Sequence Analysis of 56 Kb from the Genome of the Bacterium Mycoplasma Pneumoniae Comprising the dnaA Region, the Atp Operon and a Cluster of Ribosomal Protein Genes (original) (raw)

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Zentrum für Molekulare Biologie Heidelberg, Mikrobiologie, Universität Heidelberg

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69120 Heidelberg, Germany

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Zentrum für Molekulare Biologie Heidelberg, Mikrobiologie, Universität Heidelberg

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69120 Heidelberg, Germany

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Zentrum für Molekulare Biologie Heidelberg, Mikrobiologie, Universität Heidelberg

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69120 Heidelberg, Germany

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Zentrum für Molekulare Biologie Heidelberg, Mikrobiologie, Universität Heidelberg

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69120 Heidelberg, Germany

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Received:

31 October 1995

Accepted:

29 December 1995

Published:

01 February 1996

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Helmut Hilbert, Ralf Himmelreich, Helga Plagens, Richard Herrmann, Sequence Analysis of 56 Kb from the Genome of the Bacterium Mycoplasma Pneumoniae Comprising the dnaA Region, the Atp Operon and a Cluster of Ribosomal Protein Genes, Nucleic Acids Research, Volume 24, Issue 4, 1 February 1996, Pages 628–639, https://doi.org/10.1093/nar/24.4.628
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Abstract

To sequence the entire 800 kilobase pair genome of the bacterium Mycoplasma pneumoniae, a plasmid library was established which contained the majority of the _Eco_RI fragments from M.pneumoniae. The _Eco_RI fragments were subcloned from an ordered cosmid library comprising the complete M.pneumoniae genome. Individual plasmid clones were sequenced in an ordered fashion mainly by primer walking. We report here the initial results from the sequence analysis of ∼56 kb comprising the dnaA region as a potential origin of replication, the ATPase operon and a region coding for a cluster of ribosomal protein genes. The data were compared with the corresponding genes/operons from Bacillus subtilis, Escherichia coli, Mycoplasma capricolum and Mycoplasma gallisepticum.

Introduction

The bacteria with the smallest known genomes are found among members of the class Mollicutes (1). This class presently comprises the six eubacterial genera Acholeplasma, Anaeroplasma, Asteroleplasma, Mycoplasma, Spiroplasma and Ureaplasma (however, the term mycoplasma has been frequently used to denote any species included in the class Mollicutes). The common characteristics are the complete lack of a bacterial cell wall, osmotic fragility, colony shape and filterability through 450 nm pore diameter membrane filters (2). The relatively close phylogenetic relationship of these genera was measured by comparative sequence analysis of the 5S and 16S ribosomal RNA (rRNA) (3,4). The rRNA sequence analyses also revealed that the Mollicutes are not at the root of the bacterial phylogenetic tree, but rather developed by degenerate evolution from gram-positive bacteria with a low mol% G+C (guanine plus cytosine) content of DNA, the Lactobacillus group containing Lactobacillus, Bacillus, Streptococcus and two Chlostridium species (5). The Mollicutes lost during the process of evolution a substantial part of their genetic information. This is reflected by significantly smaller genome sizes as low as 600 kb and extending to 2300 kb (6) as compared with 2500–5700 kb long genomes of their ancestor bacteria (1). The loss of coding capacity could probably be tolerated because of the parasitic life style of the Mollicutes. They have never been found as freely living organisms. In nature, Mollicutes depend on a host cell, respectively on a host organism. For instance, Mycoplasmas and Ureaplasmas are parasites in different vertebrates, from which they obtain essential compounds such as fatty acids, amino acids, precursors for nucleic acid synthesis and cholesterol, a compound normally not found in bacteria. Only Acholeplasma and Asteroleplasma do not require cholesterol for growth (7).

Mycoplasma pneumoniae, the subject of this study, is a human pathogenic bacterium causing tracheobronchitis and primary atypical pneumonia. Associated with the host cell, surface colonization of human respiratory tract epithelial cells takes place (8). In the laboratory,M.pneumoniae can be grown without a host cell in rich medium supplemented with 10–20% horse serum. The lack of a cell wall most probably facilitates the close contact between M.pneumoniae and its host cell and guarantees the exchange of compounds, which support the growth of the bacterium. As a consequence of this bacterial surface-parasitism the host cell is severely damaged. The exchange of toxic metabolic compounds is discussed as a possible cause of cell damage (9), however, at this stage not a single toxic compound has been identified as a causative agent of cell damage.

Mycoplasma pneumoniae has an exceptional position among the Mollicutes since its DNA has the highest G+C content (41 mol%), whereas the genomes of most of the other Mollicutes have a G+C content <30 mol% (2,6). The genome size of M.pneumoniae is ∼800 kb (10,11) having a coding capacity for 700 proteins assuming an average molecular mass of 40 000 Da. Hence M.pneumoniae is among the smallest self-replicating cells known today (12).

