Nucleotide sequence of the P1 attachment-protein gene of Mycoplasma pneumoniae

217

Gene, 64 (1988) 217-229 Elsevier GEN 02347

Nucleotide

sequence

(Recombinant codons;

of the Pl

DNA;

genomic

polycistronic

and mRNA/cDNA

message;

epitopes;

Julia M. lnamine”, Timothy P. Denny Bott b, and Ping-chuan

gene of Mycoplasma

attachment-protein

libraries;

phage vectors

a*,Steven

leader peptide;

pneumoniae respiratory

14 October

Revised

and accepted

Received

by publisher

epithelium;

Loechel”, Ulrike Schaper’*,

Chien-hui Huang”‘, Kenneth F.

Huabc

Departments of ” Pediatrics and ‘Microbiology and Immunology [Tel. (919)966-SOlO], Medicine, University of North Carolina, Chapel Hill, NC 27599-7220 (U.S.A.) Received

ciliated

il and M13)

and ’ Center for Environmental

1987 11 December 20 January

1987 1988

SUMMARY

The specific attachment of Mycoplasma pneumoniae to the respiratory ciliated epithelium is mediated by a surface protein designated Pl. The nucleotide (nt) sequence of the Pl attachment-protein gene has been determined and the amino acid (aa) sequence deduced. mRNA and cDNA sequencing confirm that this gene is transcribed in M. pneumoniae. The predicted amino acid sequence matches the N-terminal 12 aa residues of Pl protein from M. pneumoniae [Jacobs et al., J. Gen. Microbial. 133 (1987) 2233-22361 beginning with Asn at aa position 60, where aa 1 represents the first codon of the open reading frame (ORF). Notably, the Trp at aa position 69 aligns with a UGA codon deduced from the nucleotide sequence, providing supporting evidence that UGA is read as Trp rather than stop in M. pneumoniae. Analysis of the first 59 aa suggests that it is probably a leader sequence that is processed to yield the mature protein. The codons of the mature Pl protein sequence represent 1568 aa with a calculated A4, of 169758. A unique feature of this protein sequence is the lack of cysteine, and this was confirmed by sodium dodecyl sulfate-polyacryamide gel electrophoresis of M. pneumoniae proteins metabolically labeled with radioactive cysteine or methionine. This study has revealed that the 4881 nt of the Pl structural gene are flanked by ORFs, and there are no obvious ribosome-binding sites or transcription termination sequences in the immediately adjacent regions. This suggests that the Pl gene is transcribed as part of a larger polycistronic message. In addition, a number of untranscribed and therefore nonfunctional Pl epitope sequences were found in the M. pneumoniae genome; their purpose remains unknown.

Corresporzdmce

to: Dr. P.-c. Hu, Department

Burnett-Womack Carolina,

Bldg.,

Chapel

CB No. 7220, Hill,

NC

of Pediatrics,

University

535

of North

27599-7220

(U.S.A.)

Tel. (919)966-2331. * Present University

addresses: of

(T.P.D.)

Georgia,

Department Athens,

of Plant Pathology,

GA

30606

(U.S.A.)

Tel. (404) 542-257 1; (U.S.) School of Veterinary

Medicine,

Carolina

27606

State

Abbreviations: complementary

University,

Raleigh,

NC

North

(U.S.A.)

triphosphate(s); ORF,

Tel. (919)829-4301. 037X-I

I1’148:$03.5(1

0

IYXX Elsevier

Science

Publishers

B.V.

(Biomedical

Division)

to RNA;

RBS,

dd, dideoxy;

kb, kilobase

open reading

phoresis; sulfate;

aa, amino acid(s); bp, base pair(s); cDNA,

frame;

dNTP,

DNA

deoxynucleotide

or 1000 bp; nt, nucleotide(s); PAGE,

ribosome-binding

polyacrylamide site; SDS,

SSC, 0.15 M NaCI, 0.015 M Na’citrate,

gel electro-

sodium

dodecyl

pH 7.6.

INTRODUCTION

alignment

The attachment of M. pneumoniue to the ciliated epithelium of the respiratory tract has been recognized as a prerequisite quent

development

for colonization of disease

and subse-

(Hu

et al.,

1977;

of its deduced

amino acid sequence

with

the N-terminal 12 aa residues of purified Pl protein (Jacobs et al., 1987). The finding of numerous, nonfunctional PI sequences in the M. pneumoniae genome may account for some of the aforementioned cloning

results.

Collier, 1980) and is thought to be one of the major virulence

factors

of this organism

(Collier,

1979).

Previous

studies have shown that a trypsin-sensitive

protein,

designated

Pl,

is involved

in the specific

attachment process (Hu et al., 1977). The involvement of P 1 protein in attachment is further supported by the demonstration

that monoclonal

MATERIALS

AND METHODS

(a) Bacteria, vectors and media

(Hu et al.,

1982) and monospecific (Krause and Baseman, 1983) anti-P1 antibodies inhibit attachment to respiratory epithelium. Immunoferritin electron microscopy reveals that Pl antigens are clustered on the differentiated tip structure of M. pneumoniue (Hu et al., 1982; Feldner et al., 1982; Baseman et al., 1982). P 1 protein is therefore an essential component involved in attachment. Recent studies have also indicated that Pl protein may be a major immunogen (Hu et al., 1981). Antibodies specific to Pl were detected in the sera of experimental animals infected by inhalation of aerosolized M. pneumoniue (Hu et al., 1981) and in patients with culture-proven infections (Hu et al., 1983). Moreover, antibodies of both IgG and secretory IgA classes were found in the respiratory secretions of human subjects (Hu et al., 1983). These findings strongly suggest that Pl protein, or its subunits, could be useful vaccine candidates. Investigations of the biological functions of Pl protein in M. pneumoniue pathogenicity and the host immune response have been approached by attempting to isolate the Pl structural gene. Recent reports have indicated that coding sequences have been cloned and expressed in Escherichia coli using nonexpression vectors (Trevino et al., 1986) as well as expression vectors (Schaper et al., 1987; Frydenburg et al., 1987). Trevino et al. (1986) reported the expression of a 140-kDa protein, designated Pl* that reacted with anti-P1 polyclonal serum, while Frydenberg et al. (1987) and Schaper et al. (1987) detected fusion proteins with monospecific and monoclonal antibodies, respectively, with much shorter translated regions. This paper presents the cloning and sequencing of a functional Pl gene as judged by its transcription in M. pneumoniae and the

