4420–4449 Nucleic Acids Research, 1996, Vol. 24, No.

22  1996 Oxford University Press

Complete sequence analysis of the genome of the

bacterium Mycoplasma pneumoniae
Ralf Himmelreich, Helmut Hilbert+, Helga Plagens, Elsbeth Pirkl, Bi-Chen Li
and
Richard Herrmann*

Zentrum für Molekulare Biologie Heidelberg, Mikrobiologie, Universität Heidelberg, 69120 Heidelberg, Germany

Received August 22, 1996; Revised and Accepted October 10, 1996 DDBJ/EMBL/GenBank accession no. U00089

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ABSTRACT of the lack of established standard genetic tools like conjugation
or transformation with self-replicating vectors (4). These disad-
The entire genome of the bacterium Mycoplasma vantages can be compensated now to a large extent by the
pneumoniae M129 has been sequenced. It has a size of methods of molecular biology.
816 394 base pairs with an average G+C content of 40.0 Morowitz pointed out in 1984, that mycoplasmas would be
mol%. We predict 677 open reading frames (ORFs) and suitable candidates for defining the genetic constitution of a
39 genes coding for various RNA species. Of the minimal self-replicating cell (5). The advantage of these bacteria
predicted ORFs, 75.9% showed significant similarity to for such studies (6,7), mainly due to their small genome size, was
genes/proteins of other organisms while only 9.9% did so obvious that several initiatives were started to sequence five
not reveal any significant similarity to gene sequences different mycoplasma genomes: Mycoplasma genitalium (8,9),
in databases. This permitted us tentatively to assign a M.pneumoniae (10), Mycoplasma capricolum (11), Mycoplasma
functional classification to a large number of ORFs and mycoides (12) and a species from the related genus Ureaplasma,
to deduce the biochemical and physiological properties Ureaplasma urealyticum (13). So far, only the complete sequence
of this bacterium. The reduction of the genome size of of the M.genitalium genome has been published (9) which, with
M.pneumoniae during its reductive evolution from 580 070 bp, is the smallest bacterial genome known so far. In the
ancestral bacteria can be explained by the loss of genus Mycoplasma, M.pneumoniae and M.genitalium are the
complete anabolic (e.g. no amino acid synthesis) and closest related species. We report in this publication the complete
metabolic pathways. Therefore, M.pneumoniae de- nucleotide sequence of the genome of M.pneumoniae, which thus
pends in nature on an obligate parasitic lifestyle which provides information on a second small bacterial genome. All
requires the provision of exogenous essential metabo- M.pneumoniae genes which had been already sequenced were
lites. All the major classes of cellular processes and reanalyzed except for the P1 operon (14). Our sequencing
metabolic pathways are briefly described. For a number strategy, early results and a detailed description of M.pneumoniae
of activities/functions present in M.pneumoniae accord- as an experimental system have been recently published (10).
ing to experimental evidence, the corresponding genes
could not be identified by similarity search. For instance
we failed to identify genes/proteins involved in motility, MATERIALS AND METHODS
chemotaxis and management of oxidative stress. Mycoplasma strain
INTRODUCTION The strain Mycoplasma pneumoniae M129 (ATTC 29342) in the
18th broth passage was used to construct an ordered cosmid library
The bacterium Mycoplasma pneumoniae has a genome size of containing the complete genome (15). This cosmid library was the
∼800 kb and completely lacks a cell wall. The bacterium is basis for the DNA sequence analysis. We selected this specific
surrounded by a cytoplasmic membrane only, which contains bacterial strain because it has been used in cytadherence and
cholesterol as an indispensable component. Mycoplasma pneumo- pathogenicity studies (2,16,17). The strain in the 20th broth passage
niae is a human pathogen, causing ‘atypical pneumonia’ (1) was still infectious in hamsters (H. Brunner, unpublished data).
usually in older children and young adults. As a surface parasite,
it attaches to the host’s respiratory epithelium by means of a DNA sequencing
differentiated terminal structure termed attachment organelle or tip
structure. For a long time, research activities mainly focused on Using the enzymatic dideoxy chain-termination method (18), the
pathogenicity-related topics such as studies on cytadherence (2), sequence data for this study were exclusively generated on a
vaccination and diagnosis (3). Mycoplasma pneumoniae was not fluorescent-based sequence-gel reader (Model 373A, Applied
considered as an organism suitable for basic studies partly Biosystems). Sequencing strategies and methods were as de-
because of its fastidious growth requirements and partly because scribed in Hilbert et al. (10).

*To whom correspondence should be addressed. Tel: +49 6221 54 68 27; Fax: +49 6221 54 58 93; Email: r.herrmann@mail.zmbh.uni-heidelberg.de
Present addresses: +QIAGEN GmbH, 40724 Hilden, Germany and
Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
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Computer assisted analysis ORFs (49.2%) were functionally assigned, based on significant
sequence similarities to genes or proteins from other organisms
Sequence assembly, map drawing and multiple alignments were with known functions (e.g. ribosomal proteins) or at least known
done with the Lasergene program package (DNA STAR). categories of function (e.g. proteins involved in cytadherence).
Other analyses were performed with the HUSAR (Heidelberg Significant similarities to proteins without known function from
Unix Sequence Analysis Resources) program package release 4.0 other bacteria, mostly M.genitalium, were shown for 181
at the German Cancer Research Center, Heidelberg, Germany. proposed ORFs (26.7%). We also included in this group those
This package is based on the GCG program package version M.pneumoniae proteins which were identified in protein extracts
Unix-8.1 of the Genetics Computer Group, Wisconsin. For of M.pneumoniae by monospecific antibodies or by the N-terminal
searching the DNA and protein databases [SWISS-PROT (19) and amino acid sequences of enriched proteins (26,27). The group of
PIR (20)] the FASTA (21) and BLAST (22) programs (BLASTX, ORFs without significant similarity or without indication for their
BLASTN and BLASTP) were used. Conserved motifs in proteins in vivo expression comprised 109 members (16.1%); 42 of them
and peptides were identified by using the program PROSITE carry characteristic motifs, which are not sufficient for defining
(23). Open reading frames (ORFs) were calculated by the a function. Examples of such motifs are the leucine zipper (29
program FRAMES allowing AUG (or GUG, UUG) as start