It was selected, mainly for this reason, as a model system for defining the minimal genetic requirements of an autonomously reproducing cell. This can be done by determining as many as possible genes and then classifying them into essential and non-essential ones. Based on these results we should be able to define a set of genes which are sufficient for the reproduction of M.pneumoniae under defined laboratory conditions. Morowitz already proposed several years ago that a mycoplasma species would be a suitable candidate for defining the essentials of a self-replicating cell (13). Apart from this model character as a genetically reduced self-replicating cell, M.pneumoniae offers a number of interesting phenomena to analyze. For instance, studies on the interaction between this prokaryotic surface parasite and its eukaryotic host cell, including the host immune reaction, might help to reveal bacterial pathomechanisms (14). Another promising area of research concerns the bacterial cytoskeleton. Despite the lack of a cell wall and other cellular appendages, M.pneumoniae exhibits a characteristic cell shape and motility. Both might be correlated to a cytoskeleton-like structure (15). Last, but not least, the evolution of the Mollicutes is, despite considerable progress in this field, still left with many unanswered questions. The large body of DNA sequence data from bacteria which are phylogenetically related to M.pneumoniae such as Bacillus subtilis might allow to reconstruct the process of degenerate evolution and to understand how Mollicutes genomes with different G+C contents, between 25 and 41 mol%, developed.

Little is known about genetics, physiology and molecular biology of M.pneumoniae in comparison with the relatively well studied bacteria Escherichia coli and B.subtilis (12). An efficient transformation system for M.pneumoniae comparable with the ones for E.coli is missing (16–18), however transposon mutagenesis has been successfully applied for the isolation of mutants (19). The dependence on rich medium for growth prevents the isolation of auxotrophic mutants and the efficient incorporation of labelled precursors. These disadvantages can be compensated to a large extent by the methods of molecular biology, for example, DNA cloning techniques, expression of genes or parts of genes in E.coli, restriction analysis and the construction of physical genome maps. Furthermore, combined with improved DNA sequencing techniques, computer aided data collection and analysis and a rapidly expanding source of information on genes and proteins in freely accessible data banks allow genes to be proposed on basis of DNA or protein sequence homology. At present ∼50–70% of DNA sequences derived from open reading frames can be defined by significant sequence homology to known genes, gene products or conserved typical motifs in proteins or DNA sequences. DNA sequence analysis is therefore the fastest way to identify a large number of genes of a given genome (20,21).

This paper introduces the M.pneumoniae genome sequencing project and describes the overall strategies and first results.

Sequencing strategy

The general strategy is to sequence both strands in an directed fashion by primer walking and to limit random (‘shot gun’) sequencing to a minimum. DNA sequence data are being generated by the Sanger method (22) using fluorescent dye labelled primers or dideoxynucleotides in combination with a semi-automated DNA sequencing unit (23–26).

An ordered cosmid library containing the complete M.pneumoniae genome in 34 overlapping cosmids, two λ phages and one plasmid (10) was the starting point for the project. The cosmid library was constructed by partial digestion of the M.pneumoniae genome with the restriction endonuclease _Eco_RI (27). The individual _Eco_RI DNA fragments from the cosmids are being further subcloned into a plasmid vector, resulting in a plasmid library consisting of clones each carrying one individual _Eco_RI DNA fragment. These fragments are between 0.1 and 28 kb long and are sequenced individually. The following methods are applied depending on the insert size. (i) Inserts up to 3 kb long are sequenced by primer walking only. (ii) Sets of nested deletions are constructed by the exonuclease III method (28) from clones with inserts between 3 and 10 kb long. A set comprising 20 nested deletions is normally sufficient to obtain the sequence from one strand of a 6 kb long fragment. The complementary strand and possible gaps in the first strand are then sequenced by primer walking. (iii) For sequence analysis of all other plasmids with inserts between 10 and 28 kb we apply a limited ‘shotgun cloning’ sequencing strategy. Suitable frequently cutting restriction endonucleases like _Sau_3A, _Alu_I or _Hae_III, are used to establish two or three different sets of ∼20 subclones carrying fragments 100–500 bp long. Both ends of individual cloned fragments are sequenced and aligned to contigs. Gaps are filled by primer walking on plasmids or cosmids carrying the _Eco_RI fragment in question.

The project is organized in such a way that many different plasmids can be sequenced at the same time and waiting for the synthesis of new primers is not a limiting step. Furthermore, sequencing efforts may be shifted to any region of interest on the genome. The speed of sequencing can be calculated since the complete genome has been cloned and therefore, the frequently painful analysis of the last few percent of the genome as a result of missing or not clonable DNA regions will not be an extra burden. The ordered cosmid library has been used to construct an _Eco_RI restriction map of the entire M.pneumoniae chromosome. Therefore, any DNA sequence can be attributed to a defined position on the physical map. This permits to establish a detailed genetic map parallel to the sequencing project.

Materials and Methods

Cloning of _Eco_RI fragments

The _Eco_RI fragments were subcloned from an ordered cosmid library comprising the complete M.pneumoniae genome (10). Standard procedures were applied (29) using as vector the plasmid pBc (Stratagene) and the E.coli strains HB101 (30) or XL1-Blue (Stratagene) for propagation of these plasmids. The plasmid clones containing the individual _Eco_RI fragments which were used for sequencing were purified by Qiagen column chromatography according to the protocol provided by the manufacturer (Qiagen).

