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Mycoplasma pneumoniae and genitalium: a comparison of two closely related bacterial species Richard Herrmann* and Berta Reiner†

The rapid progress in large quantities of DNA will species with different features at the level provide an increasing number of complete genome sequences () [7••]. Both species show a high level of similari- of closely related bacterial species as well as of pairs of ty, but also sufficient differences to make this comparison isolates from the same species with different features, such as worthwhile. This review is intended to show that a thorough a pathogenic and an apathogenic representative. This opens comparative genetic analysis will allow correlation of genetic the way to apply subtractive comparative analysis as a tool to properties with current knowledge on the physiology, mole- select from the large pool of all bacterial a relatively cular biology and microbiology of each of the two mycoplas- small set of genes that can be correlated with the expression ma species but will also provide useful information for of a certain phenotype. These selected genes can then be the planning and executing experiments designed to reveal the target for further functional analyses. function of newly proposed mycoplasmal genes that are functionally unassigned. M. genitalium and M. pneumoniae are Addresses already included in a number of comparative studies that Zentrum für Molekulare Biologie Heidelberg, Mikrobiologie, Universität also deal with the complete sequences of other bacterial or Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany archaeal (for examples, see [8•,9•,10••,11••]). *e-mail: [email protected] †e-mail: [email protected] Habitat and pathogenicity Current Opinion in Microbiology 1998, 1:572–579 are the natural for M. genitalium and M. pneu- http://biomednet.com/elecref/1369527400100572 moniae. For a long time, these were considered as surface parasites, but recent reports have indicated that © Current Biology Ltd ISSN 1369-5274 they may also enter the host cell and persist intracellular- Abbreviations ly [12,13]. This observation could explain the tendency of C cytosine M. pneumoniae and M. genitalium to survive in their hosts G guanine after treatment and clinical recovery [14]. Both IP isoelectric point possess a specialized terminal (tip ORF open reading frame structure, Figure 1), the correct formation of this tip struc- ture is the prerequisite for a successful [15,16]. Introduction As the name implies, M. pneumoniae is preferentially The genus mycoplasma is one of several genera belonging to found in the and M. genitalium in the uro- the bacterial class . The common features of all genital tract, although not exclusively, M. genitalium has mycoplasma species are the complete lack of a bacterial cell also been isolated from the respiratory tract and M. pneu- wall, the absence of a periplasmic space, a limited metabolic moniae from the urogenital tract [14]. It is beyond the potential and a parasitic lifestyle. The cells are surrounded scope of this review to evaluate the consequences of M. only by a cytoplasmic membrane which contains, uniquely among bacteria, [1,2••]. The term mycoplasma is Figure 1 used in this paper to name only the members of the genus mycoplasma and not, as frequently done, all the members of the class Mollicutes. Mycoplasmas have lost substantial parts of their genomes during evolution and are presently the bac- teria with the smallest known genomes, with sizes between 580 and 1250 kbp [3]. The cells have a diameter of 0.5 µm or even less and are too small to be clearly seen in a light micro- scope. Species of the genus mycoplasma can be found in many different hosts including humans, cattle, rodents, birds, reptiles and fish, but individual species have a strict host, organ and tissue specificity [2••].

Mycoplasmas have received new attention through the recent progress in sequencing their entire bacterial genomes. Attachment of M. pneumoniae and M. genitalium to . After [4], was electron illustrates the attachment of (a) the second bacterium for which the complete genome M. pneumoniae (mp) to hamster respiratory epithelium, and (b) sequence was published [5]. Later, when the genome M. genitalium (mg) to Fallopian tube epithelium via their respective terminal tip structure. Magnification: 28,000 X. Courtesy of Ping-Chuan sequence of was also available [6], it Hu, University of North Carolina at Chapel Hill. became possible to compare two closely related bacterial Mycoplasma pneumoniae and Mycoplasma genitalium Herrmann and Reiner 573