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cases; refered to all predicted ORFs), the typical prokaryotic
codons using the Mycoplasma translation table where UGA lipoprotein sequence pattern (46 cases) or ATP- and GTP-binding
codes for tryptophan (24). The G+C content was calculated by the sites (73 cases). In addition all predicted gene products were
program WINDOW. Codon usage was performed with the analyzed by programs for structure predictions, e.g. coiled/coiled
program CODONFREQUENCY. structures (29 cases) or transmembrane segments (275 cases).
The programs TopPred 1.1.1 (Manuel G. Carlos, Ecole The latter result supports the analysis of cell fractionation
Normale Superieure, Laboratoire de Genetique Moleculaire, experiments which indicate that the membrane fraction contains
Paris, France) and PSORT (25) (http://psort.nibb.ac.jp/) were ∼50% of the total proteins estimated by SDS-PAGE. About 8%
used for the prediction of transmembrane domains and the of the genome is composed of repetitive DNA elements RepMP1,
membrane topology of proteins. RepMP2/3, RepMP4 and RepMP5, while only 67 of all predicted
Each ORF analysis is accessible as a File Maker Pro (Claris) ORFs (9.9%) code for a product without any similarity to a
database which can be accessed at our world wide web (www) known RNA or protein.
site (http://zmbh.uni-heidelberg.de/M_pneumoniae). It contains, Finally, 58 gene families were defined comprising 298 proteins
besides genome and cosmid position of each ORF/gene, data with at least two but frequently with more paralogs; these are
about expression, availibility of antibodies, comments, literature, proteins with similarities within the same species (see www pages).
prosite patterns, amino acid composition and database search The proposed ORFs are not equally distributed over the
homology scores. All the annotations in this paper were done on genome. A lower coding density coincides with regions of lower
the basis of the highest score values. or higher G+C content than the average. There are regions with
a G+C content of up to 56 mol%. These regions code almost
Accession number exclusively for the gene P1 and gene ORF6 of the P1 operon, the
The complete M.pneumoniae sequence has been annotated in repetitive DNA sequences RepMP4, RepMP2/3, RepMP5 and
GenBank (NCBI) with the accession number U00089. tRNAs (for details see www pages).
The P1 protein, the main adhesin, is essential for adherence of
RESULTS AND DISCUSSION M.pneumoniae to its host cell (28) and the ORF6 gene product
which is only found as a cleavage product, namely a 40 and 90 kDa
The strategy and methodology for sequencing the complete protein, instead of the expected 130 kDa protein, is involved in an
genome has been described by us recently (10). A total of 2 415 202 as yet unknown manner in cytadherence (14). Gene P1 contains
nucleotides primary sequence data were provided by 6385 a copy each of RepMP2/3 and RepMP4 and gene ORF6 one of
sequencing reactions. Each strand of the genome was completely RepMP5 (29). In addition, several copies of each of these
sequenced at least once. The direct sequencing approach, repetitive DNA sequences can easily be recognized by their relative
combining primer walking with a limited shotgun strategy based high G+C content (Fig. 2).
on a complete cosmid and plasmid library considerably facilitated At the other extreme is the proposed origin of replication
the assembly of the individual sequences to the entire genome around nucleotide position 205 000 (pcosMPK05, dnaA region),
sequence. The average redundancy of the sequencing was 2.95 with a G+C content of only 26 mol% (10).
(calculated for both strands). This very low redundancy was Other regions with a low G+C content do not show a similar
achieved by the use of 5095 oligonucleotides. obvious coding pattern, but proposed ORFs coding for lipoproteins
The complete M.pneumoniae genome has a size of 816 394 bp or the hsd modification/restriction system are frequently located
and a G+C content of 40.0 mol%. Altogether 677 open reading in these regions.
frames (ORFs) and 39 genes coding for various RNA species The total length of all coding regions is 724 174 bp. The average
were predicted. All ORFs were sorted into categories according coding density of 88.7% was calculated for the M.pneumoniae
to their proposed functions (Tables 1 and 2; Fig. 1). Only 333 genome which gives an average gene size of 1011 bp. Similar

Figure 1. (Following two pages) The gene map of the complete M.pneumoniae genome. The arrows indicate the position and the size of the predicted ORFs. The colour
refers to the functional category in which the ORFs are sorted. The complete name of an ORF can be deduced by the cosmid name above the horizontal scale-line
and the number below the arrows (e.g. the ORF name of the first complete arrow in this figure is E07_orf1113). Rectangles above the scale-line indicate the size and
the position of different repetitive DNA sequences (see also Table 4).
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4422 Nucleic Acids Research, 1996, Vol. 24, No. 22
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4423 No.122
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4424 Nucleic Acids Research, 1996, Vol. 24, No. 22

Table 1. Predicted functions and classification of all M.pneumoniae ORFs

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4425 No.122
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Table 1. Continued
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4426 Nucleic Acids Research, 1996, Vol. 24, No. 22

Table 1. Continued
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4427 No.122
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Table 1. Continued
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4428 Nucleic Acids Research, 1996, Vol. 24, No. 22

Table 1. Continued
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4429 No.122
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Table 1. Continued
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4430 Nucleic Acids Research, 1996, Vol. 24, No. 22

Table 1. Continued
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4431 No.122
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Table 1. Continued
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4432 Nucleic Acids Research, 1996, Vol. 24, No. 22

Table 1. Continued
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Table 1. Continued

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MG is the name of the corresponding ORF in M.genitalium (9).

coding densities have been also estimated for the smaller M.genita- 50 and 56 mol%) and for all 50 ribosomal protein genes (42.8 mol%)
lium genome (9) and for the genome of Haemophilus influenzae as an example for frequently translated genes. Codon usage of the
which is more than twice as large (30). The length of the proposed low and high G+C content subfractions is clearly influenced by the
proteins in M.pneumoniae ranges from 37 (4.3 kDa) to 1882 (209.4 DNA composition, favouring either codons with G/C or A/T at the
kDa) amino acids (Fig. 3). One of the largest proteins is the third position. The codon usage pattern differs also for the complete
cytadherence accessory protein HMW2 (F10_orf1818) and the genome and for genes which are frequently expressed like the ones
smallest identified protein is the 37 amino acid ribosomal protein coding for ribosomal proteins.
L36 (GT9_orf37). For practical reasons we introduced at the The most frequently used codons are AUU (Ile, 4.6%); AAA
beginning of the sequence analysis a cut-off point of 100 amino (Lys, 4.6%); UUU (Phe, 4.3%); GAA (Glu, 4.2%) and UUA
acids for proposed proteins unless we found smaller proteins such (Leu, 3.9%) and the most common amino acids are Leu (10.3%),
as some of the ribosomal proteins during the initial BLASTX Lys (8.5%), Ile (6.6%), Ala (6.6%) and Val (6.5%). The high
homology search. All intergenic or non coding regions were value for Lys is in agreement with the relative high percentage of
reanalyzed with a cut-off point of 50 amino acids and searches were proposed proteins with calculated isoelectric points between pH
done for specific small proteins. However, we cannot exclude the 9 and 12 (Fig. 4). The least frequently used codons are UGC (Cys,
possibility that some of the smaller proteins, not showing similarities 0.2%); CGA (Arg, 0.25%); AGG (Arg, 0.29%); AGA (Arg,
to known proteins from other organisms, have been missed in our 0.4%) and UGU (Cys, 0.55%).
analysis. All M.pneumoniae gene products were classified (Table 1 and 2),
The codon usage of M.pneumoniae is summarized in Table 3. We with some minor modifications, in accordance with criteria
compared it for all proposed genes, for the subsets of genes with a introduced for Escherichia coli (31) and adapted for the
low G+C (content below 35 mol%) and high G+C content (between classification of putative genes from H.influenzae. We added
4434 Nucleic Acids Research, 1996, Vol. 24, No. 22