The cloning vector pBC was purified by centrifugation to equilibrium in two sequential cesium chloride-ethidium bromide gradients (29).

The following nomenclature was used for the plasmid clones as well as _Eco_RI fragments. The cosmid always had the prefix pcosMP and a letter and a number, e.g., pcosMPD2. The plasmid carrying an _Eco_RI fragment from this cosmid received a ‘p’ as prefix and the letter and number applied to the cosmid and additionally the size of the _Eco_RI fragment in kb, e.g., pD2/4.8. The EcoRI fragment alone was named D2/4.8.

Long range PCR

To determine or to check the orientation of adjacent _Eco_RI fragments the improved PCR method for the amplification of DNA fragments up to 45 kb long was used (31). The reactions were done with the GeneAmp XLPCR kit from Perkin Elmer according to the manufacturers protocol. Genomic M.pneumoniae DNA used for amplification was purified as described (27). The primers for the reactions were designed as 22mers with a melting temperature of 68°C.

Synthesis of oligonucleotides

Synthetic oligonucleotides were synthesized (R. Frank et al., ZMBH) according to phosphoramidite chemistry using a solid carrier (32) on a model 394 DNA/RNA synthesizer from Applied Biosystems. Oligonucleotides between 17 and 20 nt long were specifically designed using the program OLIGO 4.0 (National Biosciences Inc.) and were used without further purification following a standard dilution for the sequencing reaction.

DNA sequencing

The sequence data for this study was exclusively generated by the enzymatic dideoxy chain-termination method described by Sanger et al. (22). The radioactive label was substituted by a fluorescent label and Taq polymerase was used in the reaction. The protocols were adopted from cycle sequencing protocols introduced by Craxton (33) in which the basic principle of this method is the linear amplification of the target DNA with a single primer.

All data were generated on a fluorescent-based sequence-gel reader (Model 373A, Applied Biosystems). Either fluorescently labelled universal primers (-21M13, M13 RP, T3 and T7) or fluorescently labelled dideoxynucleotides were used as label.

Taq dye primer cycle sequencing and Taq dye deoxy cycle sequencing were done as provided in the manufacturer's protocol. In each sequencing reaction 1 µg plasmid DNA or 2.3 µg cosmid DNA and 10 pmol primer were used.

In a typical sequence analysis ∼500 nt were read. Primers for primer walking were selected between nucleotide 300 and 400 from such a sequence. All sequence chromatograms were visually inspected and edited by the SeqEd program (Version 1.03) from Applied Biosystems. Sequence Assembly was performed by using the Sequence Project Management program of the DNA* program package by Lasergene.

Computer assisted analysis

Computer analyses were performed with the program package HUSAR (Heidelberg Unix Sequence Analysis Resources) release 4.0 at the German Cancer Research Center, Heidelberg, Germany. This package is based on the GCG program package version Unix-8.01 of the Genetics Computer Group, Wisconsin. For searching the DNA and protein databases [SWISS-PROT (34) and PIR (35)] the FASTA (36) and BLAST (37) programs (BLASTX, BLASTN and BLASTP) were used. Conserved motifs in proteins and peptides were identified by running the program PROSITE (38). Open reading frames (ORFs) were calculated by the program FRAMES allowing AUG (or GUG, UUG) as start codons using the Mycoplasma translation table where UGA is coding for tryptophane (39). The G+C content was calculated by the program COMPOSITION. Protein sequences were aligned by using either the program GAP (pairwise alignment) based on the algorithm of Needleman and Wunsch (40) or CLUSTAL (41) for multiple alignments.

Accession numbers

dnaA region: U34816 GenBank; ATPase region: U43738 Gen-Bank; GT9/25: U34795 GenBank.

Results and Discussion

Subcloning of the _Eco_RI fragments from the cosmid library into the plasmid pBC

All but six of the 143 unique _Eco_RI fragments which have been identified in the cosmids (42) were subcloned into the plasmid pBC (43). Several attempts failed to subclone a 4.9 kb _Eco_RI fragment from the cosmid pcosMPD12 and a 10.8 kb _Eco_RI fragment from the cosmid pcosMPG7. Since these and the other four _Eco_RI fragments (D12/4.9, G7/10.8, GT9/25, H8/8.2, H8/0.43B, K8/2.5) were present in the cosmids, which are also suitable for sequencing no further attempts were made to subclone them. In addition, any region of interest can be amplified by polymerase chain reaction (PCR) if necessary. Personal experience showed that some _Eco_RI fragments could be better propagated in E.coli as part of a cosmid than as an individual fragment cloned in a plasmid.