M. pneumoniue strain M-129 (ATCC29342) was originally isolated from a patient with pneumonia (Lipman and Clyde, 1969). Monolayer cultures were grown in glass prescription bottles, rinsed, and harvested as previously described (Hu et al., 1977). E. coli strains Y 1088 and Y 1090 (Young and Davis, 1983b) were used to propagate derivatives of phage vector 1gt 11 (Young and Davis, 1983a), strain NM539 was used to propagate derivatives of phage vector AEMBL3 (Frischauf et al., 1983) and strains JMlOl (Messing, 1979) and JM109 (YanischPerron et al., 1985) were the hosts for phages M13mp18 and M13mp19 (Norrander et al., 1983) and their derivatives. All E. coli strains were cultured with Luria broth (Maniatis et al., 1982). Phage /1 derivatives were propagated in E. co/i by standard procedures (Maniatis et al., 1982). Transformation of E. coli was performed by the method of Hanahan (1983). (b) Chemicals and enzymes Restriction cndonucleases and other enzymes used in molecular cloning were purchased from a number of sources. Ml3 phage vectors were from Bethesda Research Laboratories. Plasmid pIBI25 was from International Biotechnologies, Inc. dNTPs and ddNTPs were from Pharmacia. Radioactive materials were from Amersham Corporation and ICN Biomedicals, Inc. Phage Agtl 1, and E. co/i Y 1088 and Y1090 were gifts from Dr. Clyde Hutchinson III. Primer oligodeoxyribonucleotides were synthesized in Dr. Hutchinson’s laboratory. The sources of other products and kits are given in the text. Unless otherwise stated, all were used according to the manufacturer’s instructions.

(c) Isolation of nucleic acids

primer

(see RESULTS,

quence) from M. pneumoniae

DNA was isolated dard methods

(Maniatis

combinant extraction munoaffmity

method 2 phage

buffer,

and

was

et al., 1982) and purified by

Analysis of the products from a tracer reaction on an alkaline 1.4% agarose gel showed a sharp band of

(Maniatis

et al., 1982). Re-

was isolated

of phage purified

by phenol

with LambdaSorb

(Promega

im-

Biotec).

approx.

1.4 kb (it is not known

stopped

at this point;

later analysis

of the complete

sequence

did not predict

any significant

nucleotide

of genomic libraries

Two M. pneumoniae genomic libraries were constructed. The first employed expression vector Agtl 1 (Young and Davis, 1983a) which has a cloning capacity of up to 7 kb, and the second was made with vector AEMBL3 (Frischauf et al., 1982) with a 9-23-kb cloning capacity. For both libraries, insert fragments were obtained by fractionation on 10-40:; sucrose density gradients, and phage were packaged with Packagene extracts (Promega Biotec). The Agtl 1 library was constructed and screened by the procedures of Young and Davis (1983b) with the following modifications. The EcoRI linkers were added to a pool of 2-7 kb mechanically sheared and repaired M. pneumoniae DNA fragments. Recombinant plaques were screened by incubating the nitrocellulose filters (Schleicher & Schuell) overnight with rabbit antiserum produced against M. pneumoniae Pl protein that had been extensively adsorbed with E. coli lysates prior to use. The washed filters were incubated for 2 h with 1 PCi of 1251-labeled protein A. The IEMBL3 library was constructed with M. pneumoniae DNA fragments generated by partial Sau3A digestion and a ;1EMBL3 BamHI armscloning system (Promega Biotec) and screened by standard plaque-hybridization procedures (Maniatis et al., 1982).

why the synthesis

secondary structure). The ends of the cDNA fragments were repaired with T4 DNA polymerase and ligated

(d) Construction

reaction

se-

heated at 65 ‘C for 3 min and slowly cooled to 37 o C.

DNA

adsorbent

in first-strand

c, for the primer

by stan-

CsCl equilibrium density-gradient centrifugation. Total cellular RNA was isolated by the guanidinium chloride/CsCl

section

to

phosphatase-treated,

pIBI25 by standard 1982). Transformation

HincII-digested

procedures (Maniatis et al., into library efficiency compe-

tent cells of E. coli strain DH5a (obtained from Bethesda Research Laboratories) yielded approx. 1000 colonies. Insert fragments were cloned into M 13mp 18 and M 13mp 19 for sequencing. (f) DNA manipulations Restriction fragments were purified from Seakem GTG or LE agarose (FMC Corporation) gels using Spin-X centrifuge filter units (Costar). Nested deletions of Ml3 clones were produced by the exonuclease III method of Henikoff (1984) using the Erase-a-base system (Promega Biotec). (g) Nucleotide

sequencing

Total M. pneumoniae

RNA was used as the tem-

plate for sequencing with a PI-specific primer (see RESULTS, section c, for primer sequence) using the GemSeq transcript sequencing system (Promega Biotec). Nucleotides were sequenced by the dideoxy chain-termination method of Sanger et al. (1977) using [ T-~~S]~ATP (Biggin et al., 1983). Sequencing reactions were resolved on 6 “/ polyacrylamide field gradient gels (Olsson et al., 1984; Ansorge and Labeit, 1984) cast with wedge spacers (Bethesda Research Laboratories) using an Ephortec Sequencer (Haake-Buchler). Nucleotides were analyzed with the Beckman MicroGenie program.

