circular bacterial chromosomes have recently revealed, there are still many fascinating discoveries that remain to be made regarding environmental microbes.
References
1 Huber, H. et al. (2002) Nature 417, 63 67 2 Waters, E. et al. (2003) The genome of Nanoarchaeum equitans: Insights into early archaeal evolution and derived parasitism. Proc. Natl. Acad. Sci. U. S. A. 100, 12984 12988 3 Gilbert, W.F. (1987) The exon theory of genes. Cold Spring Harb. Symp. Quant. Biol. 52, 901 905 4 Glockner, F.O. et al. (2003) Complete genome sequence of the marine planctomycete Pirellula sp. strain 1. Proc. Natl. Acad. Sci. U. S. A. 100, 8298 8303 5 Bentley, S.D. et al. (2002) Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141 147 6 Omura, S. et al. (2001) Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites. Proc. Natl. Acad. Sci. U. S. A. 98, 12215 12220 7 Brochier, C. and Philippe, H. (2002) A non-hyperthermophilic ancestor for Bacteria. Nature 417, 244
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