Revised version of the _Eco_RI restriction map

Initially 300–400 nt from the end of one strand of each cloned _Eco_RI fragment were sequenced. This required only the two primers T3 and T7 which hybridized to the left and to the right of the _Eco_RI cloning site of the vector pBC. Approximately 100 000 nt were gained from sequencing 274 ends from 137 fragments. The information was distributed over the genome in short mosaic like stretches. Based on this information, reverse primers were synthesized which permitted to sequence on the corresponding cosmid over the adjacent _Eco_RI site into the neighbouring _Eco_RI fragment and to align the correct ends of adjacent _Eco_RI fragments. Sixteen new _Eco_RI fragments between 15 and 193 bp long, which were not recognized by the restriction analysis of the cosmids, were also identified. Eight of these fragments were 87 bp long and were known to be part of the repetitive DNA sequence RepMP1 (44). The sizes of the other eight fragments were 15, 37, 42, 98, 128, 160, 168 and 193 bp.

The sequence analysis also revealed two problem regions, the overlapping cosmids pcosMPD12-pcosMPK5 and pcosMPK4-pcosMPG7 (42). It was found that a 5.4 kb _Eco_RI fragment of pcosMPK4 was a cloning artefact and had to be replaced by a 2.5 and a 0.7 kb _Eco_RI fragment which were both cloned in a phage M13 vector. The 0.4 kb fragment of pcosMPD12 could not be positioned unambiguously. Both regions were reexamined by long range amplification of genomic M.pneumoniae DNA by the polymerase chain reaction (PCR). Restriction fragment analysis of the amplified DNA with endonuclease _Eco_RI and DNA sequence analysis of the regions around the _Eco_RI sites confirmed number, size and position of the _Eco_RI fragments as shown in the map. In five other instances we had to exchange the positions of two fragments which were very similar in size in four cosmids (E7/1.85-E7/1.9; E7/0.7-E7/0.75; H8/0.43A-H8/0.43B; H91/1.9-H9/1.8; K4/8-G7/7.5). The order of all other fragments (42) was confirmed by the sequence analysis.

DNA sequence analysis of three selected regions

Based on previous experiments (42) and results of data base homology searches with the first 100 000 nt DNA sequence (one strand only) regions were selected for sequencing longer coherent stretches. Among others, the region around the dnaA gene was selected (pcosMPK5), since we expected to find there the origin of replication, as well as two other regions coding for conserved genes like the F0F1 ATPase operon (pcosMPD2) or ribosomal protein genes (pcosMPGT9). Sequences from these genes were also available from other mycoplasma species and from the phylogenetically related B.subtilis which could be used for comparative analyses. All DNA sequences published were generated from independent sequences of both strands of a given region. Discrepancies between two sequences were resolved by directly comparing the sequencing chromatograms and, if necessary, by repeating the sequence analysis of one or the other strand under different conditions, e.g., by selecting a new sequencing primer.

Figure 1

The dnaA region. Boxes represen _Eco_RI fragments in kb. Arrows indicate genes or ORFs drawn to scale and their direction of transcription. Between dnaN and soj is the untranslatable region. The G+C contents was calculated for a window of 150 bp with a 25 bp overlap (GenBank accession no. U34816).

To estimate the sequencing error rate the following two types of experiments were done with a 12 kb long region comprising the _Eco_RI fragments (D2/7.3, D2/4.8) (Fig. 4A) using the same batch of DNA preparation: (i) two persons (H.H. and R.Hi.) edited the same sequence chromatograms independently; (ii) using different primers, the 12 kb region was sequenced independently by the same two persons. The reading of the same chromatograms revealed three discrepancies, but the two independent generated sequences agreed fully.

These data do not permit to calculate an error rate for the complete project, but they hint at the fidelity of our sequencing analysis and underscore that sequencing by primer walking with low redundancy produces reliable results. The redundancy for one strand is 1.3 for a sequence generated by primer walking only, and ∼1.9 for a sequence generated by primer walking and shot gun subcloning of a larger _Eco_RI fragment like GT9/25.

The dnaA region Figure 1 shows the physical and genetic map of the dnaA region of M.pneumoniae and Table 1 summarizes the proposed open reading frames. The first obvious result is the uneven distribution of G and C in this area. Although the average G+C content of the M.pneumoniae genome is ∼41mol%, the region between nucleotide 4000 and 4850 is characterized by a G+C content well below 30mol% and by the absence of open reading frames coding for proteins >40 amino acids. The orientation of genes flanking this untranslatable region is also striking, because the genes to the right and to the left are transcribed divergently in opposite direction (Fig. 2). The genes located in the dnaA region of M.pneumoniae are also unusual. Many of them are not found in the corresponding regions of other bacteria (Table 1 and Fig. 2) and the conserved genes, gyrB, dnaN and dnaA, are in different order or orientation with respect to each other.