Table 2. Summary of the functional classification of the ORFs

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Figure 2. Distribution of the G+C content of the coding sequences of all
M.pneumoniae ORFs.

DNA replication and repair

The central enzyme for DNA replication in bacteria is the DNA
polymerase III holoenzyme (32), which consists of 10 subunits in
E.coli, a DNA polymerase subunit α and nine accessory proteins (ε,
υ, τ, γ, δ, δ′, χ, ψ and β). Mycoplasma pneumoniae codes for two
potential α subunits (the gene name in the literature is either dnaE
or polC). Both proposed α subunits, A19_orf872 and B01_orf1443,
differ in length and also in their degree of similarity to the α subunits
from E.coli and Bacillus subtilis. The protein from B01_orf1443
shares the highest similarity with the α subunit from Gram-positive
bacteria including the motif for a 3′–5′ exonuclease activity which
is typical for these bacteria. In contrast, the orf A19_orf872 is most
similar to the α subunit from E.coli and does not contain a 3′–5′
exonuclease domain. The 3′–5′ exonuclease activity in E.coli is
encoded by a separate gene (dnaQ), which has not been found in
M.pneumoniae. Of the other subunits which build the DNA
polymerase III holoenzyme in E.coli (32) only the subunits β
(dnaN), δ′(holB), γ and τ (dnaX) are present in M.pneumoniae,
indicating a simplified replication complex compared with the
Gram-negative bacteria E.coli and H.influenzae. Presently, it cannot
be excluded that other proteins replace these subunits in M.pneumo-
niae. A true comparison with a phylogenetically closer related
Gram-positive bacterium like B.subtilis is not possible since the
Bacillus DNA polymerase III holoenzyme complex has not been
defined as yet and the nucleotide sequence of the entire B.subtilis
genome has not been completed.
Mycoplasma pneumoniae does not code for a DNA polymerase
‘cytadherence associated proteins’ to the category of cell I (polA)-like DNA repair enzyme. Instead, we find a truncated
envelope–surface structures, since evidence is mounting, that polA gene (A19_orf291) comprising only the 5′–3′ exonuclease
M.pneumoniae possesses a cytoskeleton-like organization which domain, whereas in E.coli and B.subtilis the polA gene is much
stabilizes the bacterium and protects it against osmotic lysis (2). larger and codes for the 5′–3′ exonuclease and a 5′–3′ polymer-
The category of transport and binding proteins was altered by ase-specific domain.
subdivision into three groups namely, into PTS-, ABC- and other Experimental results on DNA polymerase enzymatic activities
transport systems. To facilitate the orientation on the gene map we in mycoplasmas are confusing. It was claimed that the DNA
added a list which contains all proposed ORFs and RNAs in polymerase III of Mollicutes lacks the 3′–5′ exonuclease proof-
nummerical order (Table 4). reading activity in general (33) and this was taken as an
More details on this very general analysis will be made public on explanation for the observed genetic instability of many Mollicutes
the www (http://www.zmbh.uni-heidelberg.de/M_pneumoniae). species (4). Recently, the nucleotide sequence of the polC gene of
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Figure 3. Distribution of all M.pneumoniae proteins according to their molecular weight.

Mycoplasma pulmonis and experimental results on enzyme The number of genes involved in DNA repair is considerably
purification and characterization of enzyme activities were pub- smaller in M.pneumoniae than in the ‘standard’ eubacteria E.coli
lished (34). The results indicated that the polC gene from and B.subtilis or even H.influenzae with the smaller genome.
M.pulmonis also codes for a 3′–5′ exonuclease, and that the size of Mycoplasma pneumoniae codes only for 13 of the genes known
the predicted PolC protein, 1435 amino acids, is very similar to the to be involved in excision repair of DNA, recombination and SOS
PolC homolog B01_orf1443 in M.pneumoniae and that the repair. Thus the genes recB, recC, recD, recG and ruvC involved in
polymerase could be inhibited by compounds specific for PolC recombination are missing as well as the genes recN, recO, recQ and
proteins of Gram-positive bacteria. Furthermore, the authors recR involved in SOS repair in E.coli. Nevertheless, a rudimentary
provided some experimental evidence for a second, smaller stock of enzymes has been conserved in M.pneumoniae to permit
enzyme with DNA polymerase activity. Considering the charac- homologous recombination [RecA, Ssb, PolA (see above), GyrA,
terization data of DNA polymerase activities in M.pulmonis and GyrB, RuvA and RuvB] (36), excision repair (37) and a kind of
the nucleotide sequence data on DNA polymerase genes of truncated SOS repair (38). In particular missing is the lexA gene
M.pneumoniae and M.genitalium (9,35), one can conclude that at which plays a central role in regulating the SOS response including
least these three Mycoplasma species have two DNA polymerase the expression of the recA gene in other bacteria.
(polC) genes coding for a larger protein (≈1400 amino acids) with We were also unable to find components of the so called
a 3′–5′ exonuclease activity and with the highest sequence mismatch-repair system encoded by the mutS, mutL and mutH
similarities to the Gram-positive B.subtilis polymerase III. There- genes. Since bacteria which normally carry the mut genes show
fore it is unlikely that an increased mutation frequency is caused by a reduced genetic stability, if these genes are mutated, it seems
the DNA replication process. The nucleotide sequence of the likely that the absence of these genes in mycoplasmas causes an
smaller Pol III homolog (≈100 kDa) of M.pneumoniae and increased mutation rate (65).
M.genitalium (9,35) resembles more the polC gene from the
Gram-negative E.coli. This is also emphasized by the absence of Transcription
the 3′–5′ exonuclease domain in the proposed genes. The gene for
the smaller, Gram-negative typical PolC has not yet been found in The DNA dependent RNA polymerase of M.pneumoniae is
M.pulmonis, but during the purification of the larger PolC, a second coded by the conserved genes rpoA (α subunit), rpoB (β subunit),
polymerase activity lacking exonuclease activity has been identi- rpoC (β′ subunit) and rpoE (δ′ subunit). The only sigma factor
fied. The function of the exonuclease negative DNA polymerase found (H91_orf499) shares the highest similarity with the sigma
can only be elucidated experimentally and it remains to be seen if factor SigA from B.subtilis (39). Presently, not enough experimental
it can substitute for the function of the polymerase I (PolA) in data are available for defining promoter sequences in M.pneumo-
combination with the proposed 5′–3′ exonuclease of the truncated niae. The promoter of only three genes/operons have been
polA gene (A19_orf291). This topic has been also discussed for determined experimentally by primer extension. These genes are
M.genitalium (35). the P1 operon (14), the ribosomal RNA operon (40) and
In addition to the DNA polymerase many more gene products F10_orf405 (27). The –10 region and to a lesser extent the –35
are necessary for DNA replication, e.g. initiation, elongation and region of these three examples are comparable with consensus
termination (32). The most obvious functions missing in promoters sequences in B.subtilis (41). Termination of transcription
M.pneumoniae according to the sequence analysis are an RNaseH seems to be independent of the termination factor Rho, since the
for primer removal and a protein for the termination of corresponding gene could not be found. Transcription stops on
replication. typical terminator sequences which are short interrupted palin-
4436 Nucleic Acids Research, 1996, Vol. 24, No. 22