(e) cDNA clone construction (h) SDS-PAGE cDNA was made from M. pneumoniae RNA with a cDNA synthesis system (Bethesda Research Laboratories) with the following modifications. The annealing mixture contained 6 pg of total M. pneumnniae RNA and 0.4 pmol of PI-specific

and preparation of immunohlots

The procedures for SDS-PAGE of mycoplasma proteins and preparation of immunoblots (Western blots) have been described previously (Hu et al., 1981).

220

RESULTS

AND

J. Ho, S.L., and P.-c.H., manuscript in preparation). These results accounted for the failure of AMpl-11

DISCUSSION

(a) Cloning

and expression

Escherichia

coli

of a PI

epitope

in

to be fully expressed

and indicated

could not be completely premature

termination

that the PI gene in E. coli due to

translated

at UGA codons.

The results

A P 1 epitope was cloned in E. coli from a genomic library constructed with the expression vector Agt 11. The library was initially screened with rabbit antiserum produced Plaque-purified screened clonal

against M. pneumoniae P 1 protein. P l-positive clones were then

by Western-blot

antibodies

analysis

with 23 mono-

specific to Pl protein

(Hu et al.,

1982; Clyde and Hu, 1986). The Pi/P-galactosidase fusion protein produced by one of the Agt 11 recombinants, designated AMpl-11, was recognized by one of the Pl-specific monoclonal antibodies, designated M-328 (Fig. 1). Since the monoclonal antibodies were produced and screened against whole M. pneumoniae organisms, monoclonal antibody M-328 probably recognizes native Pl protein in its membrane-associated conformation. Monoclonal antibody M-328 specifically inhibits the attachment of M. pneumoniae to hamster respiratory epithelium in tracheal organ culture (not shown). Therefore, the results indicated that AMpl-11 contained the DNA fragment coding for an epitope of native P 1 protein. In addition, the AMpl-11 fusion protein retains its antigenicity in producing P l-specific antibodies in mice (not shown). Restriction endonuclease mapping and Southernblot analysis of AMpl-11 showed that it contained an M. pneumoniae 4.4-kb DNA insert defined by EcoRI sites introduced during the cloning procedure (not shown). Since transcription proceeds through the IacZ-coding region of the Agt 11 vector, nucleotide sequencing was initiated at this junction. The results showed that the insert of ,?Mpl-11 contained six TGA (stop) codons in an otherwise open reading frame (Schaper et al., 1987). The first TGA appeared after nt position 234, corresponding to an 8-kDa size increase in the fusion protein. This was in agreement with an estimate of the size of the fusion protein vs. fi-galactosidase (Fig. 1). Since there is evidence that UGA is read as tryptophan rather than stop in another mycoplasma, M. capricolum (Yamao et al,, 1985) it seemed likely that this unusual codon usage would also be true for M. pneumoniae. Indeed, a capable of reading UGA as tryptophan tRNA,,, has since been identified in M. pneumoniae (J.M.I.,

12 Fig. 1. Expression demonstrated RIALS

1

34 of

a PI-epitope

by SDS-PAGE

washed

with acetone.

were dissolved containing

section

lysatcs

were

and subjected

5”,, p-mercaptoethanol,

separating

was performed

and

stacking

mM Tris-glycine, phoresis,

the gel was either stained

A) or the proteins

were transferred

(marked

of an immunoblot

profile

which corresponds

from a similar gel. The filter

antibody

by incubation

with

(M-328) specific to PI ‘ZsI-labeled

rabbit

in E. coli by the recombinant

I (lane 2) and the Pl protein of M. with

in E. culi

%gtll phage expressing

by arrow),

prepared

mouse IgCi. The FP produced

arrows).

filter for

shown in lane 4. (B) Radioauto-

with a monoclonal

followed

reacted

electro-

blue (panel

to a nitrocellulose

lysate from wild-type

/Jgalactosidase

AMpI-I

with Coomassie

I is indicated by the arrow in lane

to the purified p-galactosidase

both

buffer was 25

fusion protein (FP) produced

clone 1Mpl-1

2. Lane 3 contains

protein

The running

with the position of PI noted by the arrow. The

hybrid P l/p-galactosidase by recombinant

was probed

system with

3”,, polyacrylamide

(panel B). (A) Lane 1 shows the protem

ofA4. pne~toniue

graph

and 2”” SDS,

(1.5 mm x 10 cm x 14 cm)

pH 8.3, with O.lU, SDS. Following

immunoblotting

normal

Samples

HCI buffer, pH 6.8,

of 7 and

0.1”” SDS, respectively.

containing

1OY; TCA,

to SDS-PAGE.

in a discontinuous

gels

and wild-

with

IO”,, sucrose

and then boiled for 3 min. Slab-gel

(see MATE-

h). Recombinant

precipitated

4

in E. coli as

sequence

in 50 ~1 of 62.5 mM Tris

electrophoresis

3

and immunoblotting

AND METHODS,

type igtl 1 phage

2

the Pl-specific

pneumoniae

monoclonal

antiphage

(lane 1)

antibody

(see

221

also indicated contained

that the first 234 bp of IMpl-11

the M-328 epitope

ments was probably templates

sequence.

4.4-kb (b) Detection

of multiple

versions

of the M-328

epitope sequence As a prelude to cloning the entire by Southern-blot

DNA

hyb~~zation

clone

sequencing

for

AMpI-11

was actually

the

transcribed

in M. pneu~~ni~e was sought. An 18-mer deoxyribonucleotide was synthesized and used as a