The gyrB gene and the beginning of the gyrA gene have already been sequenced and analyzed (45). Our data confirm the sequencing results on the gyrB gene (650 aa), however on the basis of DNA sequence comparison it is difficult to exclude the possibility that topoisomerase IV is being dealt with rather than with gyrase. The proposed DnaA protein from M.pneumoniae shares many of the conserved homologies with the DnaA proteins from other bacteria. It can also be formally divided into four domains (46–48), with the highest homologies in domain III, whereas the homologies in domains I and II are less pronounced. Domain III contains also the conserved ATP binding site. Domain IV which interacts specifically with DNA by binding to the so called dnaA box regions shows a noteworthy modification in a nonamer located near the C-terminus that is conserved in the other bacteria (46; Fig 3). It is likely that the dnaA box region(s) is an essential element of a bacterial origin of replication. It is characterized by repeats of the conserved nonamer nucleotide sequence TTATCCACA. Sequence deviations in two positions are permitted, however the 4th, 7th and 8th positions of the nonamer are highly conserved (47). The sequence TTATCCACA does not occur at all in the 14 kb region sequenced around the M.pneumoniae dnaA gene, however sequences with up to two mismatches are 55 times represented. Three of these sequences are located in the non-translatable region (Fig. 2). This appears to be a low number and whether this is sufficient for binding of the DnaA protein to this region, must be determined experimentally (49).

Figure 2

Gene order in the dnaA gene region of E.coli, B.subtilis, M.capricolum and M.pneumoniae. The genes and ORFs are boxed. For better comparison the dnaA region of M.pneumoniae is inverted compared to its orientation in the _Eco_RI map (Fig. 1). The arrows show the direction of transcription. The untranslatable region is situated between the arrows.

Proposed genes, not located in a comparable position in other dnaA regions, which were also unusual, were orf309, orf270, orf250, orf284 and orf1882 (Fig. 2). Only orf309 and orf270 were found in the immediate neighbourhood of the dnaA gene in Mycoplasma genitalium (50; note added in proof). The proposed Orf309 protein contains a conserved DnaJ box motif at its N-terminal end (51) with the following amino acids conserved: Tyr(4), Leu(7), Glu(8), Ala(13), His-Pro-Asp(30–32), Phe(41), Ala(47), Leu(51), Asp(53) and Tyr(60) (numbers in brackets give the position in the protein). The rest of the protein does not show a significant homology to the heat shock protein DnaJ. One dnaJ gene in M.pneumoniae has already been sequenced and showed significant homology over the whole protein to other bacterial DnaJ proteins (Hilbert, data not shown), therefore, we can exclude that orf309 is the dnaJ gene of M.pneumoniae. The overall homology at the amino acid level between Orf309 and the gene products of orf311 from M.genitalium is 66% identity, the only difference of interest between both proteins might be the motif RGD (Arg, Gly, Asp) which is present in M.pneumoniae. However, this appears only modified to KGD (Lys, Gly, Asp) in M.genitalium. The motif RGD plays a role in the interaction between extracellular matrix proteins and integrins (52). It remains to be seen whether this motif has such a function in the Orf309 protein. Besides 90.3% identity with its counterpart in M.genitalium, Orf270 shares the highest homology with the Soj protein from B.subtilis (53).

No significant pattern was seen with the proposed Orf250 protein, however, a relatively high percentage of glutamine (13.2%) and proline (10.8%) is present. As a result of this high glutamine content, a 20% identity with a seed storage protein is achieved, which itself has 32% glutamine.

The Orf284 protein reveals clearly the signature of the ATP-binding domains from an ABC transporter (54). The typical patterns include the Walker motif A which corresponds to a glycine-rich loop, the extended similarity to the ATP binding domains from many other ABC transporters, the absence of a transmembrane region and the potential for building α-helices, β-sheets and loops in a characteristic order (the Walker motif B is not as well conserved). We found the highest identity/similarity scores with CysA, the FtsE protein (55) and an unspecified ABC transporter protein named abc, all from E.coli (56). FtsE, which is part of the fts operon together with the genes ftsY and ftsZ, plays a role in cell division of E.coli.

The last of the proposed ORFs codes for a protein with 1882 amino acids. The open reading frame reaches beyond the _Eco_RI site into the adjacent _Eco_RI fragment K5/6.6. No significant homologies to other proteins were found.

To summarize: the dnaA region of M.pneumoniae, so far, is unique among bacteria (57) (Fig. 2). However, by comparing the DNA sequence data fromM.genitalium and not only the proposed ORFs (see note added in proof), it appears that the types and orders of genes in the dnaA region are like in M.pneumoniae except, the equivalents of orf284 (cysA) and orf250 code only for homologous proteins with extended deletions. The equivalent (MG468) of Orf1882 is present according to the DNA sequence but not annotated. In Mycoplasma capricolum the gyrB gene is localized far away from the chromosomal origin of replication (58) and in Mycoplasma hominis the region containing the gyrB gene (59) showed no resemblance to the corresponding region in M.pneumoniae with respect to gene order. Based exclusively on the DNA sequencing data, it is presently not possible to decide whether the dnaA region is the origin of replication in M.pneumoniae. More direct experimental proof is required. But independent from this uncertainty the evidence is mounting that a conserved order of the genes rnpA-rpmH-dnaA-dnaN-recF-gyrB is not the prerequisite for a functional origin of replication.