genitalium and M. pneumoniae for individual M. genitalium and M. pneumoniae compared to subtilis cells, tissue or the complete , but it should be or Haemophilus influenzae is notable (Figure 2) and should be remembered that the ability of M. pneumoniae to cause res- considered when analyzing extracts from these bac- piratory tract disease has been confirmed by many studies, teria by methods based on charge differences, such as in whereas while M. genitalium seems to be associated with two-dimensional gel electrophoresis. nongonococcal it has not been strictly proven to cause this infection. For more details see the recent clini- This high degree of similarity raises the question of cal paper by Taylor-Robinson [14], or reviews on whether these two bacteria should belong to different mycoplasma factors and the immune responses species. According to the still accepted rules for species [17,18]. The key to understanding the species-specific tis- definition in Bergey’s Manual, a difference in G + C con- sue prevalence and pathogenic mechanisms should be tent higher than 2 mol% and DNA homology values lower found in the genetic differences between M. genitalium than 40% justify separation into two species [29], but these and M. pneumoniae that have been revealed by the com- rules were established before the prospect of the use of plete sequences of their genomes. complete genomic sequences for taxonomic purposes. Therefore, these two bacteria could serve as a good para- Sequence analysis and coding capacity digm to check, and maybe also redefine, the rules for The genome of M. genitalium has a size of 580,070 bp [5] and species definition. The DNA guanine (G) and cytosine (C) is 236,324 bp smaller than the genome of M. pneumoniae content differs by 8 mol% between the genomes of (816,394 bp) [6]. The first publications reported 470 open M. pneumoniae and M. genitalium, and this is somewhat sur- reading frames (ORFs) for M. genitalium and 677 ORFs for M. prising given the high agreement in order and coding pneumoniae. Since then a number of corrections have been capacity. Detailed studies revealed that the difference is made, including new functional assignments and the propos- mainly due to variation at the third codon position, which al of new ORFs [19–21]. None of these modifications has any is with 19 mol% difference in G + C the most variable. The significant influence on this comparative study, because it third codon position in about 32,000 codons (of a total of has been shown that all the ORFs that were proposed for M. 170,400) in M. genitalium could be altered from A/T to G/C genitalium are contained in the larger genome of M. pneumo- to increase the genomic G + C content by 5.6 mol% G + C niae [7••]. Therefore, in this review, we do not consider all without changing a single . A G + C plot of the the proposed corrections and new functional classification, complete genomes of M. genitalium and M. pneumoniae and we refer always to the publications by Fraser et al. [5] and showed a similar slightly uneven distribution, which was Himmelreich et al. [6] unless otherwise stated. mainly attributed to the repetitive DNA sequences which in both have a G + C content above average To compare the number of ORFs in both bacteria directly [7••]. Kerr et al. [30] and McInerney [31•] discovered inde- one has to make an adjustment in annotation. The list of 677 pendently that a more discriminatory difference is seen ORFs form M. pneumoniae includes 26 ORFs which are when only the G + C content of the third position of the coded for by the repetitive sequences RepMP2/3 (10 codons (GC3) is plotted with respect to the position of the copies), RepMP4 (eight copies) and RepMP5 (eight copies) proposed gene on the genome. The GC3 distribution is [22]. These repetitive sequences, called then the MgPar random in M. pneumoniae but in M. genitalium it has a max- repeat [23,24], are also contained in M. genitalium at a lower imum between positions 190,000 and 205,000 copy number (14 copies all together) but they were not and minimum around nucleotide position 450,000. Thus it incorporated in the ORF index. Therefore, the actual num- seems that codon usage and genome position are correlat- bers of ORFs to be compared are 470 and 663. The repeti- ed in M. genitalium but not in M. pneumoniae. The signifi- tive sequences are part of a genetic exchange mechanism cance of this finding is unclear. that generates, probably by gene conversion, variable copies of two which are involved in cytadherence [25,26]. Functional assignment These two proteins are coded for by two genes (gene P1, Mycoplasmas developed by degenerate evolution from ORF6) of the P1 operon in M. pneumoniae [27] and its Gram-positive-bacteria of the Lactobacillus group [32]. homologs (MG191, 192) in M. genitalium which are orga- During this ongoing process they have lost many functions, nized in the MgPa operon [28]. Only the copies that reside probably only after they had adapted to a parasitic style in the P1 operon and the MgPa operon are expressed. in an environment that is physiologically fairly constant. It has been shown by biochemical experiments [33••] and was The average identity between M. pneumoniae and M. genital- confirmed and extended by the genome sequence analyses ium orthologs is 66.1% at the nucleotide level and 67.4% at that neither M. genitalium nor M. pneumoniae code for pro- the amino acid level, and the 16S and 23S ribosomal teins involved in the of amino acids, fatty are 98% identical. Furthermore, a comparison of theoretical acids, cholesterol, or and bases. This two-dimensional protein maps, based on the calculated iso- makes them very dependent on import systems. electric point (IP) and molecular weight for each proposed Unexpectedly, the number of transport systems in the protein, showed a very similar distribution. The relatively M. pneumoniae and M. genitalium is relative low compared to high number of proteins with IPs between 10 and 13 in other bacteria, therefore one has to conclude that the 574 Genomics

Figure 2

Comparison of theoretical two-dimensional Mycoplasma genitalium protein maps of M. genitalium, M. pneumoniae and B. subtilis. The isoelectric points and the molecular weights were predicted on the basis of the results from the complete genome sequences [5,6,43••].