Table 3. Codon usage of different sets of M.pneumoniae ORFs: all 677

ORFs; ORFs with a G+C content <35 mol%; codon usage of the
adhesin P1 and ORF6 (high G+C content); ribosomal ORFs as
examples for frequently expressed proteins

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Figure 4. Distribution of all M.pneumoniae proteins according to their
predicted isoelectric point (IP).

Gene expression and regulation

Regulation of gene expression in M.pneumoniae has not been
studied so far. Therefore we do not know how this bacterium
coordinates the synthesis of those gene products which are
essential for reproduction. Also, M.pneumoniae has to sense and
respond to environmental changes. This requires a signal
transduction system. The presence of only one sigma factor
(sigA, H91_orf499) which is also the only one of all proposed
proteins showing the characteristic helix–turn–helix (HTH)
motif, suggests that the response to external stimuli is not
controlled by the level of expression of alternative sigma factors.
The presence of a cis-acting conserved palindromic repeated
sequence in front of four heat shock genes, similar to the ‘CIRCE’
element first identified in B.subtilis (43) and the identification of
the proposed repressor (C09_orf351, hrcA), indicates that the
heat shock response in M.pneumoniae is regulated by the
interaction of this repressor with the CIRCE element, and
provides an example for a negative regulation of gene expression
in M.pneumoniae.
The two-component signal transduction system (44), consisting
of a sensor and a response regulator, which has been found in
many prokaryotic and eukaryotic organisms is believed to be
essential for all cells. Nevertheless, based on sequence similarity
we were unable to detect any such system in M.pneumoniae.
Concerning other proteins with regulatory functions we
identified several GTP-binding proteins and other proteins like
the virulence associated protein vacB (K04-orf726). These
regulatory proteins act by unknown mechanisms.

Translation
The translation machinery of M.pneumoniae is rather extensive.
dromic regions followed by a run of U residues. The Nus About 15% of all proposed ORFs, are involved in translation
transcription termination factors, of which NusA (E07_orf540) including 19 tRNA synthetases, 50 ribosomal proteins, various
and NusG (D09_orf320) are present, may play a role in the factors and enzymes, 33 tRNAs, one ribosomal RNA operon with
termination of transcription. NusB and NusC are absent. NusA is one copy of each 5S, 16S and 23S rRNA (45), and a gene coding
involved in termination and NusG in antitermination in other for the 10Sa RNA. The conservation of the 10Sa RNA which
bacteria. Finally, GreA promotes elongation by the RNA functions as tRNA and mRNA and is implicated in trans-translation
polymerase by utilizing a novel transcript-cleavage reaction (42). (66), is interesting in evolutionary terms. Three exceptions are
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Table 4. List of the proposed ORFs, RNAs and REPs in numerical order starting with E07_orf540o on the gene map (Fig. 1)

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4438 Nucleic Acids Research, 1996, Vol. 24, No. 22

Table 4. Continued
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4439 No.122
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Table 4. Continued
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4440 Nucleic Acids Research, 1996, Vol. 24, No. 22

Table 4. Continued
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4441 No.122
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Table 4. Continued
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4442 Nucleic Acids Research, 1996, Vol. 24, No. 22

Table 4. Continued
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4443 No.122
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Table 4. Continued
4444 Nucleic Acids Research, 1996, Vol. 24, No. 22

Table 4. Continued the ORF6 gene of the P1 operon (40 kDa protein = C, 90 kDa
protein = B). The gene for A is still unknown. Another criterion
for a putative protein of the cytoskeleton-like structure is its
partitioning into the Triton X-100 insoluble fraction after treating
M.pneumoniae with this detergent. This fraction is ill defined and
comprises ∼50 proteins, of which only a subfraction is associated
with the cytoskeleton and/or cytadherence. The following
proteins have been identified as most likely components of a
cytoskeleton (2): HMW1 (H08_orf1018), HMW2 (F10_orf1818;
Krause, submitted), HMW3 (H08_orf672), P200
(D02_orf1036o) (49), P65 (F10_orf405) (27). These proteins,
noteworthy: the lack of the ribosomal protein S1, of the peptide
with the exception of HMW2, share some common peculiar
chain release factor 2 (RF2) and of the glutaminyl-tRNA
features, like an extended acidic proline rich domain and an
synthetase. So far, quite a number of Gram-positive bacteria
abnormal migration in SDS-PAGE (49). The adhesin P1 is mainly
including Bacillus or Lactobacillus species also lack the S1