The ATPase operon

The number and order of genes of the atp operon coding for F0F1 ATPase in M.pneumoniae are conserved. That is they are identical with those determined for E.coli (60), B.subtilis (61) and Mycoplasma gallisepticum (62). In addition, our data suggest, that the operon structure may be extended, as the proposed C-terminal section of a gamma enolase in front of the atpI gene and at least the two proposed genes orf569 and orf152 (_lac_A) (63), following immediately the atpC gene could be part of the same operon. The space between the end of the preceding gene and the beginning of the following genes is at the most 8 nt long (Fig. 4A and Table 1).

Table 1

Genes and proposed ORFs of the published DNA regions of the M.pneumoniae genome

Figure 3

Comparison of the DnaA proteins (domain IV) from six bacteria. The domain IV is shown as proposed (46). The DNA binding site is located in domain IV (49); ecoli = E. coli (84); psepu = Pseudomonas putida (85); miclu = Micrococcus lutes (86); bacsu = B.subtilis (87); mycca = M.capricolum (46); mycpn = M.pneumoniae.

Figure 4

The ATPase operon (A) and the _Eco_RI fragment GT9/25 coding for a ribosomal protein gene cluster (B). The orientation of the _Eco_RI fragments is the same as on the _Eco_RI map. The genes and ORFs are represented by arrows drawn to scale indicating the direction of transcription. The ribosomal genes on this fragment belong in E.coli to three different operons: α operon: rpsM(S13)-rplQ(L17); spc operon: rplN(L14)-rpmJ(L36); S10 operon: rplP(L16), rpmC(L29), rpsQ(S17) (accession nos: ATPase region: U43738 GenBank; GT9/25: U34795 GenBank).

By comparing the corresponding genes of atp operons from different bacteria, the following picture emerges (Table 2). The highest identities exist between the two Mycoplasma species in particular among the atpA and atpD gene and also among the atpE and atpB gene with slightly less homology. The identity scores in all other instances are rather low. In some cases the scores between E.coli and M.pneumoniae are higher (atpH, atpG) than between M.pneumoniae and the phylogenetically more closely related B.subtilis. All comparable genes of the atp operon of the four bacteria are of similar length except atpE, atpF and atpI. Of interest is the structure of the b subunit (atpF). The proposed b subunits from M.pneumoniae (Fig. 5) and M.gallisepticum carry the characteristic features of a prokaryotic lipoprotein, a signal peptide with positively charged amino acids near the N-terminus, an accumulation of hydrophobic residues within a signal peptide and a cysteine between position 10 and 35 of the proposed preprotein. This cysteine will become the first amino acid after cleavage of the signal peptide. The processed protein is associated with the membrane via the diacyl-glycerol modified cysteine and a third acyl chain bound to the free amino group of the same cysteine (64,65). Downstream of the cysteine is a potential transmembrane section which would ensure that the C-terminal part of these subunits is facing the cytosol. The b subunits of E.coli and B.subtilis do not show the lipoprotein-specific structure, instead only one transmembrane section near the N-terminal end binds the b subunit to the cytoplasmatic membrane in such a way, that the residual protein is also orientated towards the cell interior permitting interaction with the F1 portion of the enzyme. Typical signatures exist for the α and β subunits which are present in most of the F0F1 type ATPases [(Pro- [Ser, Ala, Pro]-[Ile, Val[-[Asp, Asn]-X(3)-Ser-X-Ser]. This signature is also present in the β subunit of M.pneumoniae although the α subunit is modified; Asp or Asn is replaced by His. This is also the case for M.gallisepticum, while in E.coli and B.subtilis Asp or Glu has been found. Both, the α and β subunits possess also the expected ATP/GTP binding sites.

Table 2

Comparision of length and percent identity between subunits of the ATPase operon from selected bacteria with M.p.

Figure 5

Proposed lipoproteins. The boxed Cys is the first amino acid after cleavage. Hydrophobic amino acids from the signal peptide are in bold face. The proposed hydrophobic transmembrane segment downstream of the modified Cys in C12_orf207 (b subunit atpF) is in bold face and in italics.

Figure 6

Comparison of the organization of a ribosomal protein gene cluster. In E.coli these genes are organized in three operons; α operon: S13(rpsM)-L17(rplQ); spc operon: L14 (rplN)-L36(rpmJ); S10 operon: S10(rpsJ)-S17(rpsQ). The genes rpmD(L30) of M.pneumoniae and M.capricolum and the genes rpsD(S4) of M.pneumoniae and B.subtilis (88) are located in different positions on the genome. ns = not sequenced; g.d. = gene is deleted.