Mycoplasma pneumoniae

Bacillus subtilis

Current Opinion in Microbiology

mycoplasma transport systems must have low substrate to 97% identity for conserved housekeeping proteins specifities [6,34••]. Several metabolic pathways were pre- (Table 1). The degree of identity can be used to predict sent in mycoplasmas in a truncated form or completely whether a protein with unknown function is a housekeep- absent. The pathway is completely present, but ing protein. For instance, the original annotations for only two components of the pentose-phosphate pathway MG353 and its homolog in M. pneumoniae, G12_orf109, did and one of the TCA cycle could be identified [35•]. None not predict a function despite exhibiting almost 92% iden- of the proteins involved in synthesis have been tity. Fold assignments for these genes suggested that they conserved. Instead both species have an identical set of code for the histone-like protein HU [36•]. proteins which are believed to be part of a - like structure (Table 1; [15]).These proteins do not show One of the obvious differences from other bacteria is the low significant homologies to proteins from other organisms by number of proteins detected for regulation of gene expres- standard similarity searches in databases. This is consistent sion in M. genitalium and M. pneumoniae. So far, only one with the observation that the orthologs of these proteins in sigma factor has been identified, and no members of the M. genitalium and M. pneumoniae share relatively low levels almost ubiquitous two component signal transduction sys- of similarity. With a single exception they are less than 50% tem. How these mycoplasmas regulate the synthesis of many identical at the amino acid level, compared to at least 70% gene products, which are present in different concentrations Mycoplasma pneumoniae and Mycoplasma genitalium Herrmann and Reiner 575