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distributed in the membrane fraction and to a lesser extent in the
protein and the glutaminyl-tRNA synthetase (46).
Triton X-100 insoluble fraction (50).
One of the functions of the S1 protein is to bind the mRNA to
A large number of proposed ORFs contain sequences with high
the 30S small ribosomal subunit. Therefore, it was argued that
similarities to subregions of either the P1 protein or the ORF6
ribosomal binding sites in front of many genes (47) of B.subtilis
gene product of the P1 operon. The coding DNA sequences
compensate for the missing S1 protein. The Shine–Dalgarno
correspond to the repetitive DNA sequences RepMP2/3 (P1),
sequences are so well conserved, that they could be used routinely
RepMP4 (P1) and RepMP5 (ORF6). Preliminary experiments
as a good indicator for proposing ORFs in the B.subtilis genome
indicate that the proposed ORFs are not expressed under standard
sequencing projects, but this does not apply to M.pneumoniae.
laboratory conditions. It has been observed that another indepen-
The Shine–Dalgarno sequence is in many instances not well
dent isolate of M.pneumoniae, the strain FH, carries a different
conserved or missing altogether, even in genes for which we
copy of RepMP2/3, RepMP4 and RepMP5 in its P1 operon than
know the translational initiation sites from independent studies.
the M.pneumoniae strain M129 which is the subject of this paper
Of the 20 standard tRNA-synthetases, the glutaminyl-tRNA
(51,52). All experimental data so far show that only the repetitive
synthetase is the only one not detected in M.pneumoniae. Studies
sequences which are part of the P1 operon are expressed. The
on tRNA synthetases in Gram-positive bacteria have indicated
exchange of these copies presumably takes place by gene
that this enzyme is dispensable. Bacillus subtilis solves this
conversion as was indicated by DNA sequence analysis of the
problem by charging the tRNAGln first with glutamate which is
corresponding RepMP5 sequences in M.pneumoniae strains
subsequently converted to glutamine by an amido transferase.
M129 and FH. Different is the situation with RepMP1, copies of
The glutamyl tRNA synthetase aminoacylates both tRNAGlu and
which seem to be part of several expressed proteins.
tRNAGln. The corresponding amido transferase has not yet been
RepMP1-specific antibodies recognize several proteins on west-
identified in M.pneumoniae, therefore it is still an open question
ern blots of M.pneumoniae protein extracts (26).
as to how glutamine is bound to its tRNA.
Only little is known about cell division in M.pneumoniae. The
Finally, the modified codon usage by M.pneumoniae, reading
lack of mutants, especially of conditional mutants, has prevented
UGA as tryptophan instead of a stop codon, requires the absence
a detailed analysis. So far, the two proteins FtsZ and FtsH are
of the peptide chain release factor 2 (RF2) and the presence of the
classified as cell division proteins in analogy to their function in
release factor 1 (RF1). The latter recognizes the stop codons UAG
other bacteria (53). Other genes involved in chromosome
and UAA and RF2 the stop codons UGA and UAA. Since the
partitioning or septum formation have not been identified in
UGA codon is frequently located within a gene it is essential to
M.pneumoniae. Interesting problems to study might include the
exclude RF2 to prevent the premature termination of proteins.
possible interaction of FtsZ with components of the cytoskeleton-
like structure, which seems to play a key role in cell division, or
Surface structure, cytadherence-associated proteins and the effects of cellular asymmetry on cell division and the
cell division formation of daughter cells. Other genes known to be involved in
This category comprises the adhesins and the cytadherence cell division in E.coli, the muk and min genes or additional fts
associated proteins, including the components of the cytoskeleton- genes were not found in M.pneumoniae (53).
like structure, the function of which is probably to stabilize and
maintain the shape of the wall-less mycoplasma, to direct proteins Lipoproteins
to certain regions in the membrane and to keep them in these
positions (2). Adherence to the receptor(s) of the host cell depends Altogether 46 proteins were identified as lipoproteins based on the
on the tip structure. The correct assembly of the adhesin P1 following characteristic lipoprotein-specific features (54): (i) one or
(E07_orf1627) and the 30 kDa adhesin-related protein on the tip more basic amino acids among the first 5–7 amino acids of the
structure (H08_orf274) is necessary for attachment. The tip structure N-terminus, (ii) a hydrophobic signal peptide and (iii) a cysteine
is an interesting example for bacterial cellular asymmetry (48). residue immediately downstream of the signal peptide, which is
The cytadherence-associated proteins were originally defined available for modification by the transfer of the diacylglyceryl
by hemadsorption-negative mutants which had lost certain moiety from glycerophospholipid to its sulfhydryl group. The
proteins like the so called high molecular weight proteins HMW1, precursor prolipoprotein with the modified cysteine is subsequently
HMW2 and HMW3, the adhesin P1 and the proteins named A, cleaved in M.pneumoniae by a specific signal peptidase (signal
B and C (2,28). B and C are most probably the gene products of peptidase II). The modified cysteine will then be the first amino
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acid of the processed protein. The cleavage site including the for its inability to synthesize essential compounds like amino
cysteine and the three (positions –3, –2 and –1) upstream located acids. Three different transport systems, mainly involved in
amino acids, is to some extent conserved (–3: 37×L, 6×F, 1×A, import, were found in M.pneumoniae: (i) the ABC transporter
1×V; –2: 19×S, 10×A, 8×T, 6×V, 2×I; –1: 37×A, 7×S, 1×G). system (57) consisting of two ATP-binding, two membrane-span-
The number of lipoproteins in M.pneumoniae is relatively high ning and one substrate-binding domain which are frequently
compared with the Gram-negative bacteria E.coli and H.influenzae. present on separate polypeptides, but sometimes also consist of
Even in the closely related M.genitalium only 21 putative two or three different domains located on the same peptide
lipoproteins could be found by analyses of the published data (9). (D12_orf634 or D12_orf623), (ii) the phosphoenolpyruvate:
The lipoproteins of M.pneumoniae can be divided into six carbohydrate phosphotransferase system (PTS), (58) and (iii)
subgroups based on sequence similarities; also included in these facilitated diffusion systems with transmembrane proteins func-
groups are proteins with similarities to lipoproteins but without tioning as specific carriers. Mycoplasma pneumoniae codes for
the lipoprotein signature at the N-terminal end. Quite a number 43 genes involved in the above mentioned transport systems
of these proposed genes with high similarities are organized in according to the present status of annotation. In addition, there are
tandem. For instance seven lipoproteins and one protein without several proposed proteins with 6 or 12 transmembrane segments