The subunits a, b and c which build the integral Fo membrane complex, have the required transmembrane segments: six for the a subunit, two for the c subunit and one for the b subunit. The typical signatures for subunits a and c including the binding site for _N_1_N_′-dicyclohexylcarbodiimide (DCCD) are also conserved (66). DCCD inhibits proton transport across the membrane. Downstream of the atpC gene are nine proposed ORFs of which only one, Orf152 shows significant homology to a known protein, the galactose-6-phosphate isomerase, an enzyme involved in sugar metabolism. Interesting are orf521, orf217L and orf531 which code for proteins with the proposed features of prokaryotic lipoproteins as mentioned already in connection with the b subunit (atpF) of the F0F1 ATPase. In all three instances, the proposed cleavage sites are located between Ala and Cys in the preprotein, and between Ser and Cys in the ATPase b subunit. Two other proposed lipoproteins which will be introduced in the next paragraph also carry the proposed cleavage site between Ala and Cys (Fig. 5). In five of six instances the 3rd amino acid in front of the proposed cleavage site is Leu.

Remarkable is also the high percentage of identity (46.6%) between the proposed lipoproteins from orf 521 and orf 531.

The ribosomal protein opérons a, spc and S10

The last region analyzed comprises one of the largest genomic _Eco_RI fragments, the 25 kb long GT9/25.

Approximately one third of the fragment codes for ribosomal proteins. As in E.coli (67–69) the α and spc operon and four proteins from the S10 operon are located consecutively (Fig. 4B and Table 1). The corresponding genes are in the same order except the genes coding for the S4 and L30 protein and the transition between the spc operon and the α operon contains, as in B.subtilis (70), the genes secY, adh, ampM and infA (Fig. 6).

The amino acid sequences of eight ribosomal proteins, their length (Table 3) and the codon usage (Table 4) of the corresponding genes were compared with their counterparts in M.capricolum (71), B.subtilis (72,73) and E.coli. Protein sizes are similar except S5, S14 and L29 (Fig. 7). Some uncertainty is caused by the lack of protein data for N-termini of the ribosomal proteins from M.pneumoniae. The ribosomal proteins S5 in M.pneumoniae and M.capricolum have an elongated N-terminal region and in addition M.capricolum an elongated C-terminal region compared with E.coli and B.subtilis. The differences in length concerning L29 are caused by extension of the two mycoplasmal proteins at the C-terminal end. The larger size of S14 in E.coli happens as a result of an insertion in the middle of the gene/protein. We have no unifying explanation for the observed differences in size, as there is also no systematic pattern emerging. For instance, the smaller proteins are not always found in the bacteria with the smaller genomes. It might be worthwhile to analyze those ribosomal proteins for possible modifications which interact in the ribosome with L29, S5 and S14.

Table 3

Comparision of length and percent identity between ribosomal proteins from selected bacteria

Several conclusions can be drawn comparing the codon usage in eight ribosomal protein genes between four different bacteria (Table 4). The codon usage in M.capricolum is strongly influenced by the low G+C content of the DNA as has already been pointed out several years ago by Ohkubo (71). In all instances the codons with A or U in the third position are clearly preferred. The low frequency, <10% of G or C codon usage in the third position would indicate that ∼20 codons are dispensable. On the contrary, M.pneumoniae uses all codons except AUA for Ile. Bacillussubtilis and E.coli show more preferences. Seven codons in B.subtilis and three codons in E.coli are not used at all. This bias seems to be independent of the G+C content since it is similar at least in M.pneumoniae and B.subtilis. Included in the comparison of codon usage were the P1 and ORF6 protein from M.pneumoniae, because the G+C content of these two genes is clearly higher (74). In these two examples a bias in favour of codons with G and C in the third position is observed but again except the codons for cystein, all other ones are used.

Orf274, orf303 and orf434 most likely code for components of an ABC transporter system (54). The products of orf274 and orf303 exhibit the typical pattern of the ATP binding domain (see orf284 from the dnaA region). The highest scores in protein data bank searches are shared with the GlnQ protein from B.subtilis and the CysA protein from a Synechococcus strain. GlnQ and CysA are components of glutamine and sulphate-thiosulphate ABC transporters. But since the scores of quite a number of other ATP binding domains of ABC transporters are in a similar range it is impossible to determine the specificity by DNA sequence analysis alone. The Orf434 protein shows no significant homologies to other proteins, but based on computer prediction it resembles an integral membrane protein with at least six transmembrane segments. Considering that these three ORFs are organized in an operon-like structure it is likely that they code for one ABC transporter consisting of two identical membrane domains and two different ATP binding domains. Among the residual ORFs of the _Eco_RI fragment GT9/25, the Orf243 protein reveals significant homologies to a hypothetical protein from a Bacillus species and Orf319 protein to a methylase modifying the adenine in an _Eco_RI restriction site (GAATTC). This finding is in agreement with the observation that in genomic M.pneumoniae DNA the _Eco_RI site between the _Eco_RI fragments E7/1.4 and E7/1.85 are protected from cleavage, until after cloning and amplification in a methylase negative E.coli strain (Wenzel, unpublished). Orf611 has homology to the ligoendopeptidase F from Lactococcus lactis and, in addition, a zinc protease pattern from amino acid 384 to 391 His-Glu-Leu-Gly-His-Ser-Val-His. This pattern is closest to the short consensus sequence of the thermolysin family (75). Orf798 and orf760 code for two potential bacterial lipoproteins with the same characteristic features mentioned already. The Orf798 and Orf760 protein have almost 50% identity over the first 250 amino acids and 30% identity over the entire protein but no significant homologies to the other four mentioned proposed lipoproteins. The high degree of identity between the proteins Orf798 and Orf760 as well as between Orf521 and Orf531 may indicate that these proteins arose from a common ancestor by gene duplication. Finally, orf313 and orf127 code for internal peptides of the P1 protein, the main adhesin which interacts with the receptor(s) of the host cell. It seems very unlikely that these ORFs were expressed, since antibodies against the complete P1 protein did not recognize proteins of the size of these peptides. The coding regions show a very high identity of 84.9% in a 1703 bp overlap to the middle part of gene P1 [extending from nucleotide 2317 to 4097, taking the sequence and numbering from Su _et al._ (76)]. At least seven very similar copies from this part of the P1 gene exist dispersed on the bacterial genome, of which one has been located on the _Eco_RI fragment GT9/25. They were named RepMP2/3 (77) or just ‘multiple copies’ from the P1 gene (78). Ruland et al. (77) calculated, that RepMP2/3 would be ∼1800 bp long, based on hybridization experiments with specific oligonucleotides. The DNA sequence data confirm this. These repetitive DNA elements may play a role in antigenic variation or in modifying the receptor specificity, as binding sites for adherence inhibiting monoclonal antibodies are encoded by the DNA stretch representing the RepMP2/3 region (79). Individual copies of RepMP2/3 may be exchanged by gene conversion, as proposed for RepMP5, another repetitive DNA sequence (80) found in the M.pneumoniae genome.