Table 1 transferases. The standard procedure to functionally assign a proposed ORF is to search for orthologs in other Sequence similarities of selected proteins from organism. If a genome does not encode the corresponding M. pneumoniae and M. genitalium. ortholog one has to screen whether non-orthologous Proposed ORF name Identity %/ genes could be candidates for replacement of the missing function/ M. pneumoniae M. genitalium number amino orthologs. Based on this approach Koonin et al. [20] pro- protein name acids posed a number of ORFs in M. genitalium which could Cytadherence accessory proteins code for the above mentioned missing RNaseH and kinase activities. Given the close relationship between M. HMW1 H08_orf1018 MG312 33.9%/1165 genitalium and M. pneumoniae the corresponding M. pneu- HMW2 F10_orf1818 MG218 57.1%/1828 HMW3 H08_orf672 MG317 36.0%/698 moniae ORFs should also be considered. These proposals P65 F10_orf405 MG217 44.5%/409 have yet to be verified experimentally. P200 D02_orf10360 MG368 30.5%/1628 P1 E07_orf1627 MG191 46.0%/1627 M. pneumoniae specific functions ORF6 (P1 E07_orf1218 MG192 50.7%/1230 operon) Because all the ORFs proposed for M. genitalium are pre- P30 H08_orf274 MG318 45.6%/285 sent in M. pneumoniae, it is fair to assume that they code for Proteins with the highest similarities products with identical functions. It seems very likely that the differences in physiology, tissue specificity and patho- Elongation K05_orf394 MG451 96.7%/394 genicity have to be encoded by the M. pneumoniae specific factor TK Ribosomal G07_orf139 MG087 95.7%/139 ORFs which were absent in M. genitalium. Therefore, a protein S12 careful functional analysis of the 209 ORFs that are encod- Ribosomal GT9_orf122 MG161 95.1%/122 ed by the 236 kbp additional DNA in M. pneumoniae should protein L14 explain the features specific to M. pneumoniae (Tables 2 Ribosomal P01_orf53 MG325 94.5%/53 protein L33 and 3). Surprisingly, only 110 ORFs of the 209 additional Ribosomal VXpSPT7_orf87 MG155 94.3%/87 ORFs are new and M. pneumoniae specific, whereas the protein S87 residual 99 ORFs are either gene duplications, which exist Ribosomal VXpSPT7_orf1390 MG158 92.8%/138 mainly as single copies in M. genitalium, or represent ORFs protein L16 Heat shock A05_orf595 MG305 92.4%/595 derived from the repetitive DNA sequences RepMP2/3, protein DnaK REpMP4 and RepMP5. Among the single copy genes in RNA polymerase F04_orf1290 MG340 92.4%/1289 M. genitalium that are amplified in M. pneumoniae are the β′RpoC of the murein type found in . M. genitalium codes only for 21 of these lipoproteins but M. pneumoniae for 46. The 25 additional lipoproteins in M. pneumoniae are only amplifications, at the mRNA level as well as at the protein level, is unre- which are frequently organized in sequential order. It solved and presents an interesting problem for molecular might give M. pneumoniae a greater reservoir for antigenic microbiology research. variation in these frequently surface exposed proteins, but the phenomenon of antigenic variation through differential The direction of of genes is very uniform in expression of lipoproteins, which is documented for sever- M. genitalium and M. . Starting from the pro- al mycoplasma species [37], has not yet been observed in posed origin of replication [5,6,7••] 85% of all proposed M. pneumoniae. We do not assume that the additional ORFs are transcribed bidirectionally in the same way as lipoproteins and the other gene duplications contribute to DNA replication proceeds (Figure 3). In both species the species specific features of M. pneumoniae. only 15% of genes are transcribed against this general direction (Figure 3). Preliminary transcription studies on From the 110 M. pneumoniae specific ORFs only 26 can be M. pneumoniae did not show a detectable correlation assigned functionally by sequence similarity to other pro- between the location of a gene with respect to the pro- teins (Table 3). This is a rather low value considering the posed origin of replication and the concentration of tran- relatively high number of available complete bacterial scripts in logarithmically growing cells (HWH Göhlmann, genome sequences including that of Bacillus subtilis, which personal communication). is supposed to be the closest phylogenetically and which should have homologs of all genes of M. pneumoniae except The analysis of the genome sequences of both M. genital- for those which were acquired by M. pneumoniae from other ium and M. pneumoniae failed to identify genes encoding sources (e.g. by horizontal gene transfer). A possible expla- for activities that had to be present in these bacteria; the nation for this low value could be that many M. pneumoni- most prominent examples being RNaseH, which ae specific genes have diverged far. The 16 ORFs that removes RNA primers during replication of chromosomal show the highest similarity to eukaryotic proteins are DNA, nucleoside diphosphate kinase, which is essential shared by both M. pneumoniae and M. genitalium. There is for the synthesis of nucleoside triphosphates, or glycosyl one exception which is only found in M. pneumoniae 576 Genomics

Figure 3

Comparative presentation of the organization of the DNA segments last M. genitalium ORF of each of the six segments with conserved with conserved gene order and general direction of transcription in gene order. For simplification, the ORFs that were transcribed against M. genitalium (MG) and M. pneumoniae (MP). Both genomes have the general direction are not indicated, but the region of the genome in been oriented the same way as M. genitalium was originally presented which the direction of transcription changes frequently is shown by the [5], starting with the dnaN gene as MG001. The black arrows indicate interrupted lines following the arrowhead. The bottom lines indicate the the direction of transcription, starting with dnaA and dnaN in opposite nucleotide positions in the published genome sequences. R indicates directions from the proposed origin of replication (black dot). The MG rRNA operon; ▼ indicates repetitive DNA sequences. ORF numbers at the beginning and end of each bar give the first and