which

the first 185 bp of the M-328 epitope

quence, was chosen

sequence

that

to deter-

would be useful. U59, an M 13

derived

HindIII

verification

were

mine which enzymes contained

sufficient to rule out their use as

transcription,

PI gene, restric-

tion digests of M. pneumoniae genomic analyzed

in

se-

as the probe. For convenience,

U59 will be referred to as the M-328 epitope probe. Results such as that shown in iane 1 of Fig. 2 were obtained. In this example, seven Hind111 bands hybridized to the probe (there were no Hind111 sites within the probe sequence). To determine the nature of these hybridizing fragments, each HindIII fragment was cloned directly from genomic DNA with M 13 vectors. Analysis of the Hind111 clones revealed that the seven hybridization bands were actually made up of ten different Hiad fragments with estimated sizes of 8, 2 x 6.1, 4.4, 4.1, 3.5, 2.1, and 3 x 1.7 kb. Comigrating fragments could be distinguished by restriction enzyme site differences. The 8-kb fragment was extremely unstable when cloned in E.coli; however, stable subclones could be isolated from a ~EMBL3-derived recombin~t phage (see RESULTS AND DISCUSSION, section d). The other nine fragments were stable, and all ten regions were completely or partially sequenced. Sequence analysis showed that all of the fragments contained highly homologous M-328 epitope sequences but that sequence divergence occurred immediately upstream from this epitope region (not shown). The sequence from JMpl- 11 was found in one of the 1.7-kb Hind111 fragments. The 4.4-kb Hiad fragment was the only one with a long ORF containing the epitope sequence, suggesting that it was part of the real PI gene. All of the other HindIII fragments contained TAA or TAG stop codons in all three reading frames of either the divergent region or the region 3’ of the epitope sequence. (c) ldenti~cation

of the transcribed PZ sequence

1234 Fig. 2. Southern-blot epitope

sequence

phoresed

analysis with a probe containing

(US9). HindHI-digested

on a 0.84; agarose

Screen

membrane

standard

under

procedures

(Arrand,

using

column

manufacturer’s contained containing

the Ml3

instructions

50% formamide

brane was washedat42”Cin

except

are indicated

Research

that

probe

by

primer

were removed

using

according

to the

the

elution

buffer

in a solution

et al., 1982). The mem-

0.1 x SSC + 0.1 Y; SDS for 3 times 15 min, before exposing

1, M. pnez~~oniffegenomic

from IEMBW-derived probe

universal

was at 42°C

(Maniatis

15 min and 0.1 x SSC for 3 times x-ray film. Lanes:

(NEN

& Schuell)

3 M NaCl. Hybridization

et al.,

to a Gene-

with [z-“‘P]dATP

nucleotides

(Schleicher

recombinant

and 4, 3.51-7. The genomic

Although the presence of TAA or TAG stop codons in the other nine sequenced Hind111 frag-

conditions

was labeled

1985). Unincorporated

an Elutip-d

were transferred

alkaline

The U59 probe

the M-328

were electro-

gel in TBE buffer (Maniatis

1982) at 6 V/cm. The fragments Products).

DNAs

fragments

by arrows,

given in kb on the left margin.

phage; which

DNA; 2, IJI-I;

hybridize

and their estimated

to

and DNA 3, AJI-6; to the sizes are

222

primer to sequence cDNA ducing

mRNA

as well as to synthesize

from the mRNA. For the purpose of proa long cDNA copy, 5’-CTCAAAACAAC-

GACACCG-3’

was chosen

as a consensus

se-

quence from the sequence data available for the putative 3’ end of the gene (Schaper Frydenberg, restriction quence sequence

personal

communication).

endonuclease

was downstream and just outside Direct

sequencing

Based on the

map of AMpl- 11, this sefrom the M-328

epitope

(3’) of the Hind111 frag-

ments cloned by hybridization probe.

et al., 1987; J.

to the M-328 epitope of total

mRNA yielded 176 nt of information

M. prwur~onicre

with no indica-

comprising the PI structural gene (Fig. 4). As was noted in the previous section, the mRNA and cDNA sequences

spanned

the HQzdIII site at the 3’ end of

the 4.4-kb Hind111 fragment; these sequences matched perfectly the sequence data obtained from phage iJI-7,

demonstrating

transcribed

that it did indeed contain

the

gene. The mRNA sequence was found at

nt positions

3683-3858

while the cDNA

clone ex-

tended from nt 2483 to nt 386 1. Surprisingly, a consensus

RBS sequence

1974; Moran

et al., 1982) nor a structure

(Shine

neither

and Dalgarno, indicative

of a transcription terminator (Platt, 1986) could be found in adjacent regions. Rather, the PI gene is

tion of more than one species of mRNA hybridizing to the primer. The cDNA clone was 1379 bp long and was sequenced in about 250 bp from each end. The mRNA and cDNA sequences matched that of the 4.4-kb Hind111 fragment and not the other nine sequenced fragments. These results demonstrated that the PI sequences found in the 4.4-kb fragment

flanked by ORFs: ORFl (with a size of 729 nt) terminates with TAA 12 nt before the ATG start codon of the PI gene, while ORF2 (with 1000 bp of sequence so far) starts 5 nt after the TAG stop codon of the PI gene. ORFl is preceded by promoter and RBS sequences (not shown). These data suggest that the PI gene may be transcribed as part of a larger

were transcribed in M. pneumoniae and were, therefore, part of an authentic PI gene.

polycistronic message; the complete nucleotide sequence of ORF2 is being obtained to examine this possibility further. It is of interest that the 5’ sequence of IMpI1 was found about 3.1 kb downstream from the functional PI gene (the 5’ part of the i,Mpl-11 sequence is within a Hind111 fragment of phage %JI-7 that includes 4.8 kb of the right arm of /ZEMBL3, and which happens to co-migrate with the

(d) Nucleotide The

entire

sequencing of the PI gene

PI-coding gene was cloned from by screening a genomic library constructed with vector /1EMBL3. This vector was chosen because it has a cloning capacity of 9 to 23 kb, and size estimates of Pl protein indicated that about 5 kb would be required to encode the structural gene. Recombinants were selected by plaque hybridization to the U59 (M-328 epitope) probe and characterized by Southern-blot hybridization with the same probe. Such an analysis of Hind111 digests of three of the U59-positive clones is shown in lanes 2-4 of Fig. 2. Phage 3,JI-7 (lane 4) was chosen for further study because it contained the 4.4-kb Hind111 fragment which is transcribed in M. pneumoniae (as did phage AJI-6, lane 3). A restriction map of the insert of phage AJI-7 was constructed (Fig. 3, map A) and appropriate fragments were subcloned (Fig. 3, map B) and used for nucleotide sequencing by the strategy shown in map C of Fig. 3. Included in this figure are the 4.4-kb Hir?dIII fragment, which was cloned directly from genomic DNA, and the cDNA clone described above. The sequence data revealed an ORF of 4881 nt M. pneumoniae