Table 4

Codon usage for the eight ribosomal proteins rpmC, rpsQ, rplN, rplX, rplE, rpsN, rpsE, rplO

One of the problems of the DNA sequence analysis is the unambiguous assignment of the first amino acid for a proposed protein. The following criteria were used. First, all open reading frames were considered starting with ATG and predicting a protein >100 amino acids. If the intergenic regions could be filled with ORFs >100 amino acids using either the codons GTG or TTG these triplets were also considered as start codons. Whenever the protein extension caused by GTG or TTG showed significant homology to the same protein in the data base as the ORF starting with ATG then the start codon which gave the longest protein was selected. ORFs <100 amino acids were considered when the BLASTX program indicated homology or insight to smaller proteins. To minimize the loss of proteins <100 amino acids, the translation products of any sequenced DNA were analyzed routinely by the BLASTX program which screens for homologies at the protein level neglecting stop and start codons. This program picks up short proteins for example some of the ribosomal proteins, however all the peptides without significant homology to other known proteins are excluded. Presently, no reliable method to identify those small proteins is available. In other bacteria, e.g. B.subtilis, gene expression signals are valuable tools for the assignment of protein start sites. Since gene expression signals in M.pneumoniae are not extensively experimentally studied, this method can not be applied. The conserved sequence of the 3′ end of the 16SrRNA from M.pneumoniae M129 (R. Himmelreich, unpublished) suggests that a Shine-Dalgarno (SD) like ribosomal binding site (81) should promote translation initiation, however, unlike in B.subtilis, many proposed genes do not have a Shine-Dalgarno site at the correct position. Studies on the closely related M.genitalium indicate that signals other than a SD-sequence may function as a ribosome binding site (82). Likewise, Sprengart et al. (83) proposed that a region downstream of the start codon specifies a stimulatory interaction between the mRNA and 16S rRNA. Therefore, the best proof for a protein start is the amino acid sequence of the N-terminal region of the protein in question. The small number of proteins encoded by the M.pneumoniae genome and the improved methods in protein biochemistry make it likely, that a substantial part of N-termini will be sequenced within a reasonable period of time.

Figure 7

Comparison of ribosomal proteins with differences in length. (A) Ribosomal protein L29; (B) ribosomal protein S5; (C) ribosomal protein S14; bacsu: B.subtilis; ecoli: E.coli; mycca:M.capricolum; mycpn:M.pneumoniae.

Acknowledgements

We thank E. Pirkl for excellent technical assistance, R. Frank and A. Bosserhoff for synthesis of oligonucleotides, B. Reiner for her expertice in computer data analysis, V. Wasinger for critical reading of the manuscript, I. Schmid for preparing the manuscript, J. Pyrowolakis for subcloning the _Eco_RI fragment GT9/25 and last, but not least, H. Schaller for financial assistance during a critical period and his encouragement throughout our work.

This research was supported by a grant from the Deutsche Forschungsgemeinschaft (He 780/5–1).

Note Added in Proof

Since this paper was submitted the complete nucleotide sequence of the Mycoplasma genitalium genome has been published [Fraser et al. (1995) Science, 270, 397–403].

At this stage we could not consider anymore all the data from this publication. But, since there were some discrepancies to the data from Bailey and Bott (50) on the order and direction of genes of the dnaA region of M.genitalium, we included the results of Fraser et al. in our comparative analysis on bacterial dnaA regions. (Latest accessible database version, January 1996.)

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