(CO9_orf600, carnitine palmitoyltransferase II precursor tial to synthesize ATP from , and one is an NADP- [cpt2]). From the 26 functionally assigned M. pneumoniae dependent alcohol dehydrogenase. specific ORFs, 12 ORFs code for components of a hsd- type restriction-modification system. Two are components In correlation with the absence of the restriction modifica- of the phosphoenolpyruvate/carbohydrate phosphotrans- tion system and the arginine dihydrolase pathway in ferase system (PTS), five are comprising the argi- M. genitalium, the restriction endonuclease and two pro- nine dihydrolase pathway (arginine deiminase, ornithine posed arginine deiminase genes are truncated and proba- carbamoyltransferase, carbamate kinase), with the poten- bly not active in M. pneumoniae due to frame shift . It is unclear whether the full length ORF Table 2 (H03_orf438), coding for a arginine deiminase synthesizes, is an active in vivo, as such an activity has not be Differences between M. pneumoniae and M. genitalium. shown for M. pneumoniae [33••], and hence M. pneumoniae Feature Excess in M. pneumoniae has been classified as an arginine nonfermenting species. compared to M. genitalium Some of these M. pneumoniae specific functions (e.g. the transport system) may explain why M. pneumoniae is easier 236 kbp to grow in the than M. genitalium, but none of Number of ORFs 209 Number of unique M. pneumoniae 110 them provides convincing indications for the observed dif- specific ORFs ferences in pathogenicity and tissue specificity of the two Gene duplications 99 mycoplasmas. We assume that the key to understanding these phenomena might be found in the 37 M. pneumoniae specific ORFs that do not show any significant homologies to other genes/proteins in databases. To proof this and to Table 3 do functional analyses, one has now to apply the methods of biochemistry, biophysics and genetics. Classification of M. pneumoniae specific ORFs.

Classification Number of ORFs Genome organization The complete sequences so far pub- Function assigned 25 lished show convincingly that there is no long range colin- Paralogs or conserved motifs 27 earity between genomes from distantly related bacteria Repetitive DNA derived 21 [38] except in the closely related pair M. pneumoniae and Without homology to other genes/proteins 37 M. genitalium [7••]. The gene order in both mycoplasmas is Mycoplasma pneumoniae and Mycoplasma genitalium Herrmann and Reiner 577

Figure 4

The dnaA/dnaN region of M. genitalium (MG), M. pneumoniae (MP) and B. subtilis (BS). The ORFs in M. pneumoniae are oriented according to the order of ORFs in M. genitalium [5]. The untranslated region is indicated by IG. ABC indicates proposed transporter protein. The arrows show the direction of transcription. very well conserved; not over the entire genome, but with- This is not true for M. genitalium [5,41] and M. pneumoniae in six segments which are in different order on each [42]. We find here, so far, a unique divergent organisation of genome. Within each segment the order of homologous the dnaA region but with an identical gene order in both genes with respect to each other is conserved, with the mycoplasma genomes (Figure 4). The identical organisation additional M. pneumoniae specific ORFs interspaced is not obvious from the original publications, because the between them (Figure 3). The direction of transcription annotation for several ORFs differed and some ORFs were does not change in the genomic segments despite their dif- proposed for M. pneumoniae but not for M. genitalium. The ferent location on the genomes of both bacteria. This comparative analysis at the nucleotide level shows convinc- could indicate that an advantage exists in maintaining the ingly that the dnaA/dnaN clusters contain the same ORFs strongly biased direction of transcription. Such a strong organized in the same order, except that the homolog of bias in the direction of transcription has not been found in ORFMP155 is truncated in M. genitalium. other bacteria. The translocation of the genomic segments can be explained by homologous recombination between This organization, however, is not conserved among individual copies of the repetitive DNA sequences which mycoplasmas, as the cluster and their order of genes in flank these segments. Not all the copies of the repetitive Mycoplasma capricolum differ from those in M. pneumoniae DNA sequences seem to be involved in translocation of and M. genitalium and appears more similar to the cluster in segments (see Figure 3). It is obvious that in the larger part B. subtilis [40,43••]. of both genomes the order of genes is colinear although four repetitive sequences are located in this region. We Mycoplasmas as experimental systems assume that a selection pressure exists against interruption The main advantage of mycoplasmas as research models is of this part of the genome by translocation of segments their small genome size and accordingly the rather limited from outside, as they would interfere with the predomi- number of gene products. M. genitalium in particular, but nantly bidirectional transcription. also M. pneumoniae, could serve as model organisms [44] to define experimentally by further genome reduction the Siefert et al. [39] identified 16 gene clusters, categorized in essential functions for a self replicating cell under a partic- five functional groups, which are conserved in many bacteria ular set of environmental conditions [45]. In addition, the including both mycoplasmas. Among these clusters are sev- two species are excellent model organisms to test and eral ribosomal protein operons, including the FoF1 ATPase develop strategies for global expression analyses, because operon, the ribosomal RNA operon, an ABC transporter with transcription profiles or protein maps based on two-dimen- a proposed specificity for spermidine and putrescine. In sional gel electrophoresis are much easier to establish for addition, these authors identified a set of clusters which were 500–700 transcripts or proteins than for organisms which found in either the Haemophilus/Mycoplasma comparison or code for several thousand gene products. the Haemophilus/Synechocystis comparison, but not in both. Of the 14 proposed clusters only one, the dnaA/dnaN region, is A clear disadvantage for many experimental approaches is relevant in context of this review. The dnaA/dnaN region is the lack of an established gene transfer system and of a part of the proposed origin of replication (Figure 4). The defined culture medium. Transposon mutagenesis and genes dnaA and dnaN are located next to each other in many integration of foreign DNA into the genome via trans- bacterial genomes and transcribed in the same direction [40]. posons can be achieved albeit with limited efficiency 578 Genomics

