Microbial Communities

The recently sequenced genome of the parasitic bacterium Mycoplasma genitalium contains only 468 identified protein-coding genes that have been dubbed a minimal gene complement [Fraser, C. M., Gocayne, J. D., White, O., Adams, M. D., Clayton, R. A., et al. (1995) Science 270, 397-403]. Although the M. genitalium gene complement is indeed the smallest among known cellular life forms, there is no evidence that it is the minimal self-sufficient gene set. To derive such a set, we compared the 468 predicted M. genitalium protein sequences with the 1703 protein sequences encoded by the other completely sequenced small bacterial genome, that of Haemophilus influenzae. M. genitalium and H. influenzae belong to two ancient bacterial lineages, i.e., Gram-positive and Gram-negative bacteria, respectively. Therefore, the genes that are conserved in these two bacteria are almost certainly essential for cellular function. It is this category of genes that is most likely to approximate the minimal gene set. We found that 240 M. genitalium genes have orthologs among the genes of H. influenzae. This collection of genes falls short of comprising the minimal set as some enzymes responsible for intermediate steps in essential pathways are missing. The apparent reason for this is the phenomenon that we call nonorthologous gene displacement when the same function is fulfilled by nonorthologous proteins in two organisms. We identified 22 nonorthologous displacements and supplemented the set of orthologs with the respective M. genitalium genes. After examining the resulting list of 262 genes for possible functional redundancy and for the presence of apparently parasite-specific genes, 6 genes were removed. We suggest that the remaining 256 genes are close to the minimal gene set that is necessary and sufficient to sustain the existence of a modern-type cell. Most of the proteins encoded by the genes from the minimal set have eukaryotic or archaeal homologs but seven key proteins ofDNA replication do not. We speculate that the last common ancestor of the three primary kingdoms had an RNA genome. Possibilities are explored to further reduce the minimal set to model a primitive cell that might have existed at a very early stage of life evolution. The sequences of two small genomes of parasitic bacteria, Haemophilus influenzae and Mycoplasma genitalium, have been reported recently (1, 2). There is a qualitative difference between complete bacterial genomes and any sequences, including viral and organellar genomes, that have been available before. However small, a cellular gene set has to be self-sufficient in the sense that cells generally import metabolites but not functional proteins; therefore, they have to rely on their own gene products to provide housekeeping functions. Analysis of protein sequences encoded in the first two complete genomes based on this simple notion resulted in the theoretical reconstruction of unknown bacterial functional systems (3, 4). Here we systematically compare the M. genitalium and H. influenzae protein sequences in an attempt to define the minimal gene set that is necessary and sufficient for supporting cellular life. M. genitalium that has a 0.58 megabase genome, with only 468 protein-coding genes, has been proclaimed the minimal gene complement (2). However, while this is the cellular life form with the smallest known number of genes, there is no evidence that it is indeed minimal. Clearly, the M. genitalium genes are sufficient to support a functioning cell but there is no indication as to what fraction of them is necessary. M. genitalium and H. influenzae belong to Gram-positive and Gram-negative bacteria, respectively (5), and are likely to be separated from their last common ancestor by at least 1.5 billion years of evolution (6). H. influenzae is also a parasitic bacterium with a relatively small genome that is 1.83 megabases long and contains about 1700 protein-coding genes; its evolution apparently included a number of gene elimination events (1, 3). Therefore, the genes that are conserved in these two bacteria are almost certainly essential for cellular function and are likely to approximate the minimal gene set. The original analysis of the H. influenzae and M. genitalium proteins included only the most obvious sequence similarities and the respective functional assignments (1, 2). We performed an in-depth reanalysis of the H. influenzae and M. genitalium protein sequences (3, 4) using the strategy developed in the recent studies on the Escherichia coli genome (7, 8). Here we use the results of a detailed comparison of M. genitalium and H. influenzae proteins to derive and characterize the minimal gene set compatible with modem-type cellular life. We then discuss possible directions of a further reduction that may be undertaken to model a primordial cell. MATERIALS AND METHODS Sequences and Data Bases. The nucleotide sequences of the H. influenzae and M. genitalium genomes were from refs. 1 and 2, respectively. The gene complement of each of the bacteria was reevaluated. It has been reported that H. influenzae possesses 1727 protein-coding genes (1). By merging overlapping open reading frames that apparently belong to the same gene and that have been separated because of frameshifts, and by eliminating short genes whose existence could not be corroborated, we have arrived at a set of 1703 predicted genes (3). The M. genitalium genome has been reported to contain 470 protein-coding genes (2). Our analysis detected 468 genes, two of which have been missed in ref. 2, while four of the open reading frames reported in ref. 2 could not be confirmed in our study. In addition, coding regions for two genes were extended. All data base screening was against the protein and nucleotide versions of the daily updated nonredundant sequence data base maintained at the National Center for Biotechnology Information. Abbreviations: Ndk, nucleoside diphosphate kinase; PTS, sugar phosphotransferase. *To whom reprint requests should be addressed at: National Center for Biotechnology Information, National Library of Medicine, Building 38A, National Institutes of Health, Bethesda, MD 20894. e-mail: [email protected]. 10268 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1a. What is a core-genome? Haemophilus influenza 1743 genes Mycoplasma genetalium 468 ge es 256 g es = ‘core’ 1743 genes +! 468 ge es -! 256 ‘core’! = 1995 ‘pan-genes’