U59-positive 6. I-kb Hind111 genomic fragments as shown in Fig. 2). It is notable that, while the M. pneumohc~ genome has a low G + C content (38.6 to 40.8 mol”,; Razin and Tully, 1983), the G + C content of the PI gene is 53.50,b. Similar contents have been obtained for mycoplasma tRNA and rRNA genes (reviewed by Razin, 1985). In the case of the tRNA genes, the intergenic regions are A + T-rich (over 80 mol%) while the genes themselves have a G + C content of about 55 mol?:, (Razin, 1985). Mycoplasmas have the smallest genomes known for free living organisms; the genome of M. pneumoniae has been estimated to be only about 750 kb (Bak et al., 1969). It is tempting to speculate that the high G + C content of these mycoplasma genes serves to maintain their functional integrity. (e) The Pl amino acid sequence The amino acid sequence predicted from the nucleotide

of PI protein was sequence (Fig. 4). A

C

II

EBH II

I I

.4400

-

__

--

X

I1 I1

E

A

--

Sa

cc

II 1 I, 1

SaE

E

II

EE

--A-

+-

-bB_eh_-_c_------

bp--4346

endonuclease

sites are designated

except

required

sequence

B, BumHI;

which was cloned

directly

of both strands

is depicted.

c

1

-

strategy.

cc_

the region of Pl protein

of this sequenced

bp

-

Sa

antibody

is the cDNA

determination;