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the lipobox but with otherwise extended similarities are located which are candidates for membrane-spanning domains of trans-
between genome positions 249 627 and 256 463 (cosmid port systems. The relatively low number of proteins listed in
pcosMPE09). A gene family, with 13 proposed ORFs including Table 1 indicates that at least some of the systems might not be
five lipoproteins, is located between 306 862 and 320 524 very substrate specific, e.g. the transport systems for amino acids.
(cosmid pcosMPD02). Presently it is unclear whether all of the Transport systems for histidine, glutamine, an ORF showing
proposed genes are expressed. significant similarity to a probable aromatic amino acid permease
In vivo labelling of M.pneumoniae with 14C-labelled palmitic from yeast and an ABC transport system for oligopeptides were
acid and protein analysis by SDS–PAGE reveal, instead of the identified based on similarity of the ATP-binding domains of
expected 46 lipoproteins, only between 20 and 25 lipoproteins ABC transporters.
(Pyrowolakis, unpublished data). This discrepancy could be Surprisingly, we could not identify a transport system for the
explained either by a regulated expression which only allows precursors for RNA and DNA synthesis, namely adenine,
some of the several tandemly organized lipoproteins to be guanine, uracil and thymine which are essential components of
synthesized or that the labelling with palmitic acid was not mycoplasma growth media.
sensitive enough or that some lipoproteins carry fatty acids other In this context one has to be aware of the ambiguity in the
than palmitic acid. Only four of all the proposed lipoproteins identification of ABC transport proteins on the basis of sequence
show significant similarities to other bacterial genes beside the similarity of the ATP-binding proteins with respect to the
ones from M.genitalium. These include A05_orf380V [high predicted substrate to be transported, since database searches
affinity transport system P37 with unknown specificity from indicate numerous candidates with different specificities but with
Mycoplasma hyorhinis (55)], D09_orf384 (aerobic glycerol-3- very similar, high score values. All the annotations in this paper
phosphate dehydrogenase, glpD), H03_orf213 (uridine kinase) were done on the basis of the highest score values. Therefore it
and D02_orf207 (ATP synthase b subunit (atpF). might be possible that the predicted specificity disagrees with the
The processing of the prolipoprotein to the mature lipoprotein in in vivo activity in M.pneumoniae. Additional information from
E.coli requires the three enzymes prolipoprotein diacylglyceryl similarities to transmembrane domains or the substrate-binding
transferase, prolipoprotein signal peptidase and apolipoprotein proteins is only rarely at hand, since, in general, similarities
transacylase. We find in M.pneumoniae only the transferase which among these domains are not well conserved. Even in positive
catalyzes the thioether linkage between the diacylglycerol and the examples, the score values are relatively low. Sometimes
cysteine and the peptidase which cleaves in front of the cysteine additional circumstantial evidence is derived from an operon-like
following the signal peptide. The transacylase could not be identified organisation of the genes coding for ABC transporters, e.g. the
either in M.pneumoniae nor in M.genitalium (9). Therefore it is still unspecified ABC transporter consisting of the proteins P69, P29
an open question if a third fatty acid is linked to the cysteine by an and P37 from nucleotide 519 560 to 523 050 (A05_orf542,
amide bond as has been found for lipoproteins of E.coli. A05_orf244 and A05_orf380V). A05_orf542 could act as the
The absence of a periplasmic space provides reasons for the membrane-spanning domain, A05_orf244 as the ATP-binding
existence of a large number of lipoproteins. For surface-exposed domain and A05_orf380V, as a putative lipoprotein which could
proteins which have to function on the outside, anchoring them via function as a substrate-binding protein. These proteins were also
long chain fatty acids at the M.pneumoniae cell membrane is an identified by their significant similarity to the corresponding
efficient way. Already known examples are substrate-binding genes in M.hyorhinis (55).
proteins of transport systems or proteins possibly involved in In M.pneumoniae the ABC transport system for oligopeptides
antigenic variation for evasion of the immune system of the host, as consists of two different transmembrane [G07_orf376 = amiD (=
has been shown for other mycoplasmas (56). Nothing is known oppC in B.subtilis); G07_orf389a = oppB] and ATP-binding
about the fate of the cleaved signal peptides, as to whether they are domains (G07_orf851 = oppF , G07_orf423 = oppD). It is also
degraded or recycled. organized in an operon-like arrangement from nucleotide 750 865
to 756 948. In striking contrast to B.subtilis, the substrate-binding
Transport systems domain (oppA) is absent in M.pneumoniae. Since an oppA
homolog is also absent in M.genitalium a sequencing or
In light of the scarcity of metabolic pathways and the marked annotation error seems unlikely. It remains to be experimentally
dependence on exogenous nutrients (Table 1, Fig. 5), we expected determined whether the substrate-binding protein is dispensable
M.pneumoniae to code for many transport systems to compensate or is part of one of the transmembrane or ATP-binding proteins.
4446 Nucleic Acids Research, 1996, Vol. 24, No. 22

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Figure 5. Schematic diagram of the metabolic pathways of M.pneumoniae deduced from Table 1. Shaded arrows with question marks indicate missing enzymatic
activities.
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It is also possible that one or more of the lipoproteins function as Mollicutes. These components can be used for the synthesis of
substrate-binding proteins. ribonucleotides by the salvage pathway as predicted from the
There is also evidence for bacterial ABC export systems in enzymes listed (Table 1, Fig. 5). The ribonucleotides are converted
M.pneumoniae (59). For example D12_orf634 (msbA), to deoxyribonucleotides by ribonucleoside–diphosphate reductase,
D12_orf623 (pmd1) and D02_orf660 (lcnDR3) have the con- an enzyme complex formed by the gene products of nrdE
served ATP binding motif and the membrane-spanning domains (F10_orf721) and nrdF (F10_orf339). Adenine, guanine and uracil
on the same polypeptide. In addition D12_orf623 and can be metabolized directly to the corresponding nucleoside
D12_orf634 show also significant similarities to multidrug monophosphates by the enzymes adenine phosophoribosyl-
resistance proteins of different organisms. transferase (apt, F11_orf133), hypoxanthine-guanine phosphoribo-
Among the proposed PTS transport systems, we identified one syltransferase (hpt, K05_orf175) and uracil phosphoribosyl-
for glucose and one for mannitol. They are similar to the transferase (upp, B01_orf178). Uridylate, adenylate and guanylate
homologous systems from several Gram-positive bacteria, with kinases catalyze the generation of ADP, GDP and UDP.
a EIIA and EIIBC domains on two separate polypeptides for the Surprisingly, we could not find the nucleoside diphosphate kinase
mannitol transport system and with three domains (EIIABC) of (ndk), the key enzyme for the conversion from NDP to NTP. This