[46–48]. For growth both M. genitalium and M. pneumoniae Recommended for specialists and general readers. A very clearly written need a rich medium which contains 5–20% serum making comprehensive summary. pulse-labelling experiments and growth under defined 3. Neimark HC, Lange CS: Pulse-field electrophoresis indicates full- length mycoplasma chromosomes range widely in size. Nucleic conditions impossible. In addition, the medium is expen- Acids Res 1990, 18:5443-5448. sive and the yields of mycoplasma cells are low compared 4. Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, to E. coli yields. Last but not least, the codon usage in Kerlavage AR, Bult CJ, Tomb JF, Dougherty BA, Merrick JM et al.: Whole-genome random sequencing and assembly of mycoplasmas differs from that in E. coli. The codon UGA Haemophilus influenzae Rd [see comments]. Science 1995, is read as in mycoplasmas [49] but as a stop 269:496-512. codon in most other bacteria. Expression of mycoplasma 5. Fraser CM, Gocayne JD, White O, Adams MD, Clayton RA, proteins in E. coli therefore requires a UGA-suppressor Fleischmann RD, Bult CJ, Kerlavage AR, Sutton G, Kelley JM et al.: The minimal gene complement of Mycoplasma genitalium [see gene [50] and is further improved by using an E. coli comments]. Science 1995, 270:397-403. mutant with an inactive release factor 2 [51]. Even then, 6. Himmelreich R, Hilbert H, Plagens H, Pirkl E, Li BC, Herrmann R: the suppression of several UGAs in one gene is limited Complete sequence analysis of the genome of the bacterium and the synthesis of full length proteins is severely ham- Mycoplasma pneumoniae. Nucleic Acids Res 1996, 24:4420-4449. pered. Considering the advantages and disadvantages it 7. Himmelreich R, Plagens H, Hilbert H, Reiner B, Herrmann R: •• Comparative analysis of the genomes of the bacteria seems obvious that mycoplasmas are not well suited to Mycoplasma pneumoniae and Mycoplasma genitalium. Nucleic study those genes or functions which can be analyzed in Acids Res 1997, 25:701-712. bacteria like E. coli or B. subtilis with highly developed Detailed comparison between two closely related species. genetic tools and which also give high yields of cells. 8. Coissac E, Maillier E, Netter P: A comparative study of duplications • in bacteria and : the importance of telomeres. Mol Biol Evol 1997, 14:1062-1074. The genomes of three bacteria (including M. genitalium) and two Conclusions eukaryotes were compared with respect to gene duplications. All genomes The comparative analysis of M. genitalium and M. pneumoni- exhibit between 7–30% duplicated open reading frames. ae on the basis of the information provided by their com- 9. Karlin S, Mrazek J, Campbell AM: Compositional biases of bacterial • genomes and evolutionary implications. J Bacteriol 1997, plete genome sequences showed that the two bacterial 179:3899-3913. species were much more similar than anticipated from a dif- The distribution of dinucleotides and tetranucleotides across 15 diverse ference of 30% in genome size, 8 mol% in DNA G + C con- is uneven. The possible causes and implications are discussed. tent, and their different tissue specificity and pathogenicity. 10. Koonin EV, Galperin MY: Prokaryotic genomes: the emerging •• paradigm of genome-based microbiology. Curr Opin Genet 1997, Not only were all the proposed ORFs of the smaller M. gen- 7:757-763. italium genome found in M. pneumoniae, but genome orga- The comparative analysis of seven bacterial and three archeal genomes shows that there is little conservation of genome organisation and the nization and the rather uniform direction of transcription number of universal conserved gene families is small. were highly conserved. The additional 236 kbp DNA of the 11. 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