only the subset

AND

from phage in RESULTS

sites were subcloned clone described and extent of each sequence

C-DNA the direction

labeled

defined by restriction

Na, NueI; NC, NcoI; Nd, NdeI; N, NruI; Ps, PstI; Pv, PvuI; Sa,

and are not

by the sizes of three

U59 (see RESULTS

monoclonal

digestions

part of phage iJI-7 is indicated

was designated double

section boxes show the locations by Pl-specific

by appropriate

I

---___ H

1 1

H

AND DISCUSSION,

-

--__

The hatched

in RESULTS

----

2084

that is recognized

were located the Pl gene. The fragments The fragment The arrows indicate

DNA.

~~~~~

phage AMpl-11.

sequenced region

II I,

EH

,,,,,..........~,,,.,._....

I-.-___ ---__ ---___ --._--._ -_

I

the first 185 bp ofthe M-328 epitope sequence

Ev, EcoRV; H, HindIII;

outside

from genomic

a---c

Region

Sa

phage IJI-7 (described

box on the right. The completely

E, EcoRI;

sites shown

encoding

cloned into Ml3 vectors for sequencing

by letters:

--

H -I-

I

H

S

E

H

II II II I1

SmSH

I

H

Sa

Ev

bpe

( Epitope

S

S

,,,, III,

SH

2084

"""'..........M-328

jORF2

and the cloned insert of recombinant containing

sequence

iMpl-11

to the hatched

Pl gene region showing the sequencing

Hind111 fragment

section c. (Map C) Expanded

for the 4346-bp

for the complete

DISCUSSION,

iJI-7,

SalI; Sm, SmaI; Ss, Π S, SstI; X, XbaI. (Map B) Fragments

Restriction

of its insert corresponds

ofphage

(4400, 4346 and 2084 bp). The restriction

section b), and the location

comprehensive.

--

---_c-_-_A--

I1

Sm

C-DNA

PV

I,

Ss

w

Gene

map ofthe cloned insert ofrecombinant

as the nucleotide

section a). An Ml3 subclone

AND DISCUSSION,

Hind111 fragments

--

and the Pl gene as well as the 5’ ends of 0RF2

AND DISCUSSION,

corresponding

E

Nd

Na

I

PI

bp --

NC

I11 111 III

Ps BPv

181 ,,I

NSm

endonuclease

---c_-

H

S

II, III

HSE

IORF 11

The M-328 epitope region is defined

of ORFl

M-328 (see RESULTS

of the M-328 epitope regions.

d). Open boxes indicate the positions

Fig. 3. Analysis ofthe Pl gene region. (Map A) Partial restriction

Sa

A

computer search of the National Biomedical Research Foundation Protein Data Bank detected no significant homology with sequences in the database. The deduced aa sequence was compared to the N-

terminal end of Pl protein from M. pneumoniue strain FH that has been sequenced up to 12 aa (Jacobs et al., 1987). There was a perfect match beginning with Asn at aa position 60, where aa 1 repre-

2670

2700

273”

2780

265G

2660

2970

3000

CCTCGAAGAACCCTCGACCAGCCAACCTCCTCCAGCTGPGTTCACGAGCGCTACGGGG ProArgArgThrLe”AspGl~Al~AsnLeuClnLeuTrpThrGlyAlaGlyTr’PArgAsnAspLysAlaSprSerGlyGlnSerAspGluAsnHisThrLysPheThrSerAlaThrGJy 2790

262”

ATGGACCAGCAGGGAC~TCAGGTACCTCCGCGGGGMTCCCGACTCGTT~GCA~GATAATATTA~TAAGA~TGGGGATAGTTT~CCACGCAGGACGGC~TGCGATCGATC~C~ WetAspGInGlnGlyGlnSerGlyTh~SerAJaGlyAsnPrnAspSPri.P~SS~i~AsPA~nII~SierLysS~rGlyAspSPrLPuThrThrGJnAspGJyA~nAlalleAspGlnGln 2910

2940

GAGGCCACC~CTACACCACCTCCCCCCCAACCTCACCTCACCCCCACCGCTGATTGACcGAACGCGCTGTrATTCACCAACMGAACAACGCGCAGCGCGrCCAGCT~TTCCTCCGCGGCTTG GluAJaThrAsnTyrThrAsnLeuProProAsnL~”ThrProThrAlaAspTrpP~~AS~A~~Le~,S~~P~~ThrAsnLysAsnAsnA~~GlnAr~AlaGJnl,~“PheLe~~Ar~GlyLeu 3030

3”6”

3090

3120

3210

3240

3330

3360

TTGGGCAGCATCCCGGTGTTGGTGMTCGAAGTGGGTCCGATTCCAACAAATTCC~GCCACCGACCA~ATGGTCCTACACCGACTTACATTCGGACC~CC~ACTGMCCTCCC~ LeuGJySerIleProValLe”ValAsnAr~SPrClySPrAspSer*snLysPhe~lnAlaThrAspFlnl.ys’~rpSe~TyrThrAspLe”HisSprAspGlnThrLysLe~~A8nLe”Pro 3150

3180

GCTTACGGTGAGGTGAATGGGTTGTTGAATCCGGCGTTGGTGGAAACCTATTTTGGGAACACGC~AGC~GGTGGTTCGGGGTCC~CACGACCA~TTCACCCGGTATCGGTTTT~TT AJaTyrGJyGJuVaJAsnG1yLeuLeuAsnProAJaL~uValGluThrTyrPheG~yAsnThrAr~4J~Gly~lySerGlySerAsnThrThrS~rS~rPrOGJyIleGJYPheLysJl~ 3270

3300

CCCGAACAAAATAATGATTCCAAAGCCACCCTGATCACCCCCGGGTTGGCTTGAACGCCCCAGGACGTCGGTMCCTCGTTGTCAGTG~CACCACGGTGAGCTTCCAGCTCGGCGGGTGG ProGluGJnAsnAsnAspSerlysAlaThrLe”JleThrProGlyLeuAJaTrpThrPrn(;lnAsJ~VaI~~lyAsnl.e~~ValValSerGJyThrThrVaJS~rPh~Gl”L~uGJyGJyTrp 3390

3420

3450

3460

CTG(;TChCCTTCACGGAC~GTCA~CCCCGCGC~GTTACCTC~TCTCCAGTTAACGGGCTTGGATGCAAGT~ATGCGACGCAGCGCGCCCTCAT~~GCCCCCCG~CCCTGAGCG Le”YaJThrPheThrAspPheVaJLysPfoAr~AlaClyeu~iyLe~tFlnLe”ThrGIyL~uAspAl~SerAspAl~ThrGInAr~AlaLe”JleTrpAIaProAr~ProTrpAla 3510

-

3540

3570

3600

GCCTTTCGTQCCAGTTGGGTCAPlCCGGTTGGCCGCCGCGTGGAGAGTGTGTGGGATTTGAAGGGGGTGTGGGCGGATCAAGCTCAGTCCGACTCGC~GGATCTACCACCACCGCMCAAGG AlaPheAr~GJYSerTrpValAsnAr~i.euGlyAr~ValGl”S~rVaJTr~ASpLeuLYs~lyVaJTr~AlaAspGJnAlaGlnS~rAspS~rGJnG~ySerThrThrThrAlaThrAr~

3630

36RG

3680

3120

3810

3640

3930

3960

~CGCCTTACCGGAGCACCCGAATGCTTTGGCCTTTCAGGTGAGTGTGGTGGAAGCGAGTGCTTAC~GCCAAACACGAGCTCC~GCCA~CCCMTCCACTAACAGTTCCCCCTACCT~ A~nAlabe”PrOD1uH1sProAsnAlaLe”AlaPheGInVa~SerVaJValG~“A~aSe~AlaTyrLy~~roAsnThrS~rS~rGlyGInThrG1nS~fThrAsnSerSerProTyrJ.pu 3750

3760

CACTTGGTGAAGCCTMGAAGTTACCCAATCCOACMG~AGACGACGATCTT~ACCTGTTGGACCCCAACCAGGTTC~CACC~GCTGCGCCMAGCTTTGGTACAGACCATTCC HlsLeuValLysProLysLysValThrGJnSerAs~ysLe”AspAspAspLe”LysAsnLeuLeuAspProAsnGlnValAr~ThrLysL~uAr~GlnS~rPh~GlyThrAspHisSer 3670

3900

ACCCAGCCCCAGCCCCAATCGCTCAAAACAACGACACCGGTATTTGGGACGAGTAGTGGTAACCTCAGTAGTGTGCTTAGTGGTGGGGGTGCTGGAGGGGGTTCTTCAGGCTCAGGTCM ThrGlnPr”GInProGAnSerLe”LysThrThrThrProValPheGlyThrSerSerGlyAs”I~~uS~rSPrValLe”S~rGlyGly~lyAlaGlyGJyGIyS~~S~rGlyS~rGlyG~” 3990

4020

4050

4080

417”

4200

4290

4320

4410

4440

4530

4560