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enzyme II in one polypeptide for the glucose transport system. finding is in agreement with data from the genomic sequence
Besides glucose and mannitol, fructose also seems to be analysis of M.genitalium.
imported by the PTS system. According to our data the Another important enzyme, the CTP synthetase which converts
fructose-permease II component R02_orf694 (fruA) contains all UTP to CTP is also missing. Therefore the only route for the
three domains of enzyme II in one gene (EIIABC). In addition, synthesis of CTP appears to be from cytidine to CMP by uridine
R02_orf694 and the 1-phosphofructokinase (fruK, R02_orf300) kinase (H03_orf213) and to CDP by cytidylate kinase
are probably in one operon, but we do not find fruF which is also (P01_orf217). Deoxythymidine monophosphate (dTMP) could
part of the fructose operon in enteric bacteria (58). be either synthesized by thymidine kinase (tdk, B01_orf191) or
by thymidylate synthase (thA, F10_orf328).
It will be of special interest to experimentally identify the
Protein secretion
enzyme(s) of M.pneumoniae which convert NDPs to NTPs, since
Both, Gram-positive and Gram-negative bacteria have a well such an enzymatic activity seems to be essential.
conserved protein translocation system. The components identi-
fied which are part of the well characterized E.coli system (60)
include cytosolic chaperones or regulators [trigger factor, SecB, Carbohydrate metabolism and energy conservation
DnaK, SRP (a ribonucleoprotein composed of 4.5 S RNA and
Ffh) and FtsY] which deliver the protein to a membrane receptor The ability to metabolize glucose and/or arginine and use it for the
(SecA). The receptor is also supposed to function as a motor, ATP synthesis is one of the key features in classification of
pushing the protein across the membrane via specific protein Mollicutes. Mycoplasma pneumoniae is listed in Bergey’s manual
channels (SecY, SecG, SecE, SecD and SecF). The secreted of systematic bacteriology as a glucose fermenter but not as an
proteins to be transported carry an N-terminal signal peptide arginine-hydrolyzing species (61). This contrasts with our
which will be removed by a signal peptidase (SPaseI). Two routes sequencing results, since the three enzymes involved in the
of export have been proposed either via SecB and SecA or by arginine degradation pathway, arginine deiminase (H03_orf438),
SRP. The protein secretion system in M.pneumoniae is less ornithine carbamoyltransferase (H10_orf273) and carbamate
complex (Table 1). So far, the trigger factor, DnaK, SRP, FtsY and kinase (F10_orf309) are present according to our sequence data.
SecA have been identified. From the channel-forming proteins The arginine deiminase gene occurs twice but one copy is inactive
only SecY is present but SecG, SecF, SecE, SecD and the due to a raster-mutation resulting in two proposed ORFs
cytosolic receptor protein SecB are missing. Also absent is the (H10_orf198 and H10_orf238) corresponding to the N-terminal
signal peptidase SPaseI although computer-assisted motif predic- and C-terminal halves of a complete deiminase. The change in
tion programs indicate the presence of corresponding substrates reading frame was also confirmed by sequencing of directly
(signal peptides). The simplified protein export system might be amplified genomic DNA. All these proposed ORFs are organized
a reflection of the fact that M.pneumoniae is only surrounded by in an operon-like arrangement except for the deiminase
a cytoplasmic membrane. Another problem concerns refolding of (H03_orf438) which seems to be expressed as a single gene
secreted proteins which are normally exported in an unfolded located far away from the mentioned operon. Included in this
stage. Refolding might be catalyzed by chaperones which have to operon is a proposed protein (F10_orf565) with 12 predicted
function on the cell surface (60). This might impose a special transmembrane domains indicative of a putative permease.
problem on the wall-less bacteria in general, since they do not Glucose, fructose and mannitol are transported by the PTS
possess a periplasmic space which could prevent proteins from system into the cell and further degraded by the Embden–Meyer-
diffusing. To anchor the proposed chaperones on the cell surface hof–Parnas (EMP) pathway to pyruvate. All enzymes required
as lipoproteins would be a possible way to solve this problem. for this pathway have been identified. The second pathway for
metabolizing glucose, the pentose phosphate pathway, is incomplete
Nucleotide synthesis: purine and pyrimidine salvage in M.pneumoniae. We found only the enzymes ribulose-5-
pathways phosphate-3-epimerase and transketolase (Fig. 5). Glucose-6-
phosphate dehydrogenase (G6Pde), 6-phospho-gluconate dehydro-
Guanine, guanosine, uracil, thymine, thymidine, cytidine, ade- genase (6PGde), and a transaldolase are missing. These data agree
nine and adenosine may serve as precursors for nucleic acids and with enzymatic studies showing that G6Pde and 6PGde are absent
nucleotide coenzymes, as determined in nutritional studies of in mycoplasmas (62).
4448 Nucleic Acids Research, 1996, Vol. 24, No. 22

Pyruvate can be further metabolized by two alternative reactions, find any indication for a number of genes/proteins, which should
either to lactate by lactate dehydrogenase (K05_orf312) or to be there based on experimental evidence. Mycoplasma pneumoniae
acetyl-CoA by the pyruvate dehydrogenase complex and further has been shown to be motile and to exhibit chemotactic behaviour
to acetate by the phosphotransacetylase (A05_orf320, pta) and the (64). Motility genes are difficult to identify since the motility in
acetate kinase (G12_orf390, ackA). The pyruvate dehydrogenase M.pneumoniae is independent of pili or flagella and it is not yet
complex consists of E1α (F11_orf358a) E1β (F11_orf327), the known which are potential candidates. Therefore, any progress in
two subunits of the pyruvate dehydrogenase, the dihydrolipoamide this field depends on the isolation of mutants. Furthermore, none
acetyltransferase E2 (F11_orf402) and the dihydrolipoamide of the components of the chemotactic signal pathway, the Che
dehydrogenase E3 (F11_orf457). The corresponding genes are proteins, which are well conserved among bacteria, or any other
clustered (nt 549 943–557 431; pcosMPF11); part of this cluster ‘two-component signal transduction system’ could be detected.
also contains the genes coding for NADH oxidase (nox, Chemotactic behaviour in M.pneumoniae is difficult to study.
F11_orf479) and lipoate protein ligase (lplA, F11orf339). The While it might be possible that these bacteria are chemotaxis
later enzyme joins lipoic acid in an amide linkage to the ε amino negative, only additional experiments will clarify this point.
group of a lysine residue of the dihydrolipoamide acetyltransferase. It has been reported that M.pneumoniae produces hydrogen