TCTGGCGTGGATCTCTCCCCCGTTGAAAAIIOTGAGT~GTGGGTGGCTTGTGGGGCAGTTACC~GCACGAGT~ACGGMACACCTCCTCCACCMCAACCTCGCGCCT~TACT~TACGGGG SerClyVaJAspLeuSerProValGJ”Ly8V~lSerGlYT~PLe”V~~GJyGJnLe”ProSerThrSeFAspGJyA~nThrSerS~rThrAsnAsnLeuAJaProAsnThr.AsnThrGly 4110

4140

AATGATGTGGTGGCGGTTGGTCGACTTTCTG~AGC~CGCCGC~AGATG~TGACGATGTTGATGGTATTGTACGCACCCCACTCGCTGAACTGTTAGATGGGGMGGACMACAGCT AsnAspVaJVal~lyVaJGJyAr~l.P”S~rGl”SPrAs~A~aAJaLy~HetA~~AspASpValAspGlylleVa~Ar~ThrProL~“Al~Gl”L~“J,,~“AspGlyGl”~JyGl~ThrAl~ 4230 4260 GACACTGGTCCAC~Z~GCGT~GTTCMGTCTCCTGACC~TT~~CTTC~CCGC~GTTTACCCACC~AGTCACC~ATCTGTTTGATCCGGTAA(:TATGTTG~T~TATGACCAGTAC AspThrGJyProGlnSerValLysPh~LYsS~rProAspGl~Il~ASpPh~AS~ArgLe”PheThrHisProVaJThrAspLe~tPheAspProVaIThrM~tLe”ValTyr~AspGl~Tyr 4350 4360 ATACCGCTGTTTATTGATATCCCAGC~~CTOTGTGMCCCT~TGGTTCGTTT~GGTCTTGAGCTTTGACACCMCG~CAGAGCTTA~GT~~TCC~CTTAGAGTTCTTTAAACCTGAT IleProLeuPheJleAspIleProAJaSerVaJAsnPr”J.YS~etV~JA~~L~ul.ysl,~~,~;loPhePt~el.ysPr~Asp 4500

4470

CAAGATACCCAACCA~PICPC~CGTTCAGGTCAATCCGMT~CGGTGACTTCTTACCACTGTTAACGGCCTCCAGTCAAGGTCCCC~~CTTGTTTA(;T(:CGTTTAACCAGTGACCT GInAspThrGlnProAsnAs~A~“ValGlnValA~nPrOAs”AsnG~YA~p~h~L~UPrOL~”L~UThrAJ~SerSerGJnGJyProGinThrL~”Ph~~S~rP~~Ph~As~Gi~TrpPr~ 4620 GATTACGTGTTGCCGTTAGC~ATCACTGTACCTATTGTTGTGATTGTGCTCAGTGTTACCTTAGGACTTGCCATTGGAATCCC~T~CAC~G~C~CAGGCCTTG~~GCTGGGTTT

4590

AspTyrValJ.euProL~“Al~JleThrValProXleValVnlIleYalI.PuSrrVaJThrJ.e~lGJYLeliAJ~i1~Glyl

4650

4680

JePr~M~t”iSl.ysAsni.ysT,lnA,iJ,~,,J,ysA,~G,yPh~

4710 4740 GCGCTATC~~ACCAAAAGGTTGATGTGTTGACC~GCGGTTGGTAGTGT~TTTAAGG~TCATT~CCGCACAGGTATCAGTCAAGCGCCM~C~CTTGAAACAAACCA~TGC~G~T

4770

4600

AlaLeuSerAsnGlnLysVaJA~pValLeuThrLysAlaVaJGJYSerValPheLySGJ”~JelleA~~Al‘2ThrGlylJeSerGlnAJaPr”LysAraLeuLysClnfhrSerPllaAl~ 4660

4630

AAAAAACCCGCTTAG

~CCAGGAGCACCCCGCCCACCAGTACCACC~GCCAGGGGCTCCT~GCCACCAGTGCMCCACCT Ly~ProGlyAJaProAr~ProProVafProProi.ysProGlyAlaProLysProProVnfGJnProProLysLysProAl~

Fig. 4. The nucleotide sequence.

sequence

of the PI attachment

Only the nt are numbered,

codon. The 12 aa that match the N terminus to the TGA codons. nt position

in parentheses

(907). BarnHI

referring

gene. The deduced

ofP1 protein (Jacobs

sites shown

et al., 1987) are underlined,

in Fig. 3, Map A, between

to the first 5’-base

amino acid sequence sequence

in the recognition

is shown below the nucleotide

and 4884 being the last nt of the TAG stop as are the 2 1 Trp residues which correspond

the unique NruI and EcoRV sites are as follows, with the sequence:

Nrul (71), .%a1

(256 and 3280), Noel (304), PstI

(I 182). Paul (1436 and 2867). Ndei (1622), NcoI (1778), SspI (2821), Sstl (3678). IiindIII

(4336). The Nindlll deoxyribonucleotide, sequence

The restriction

protein

with 1 being the first nt of the Pl coding

corresponds

site between

the 4346

and 2084-bp

used as the primer to sequence to nt 3683-3858,

fragments

mRNA

while the cDNA

and synthesize

sequence

(3819), Sal1 (4100), EeoRV

shown in Fig. 3, map C, is at nt 3819. The sequence extends

cDNA

from the mRNA,

from nt 2483 to nt 3861.

is at nt 3862-3879.

of the 18-mer The mRNA

226

sents the first codon of the ORF. at aa position

Notably,

the Trp

(f) Godon usage of the

PI gene

69 aligns with a UGA codon deduced

from the nucleotide sequence. This further supports the evidence that UGA is read as tryptophan in

Analysis of the codon usage of the mature PI gene showed that all possible sense codons were used

M.pneumoniae

with the exception

(noted

in

section a). Jacobs

CUSSION,

both Be and Gly in position position

RESULTS

AND

DIS-

et al. (1987) reported 3 (corresponding

62 of the ORF) and the deduced

to aa

sequence

was Ile; this aa has now been clearly determined be Ile in five analyses of different preparations protein (E. Jacobs, personal communication). Pl

protein

bound,

is surface-exposed

and

to

of PI Since

In addition, phan.

of UGU

The absence

confirmed

and UGC for cysteine.

there were 21 UGA codons of cysteine

by labeling

in Pl protein

M. p~e~~~~~ae

cysteine or [ i5S ~methionine, teins on a polyacrylamide

for tryptowas

with [ %I-

and separating

the pro-

gel: an autoradiograph

of

membrane-

one would expect a signal peptide to be in-

volved in its transport across the membrane of M. pneumnnirre. Although most prokaryotic signal peptides are 20-40 aa long, mitochondrial and chloroplast signals can be polypeptides of up to 65 aa (Watson, 1984). The nature of the first 59 aa of the deduced sequence of P 1 was examined by computer analysis of the hydrophilicity values and by comparison to published signal sequences (Watson, 1984). Features of the sequence include two positively charged residues at the N terminus followed by a hydrophobic core and then a hydrophilic core. Fourteen of the residues are Thr (24%). A possible cleavage site based on the (-3, - 1) rule (von Heijne, 1986) is not found immediately before the Asn at position 60, but there is one located after the hydrophobic core; Arg-His-Thr at positions 57-58-59 breaks the -3 rule while the Ala-Thr-Ala at positions 20-21-22 conforms to the most common consensus sequence (Perlman and Halvorson, 1983). At least two possible interpretations can be made from this analysis. First, that the cleavage site of Pl protein is an exception to the rules and that M. ~?le~~~)niae contains a signal peptidase that cleaves the Arg-HisThr following the hydrophilic core. Second, that a conventional signal peptidase removes the first 22 aa and that secondary processing occurs to cleave off the remaining leader sequences to generate the mature Pl protein. Further work will be necessary to identify the processing step(s). The codons of the mature Pl protein sequence represent 1568 aa with an M,. of 169758. This is in good agreement with previous size estimates determined by SDS-PAGE: 165 kDa by Baseman et al. (1982), 168 kDa by Jacobs et al. (1986), 170 kDa by Vu et al. (1987), and 190 kDa by Hu et al. (1981).

Fig. 5. Comparison

of the [?3]cysteine-

and [‘“S]methionine-

labeled protein profiles of M. pneumoniae. Monolayer

cultures

M. pnemmonicre were grown

(Hu et al.,

1977) to early log-phase, (MEM),

and labeled

pCi;mI) in cysteinelabeled

cultures

SDS-PAGE

as previously

described

rinsed with minimal essential

with [““SJcysteine

were

rinsed,

(described

harvested,

and

processed

in labeling

radioautographs efticiency,

produced

[ “S]methioninc

panel

(panel A)

(panel B). Because of B is a composite

from the same gel after 60 h (lane

and 24 h (lane 2) of exposure.

The PI protein

by arrows.

of

f)

band is labeled by

(panel B, lane 2) but not [‘5S]cysteine

lane l), as indicated

for

in Fig. 1). The Coomassie-blue-stained

and then used to produce radioaut~graphs

(5

MEM for 12 h. The

gel was dried down onto filter paper and photographed differences

medium

or [~~S]methionine

or methionine-deficient

of

(panel B,

221

the gel was then compared

to the protein

the Coomassie

gel. No incorporation

[ 35S]cysteine

blue-stained

profile of

quences

alone

of

M. pneumoniae

into P 1 could be detected, while the P 1

is no evidence

band was labeled with [ 35S]methionine

total genome.

kb

or

As mentioned

antigenic

variation.

been presented.

the

above, there Current

see if this suggests any evolutionary attachment-protein

of

species efforts

are being made to determine the organization of these P 1 sequences in the M. pneumoniae genome to

(g) Conclusions The cloning

5%

for more than one Pl protein

and no apparent

(Fig. 5).

40

and sequencing

of a functional

Pl

gene from A4. pneumoniae

has

The authenticity

of this gene has

been demonstrated by the following. (1) Ten Hind111 fragments containing epitope region were completely

or partially

the M-328 sequenc-

ed, and only the 4.4-kb fragment contained a long ORF. (2) The mRNA and cDNA sequences match the PI sequences found in the 4.4-kb Hind111 fragment and phage AJI-7, confirming that this gene is transcribed in M. pneumoniae. (3) The N-terminal amino acid sequence of Pl protein determined by Jacobs et al. (1987) aligns perfectly with the deduced amino acid sequence beginning with Asn at aa position 60. The surfaceexposed and membrane-bound nature of P 1 protein is compatible with the first 59 aa being a signal sequence that is processed in one or two steps to yield the mature protein. As the data in this paper indicate, the complex P 1 story is just beginning to unfold. More questions than answers have been generated which this laboratory is actively seeking to answer. (i) There is no evidence for more than one PI protein as judged by one- and two-dimensional PAGE (Hansen et al., 1979). In addition, there is no apparent antigenic variation in Pl protein over time as shown by Vu et al. (1987) in immunoblot analyses of twelve M. pneumoniae isolates collected during a IO-year period. Despite this compelling support for our belief that we have cloned and sequenced the only functional PI gene in M. pneumoniae, we cannot rule out the remote possibility that an additional PI gene exists since other co-migrating fragments could have escaped detection. We are determining the relative chromosomal locations of the ten characterized Hind111 fragments and should identify any additional fragments during this analysis. (ii) The purpose of the untranscribed and therefore nonfunctional PI sequences is a mystery. The Hind111 fragments containing the M-328 epitope se-

significance.

(iii) The ORFs flanking the Pl gene, together with the absence sequences

of RBS and transcription in the

immediately

termination

adjacent

regions,

suggest that the Pl gene is transcribed as part of a larger polycistronic message. The ORFs could encode P 1 processing enzymes, regulatory functions, or other proteins involved in the attachment of M. pneumoniae to the respiratory epithelium. Complete nucleotide sequencing of these ORFs will aid in devising experiments to study this question. (iv) The signal sequence hypothesis could be tested in the proper genetic system. Studies of gene expression in M. pulmonis and Acholeplasma laidlawii (Dybvig and Cassell, 1987) or M. hominis (Roberts and Kenny, 1987) may be possible in the future. An alternative approach would be to ‘correct’ the UGA codons by in vitro mutagenesis and study the expression of P 1 protein with some well-established prokaryotic host/vector systems.

ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health grants AI-20391 and HL-19171 and by Cooperative Agreement CR807392 from the Environmental Protection Agency. We thank Dr. C.A. Hutchinson III for helpful suggestions, vector DNA and E. coli strains, and the synthesis of oligodeoxyribonucleotide primers; Dr. P. Bassford Jr., for helpful discussions about signal sequences; S.C. Hu for preparation of radioautographs; B. Noel1 for technical support; and A. Thomas and B. Shambley for secretarial assistance.

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1N PROOF

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Communicated

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by R.E. Yasbin