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peroxide considered to be a pathogenicity factor (17). Therefore,
Membrane phospho- and glycolipid synthesis to protect itself from oxidative stress one would expect to find the
standard enzymes dealing with these stress factors like catalase,
In M.pneumoniae strain FH the following membrane phospho- superoxide dismutase or peroxidase, but we have no similarity
and glycolipids have been found: digalactosyldiacylglycerol, based evidence that these enzymes exist in M.pneumoniae.
trigalactosyldiacylglycerol, glucosylgalactosyldiacylglycerol, Experimental data on this topic are also inconsistent (62).
phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) The results of our sequence analysis explain quite well the kind
(63). Since M.pneumoniae FH and M.pneumoniae M129 are very of changes which have led to the observed reduction of the
similar we assume that both strains carry essentially the same genome size in M.pneumoniae from the presumed genome size
genes for phospho- and glycolipid-synthesis. of several million base pairs of the ancestral bacteria. The main
About 10 genes are required for the synthesis of the above- cause is the loss of complete anabolic (no amino acid synthesis)
mentioned lipids; but according to our DNA sequence analysis and metabolic pathways and of genes for the synthesis of complex
only three of the expected genes could be unambiguously structures like the bacterial cell wall which requires a large
identified. They code (Fig. 5) for the enzymes 1-acylglycerol-3-
number of genes. In addition, for several processes like DNA
phosphate acyltransferase (plsC; gene name in Saccharomyces
repair, DNA recombination, cell division or protein secretion, the
cerevisiae is slc1), phosphatidic acid cytidyltransferase (cdsA)
number of genes involved is smaller than in the more complex
and glycerolphosphate phosphatidyltransferase (pgsA). These
enzymes are involved in the biochemical pathway for the bacteria.
synthesis of PG and DPG. Missing are the glycerol-3-phosphate No significant changes were observed in the size of individual
acyltransferase (plsB) catalysing the synthesis of 1-acylglycerol-3- genes which resemble more or less their counterparts in E.coli or
phosphate (acyl-G3P) from glycerol-3-phosphate (G3P), the B.subtilis. The occasionally observed smaller intergenic regions,
phosphatidylglycerol phosphate phosphatase which converts like those found in the ATPase operon, do not appear to
phosphatidylglycerol-3-phosphate to PG and finally the cardiolipin significantly contribute to the overall genome size reduction.
synthetase (cls) which synthesizes DPG from PG. Interestingly, In contrast with the loss of complete pathways we frequently
we find a gene homologous to the plsX gene from E.coli which observed the amplification of complete genes or segments of
is involved in membrane lipid synthesis in an undefined manner. genes (see sections on lipoprotein families or on the repetitive
The glycolipid synthesis could start with phosphatidic acid and DNA sequences RepMP2/3, RepMP4 and RepMP5). In these two
would probably require a phosphatidic acid phosphatase and instances the obvious advantage would be the potential of
several UDP-glucosyl- or galactosyltransferases. None of these expressing antigenic variants of surface-exposed proteins.
enzymes could be identified by similarity searches in databases. The various truncated genes which are also present in full
As expected from biochemical studies no gene involved in fatty length copies e.g. arginine deiminase (H03_orf438 and
acid or cholesterol synthesis was determined in the sequence H03_orf238), DNA primase (H91_orf620 and D12_orf212) and
analysis. These components are incorporated as such from the the dihydrofolate reductase (H10_orf506 and F10_orf160) might
medium. be relics of recombination events which took place in the course
An interesting enzyme is the proposed carnitine palmitoyl- of the process of evolution.
transferase encoded by C09_orf600, which might be involved in Finally among the many proposed proteins are a few which
the modifacation of exogenous phosphatidylcholine (67). share the highest similarity over their entire length with a
eukaryotic protein. The most prominent examples are the pre-B
CONCLUSIONS cell enhancing factor (pbeF, D09_orf451) and the carnitine
palmitoyltransferase II precursor (cpt2, C09_orf600). Both might
It is impossible to address each proposed M.pneumoniae gene in be candidates for examples of horizontal gene transfer, but at the
this paper. We have tried to cover the most important categories present state of analysis a definitive answer cannot be given.
of functions and point to genes which should be present, but could It will be the main task of future studies to reconcile the
not be found by our applied methods. Typical examples are the experimental evidence and the DNA sequence-based predictions,
missing diphosphonucleoside kinase for the conversion of i.e. to indentify the genes for observed functions and vice versa,
(d)NDPs to (d)NTPs, and the substrate binding domain (oppA) and to assign functions to proposed open reading frames with
for the oligopeptide ABC transporter. In addition, we could not hitherto unknown functions.
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One obvious topic is the comparative analysis between the 26 Proft, T. and Herrmann, R. (1994) Mol. Microbiol., 13, 337–348.
completely sequenced genomes of the closely related species 27 Proft, T., Hilbert, H., Layh Schmitt, G. and Herrmann, R. (1995)
J. Bacteriol., 177, 3370–3378.
M.pneumoniae and M.genitalium (9). Since the present paper is 28 Razin, S. and Jacobs, E. (1992b) J. Gen. Microbiol., 138, 407–422.
already very voluminous we decided to publish this analysis in an 29 Ruland, K., Wenzel, R. and Herrmann, R. (1990) Nucleic Acids Res., 18,
additional paper (Himmelreich et al., in preparation). 6311–6317.
30 Fleischmann, R. D., Adams, M. D., White, O., Clayton, R. A., Kirkness, E.
F., Kerlavage, A. R., Bult, C. J., Tomb, J. F., Dougherty, B. A., Merrick, J.
ACKNOWLEDGEMENTS M. et al. (1995) Science, 269, 496–512.
31 Riley, M. (1993) Microbiol. Rev., 57, 862–952.
We thank R. Frank and A. Bosserhoff for the synthesis of 32 Baker, T. A. and Wickner, S. H. (1992) Annu. Rev. Genet., 26, 447–477.
oligonucleotides, B. Reiner for her expertise in computer data 33 Mills, L. B., Stanbridge, E. J., Sedwick, W. D. and Korn, D. (1977)
analysis, Raphael Mosbach for his technical assistance concern- J. Bacteriol., 132, 641–649.
ing hardware problems, U. Leibfried for technical assistance, I. 34 Barnes, M. H., Tarantino, P. M., Jr., Spacciapoli, P., Brown, N. C., Yu, H.
Schmidt for preparing the manuscript, D. Hofmann and H. and Dybvig, K. (1994) Mol. Microbiol., 13, 843–54
35 Koonin, E. V. and Bork, P. (1996) Trends Biochem. Sci., 21, 128–129.
Göhlmann for reading of the manuscript and H. Schaller for 36 Camerini-Otero, R. D. and Hsieh, P. (1995) Annu. Rev. Genet., 29,

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financial assistance and his encouragement throughout our work. 509–552.
We thank S. Razin, A. Wieslander, K. Dybvig, K. Sitaraman, R. 37 Demple, B. and Harrison, L. (1994) Annu. Rev. Biochem., 63, 915–948.
Walker, H. Neimark and R. Miles who read drafts of this 38 Sancar, A. and Sancar, G. B. (1988) Annu. Rev. Biochem., 57, 29–67.
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