A previously identified trypsin-resistant surface protein of Mycoplasma pneumoniae clusters at the tip organelle of virulent mycoplasmas and appears to be essential for cytadherence and virulence. Monoclonal antibodies generated against this protein were used to identify positive recombinant clones from M. pneumoniae genomic DNA libraries. The structural gene was sequenced and contained an open reading frame of 825 nucleotides that encoded a protein of 275 amino acids with a calculated

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... mass of 29,743 Da. This protein (P30) contained three types of repeat sequences at the carboxy end, each consisting of six amino acids. In addition, the protein was proline rich (20.7%) and exhibited significant amino acid homology with the P1 cytadhesin of M. pneumoniae and with several matrix-associated eucaryotic proteins. Mycoplasma pneumoniae is a cell wall-less procaryote that colonizes the human respiratory tract and causes primary atypical pneumonia. Successful tissue parasitism results from the tip-oriented adherence of mycoplasmas to respiratory epithelial cells, and studies using isogenic mutants have established that the cytadherence event requires a complex interaction of several mycoplasma proteins (2, 15). The surface protein designated P1 (170 kDa) is considered a major adhesin of M. pneumoniae (1, 2, 7, 9) , and this protein and its structural gene have been extensively studied (1, 2, 4, 5, 10, 12, 25, 26) . We have implicated an additional surface protein, designated P30, in cytadherence and virulence of M. pneumoniae (2, 3). Spontaneous hemadsorption-negative (HA-) mycoplasmas that lack this protein are nonadhering and avirulent and are termed class II mutants (3, 15). Hemadsorption-positive (HA') revertants of class II mutants resynthesize the P30 protein and regain cytadherence and virulence capabilities (2, 3, 14) . In addition, antibodies generated against the P30 protein localize at the tip organelle of M. pneumoniae and block adherence (3, 21). Furthermore, convalescent-phase sera from humans and experimentally infected hamsters exhibit strong immunoreactivity against this protein (3, 16). In the study described here a Agtll library was constructed by using wild-type HA' M. pneumoniae genomic DNA. Recombinants were screened with a pool of monoclonal antibodies reactive against the P30 protein, and the structural gene was subcloned and sequenced. M. pneumoniae wild-type HA' strain M129-B16 was grown in 32-oz (ca. 950-ml) glass prescription bottles in 70 ml of modified Edward medium (6) at 37°C for 72 h. The glass-adherent mycoplasmas were washed four times with phosphate-buffered saline (pH 7.2) and collected by scraping prior to lysis by addition of 0.3 ml of 10% sodium dodecyl sulfate. The lysate was incubated with 50 ,ug of RNase (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) per ml for 30 min at 37°C and then with 0.05 M EDTA (pH 8.0) and 50 ,ug of proteinase K (Boehringer Mannheim) per ml for 30 min at 37°C. DNA preparations were obtained by extracting the digested lysate three times with equal volumes * Corresponding author. of phenol equilibrated with 1 M Tris (pH 8.0), once with phenol-chloroform (1:1), and once with chloroform-isoamyl alcohol (24:1). Sodium acetate (3 N, 0.1 volume) was added to the solution, and the DNA was precipitated with ethanol. The M. pneumoniae genomic DNA libraries were constructed in the expression vector Xgtll by the procedures of Young and Davis (29, 30) with minor modifications (4, 5) and screened with a pool of anti-P30 monoclonal antibodies (clones H13.13B2, H13.1C8, and H13.4F4 [3]). Twelve immunoreactive phage clones that produced fusion proteins containing between 35 and 227 amino acids of the P30 protein were identified. DNA was extracted as previously outlined (5, 27), and insert DNA from two positive clones was subcloned. Sequence data indicated that these two inserts overlapped and that there was a stop codon at a position consistent with the size of the expressed fusion proteins. Since differences in molecular masses (17.5 and 25 kDa) existed between the peptides associated with the recombinant clones and the native P30 protein, the recombinants were used to probe the M. pneumoniae genome at high stringency (68°C). Southern blot analysis revealed hybridization patterns as shown in Fig. la . The 4.6-kbp PstI fragment that hybridized to insert DNA from all positive recombinants was separated by gel electrophoresis, ligated to the PstI site of pUC19 (18) , and used to transform library-efficient competent cells of Escherichia coli DH5a according to the instructions of the manufacturer (Bethesda Research Laboratories, Inc., Gaithersburg, Md.). Twenty transformants were selected on LB agar plates containing 50 ,ug of ampicillin per ml, and plasmid DNA was isolated from overnight cultures by the alkaline lysis method (18). The 4.6-kbp PstI insert was mapped (Fig. lb) , and a 1.4-kbp HinclI DNA fragment that hybridized to the positive recombinant clones was isolated, cloned in pUC19, and subsequently sequenced by subcloning into the M13 phage vector (19, 24) . Analysis of the sequence data identified an open reading frame of 825 nucleotides (Fig. 2) , and the deduced amino acid sequence encoded a protein of 275 amino acids with a calculated molecular mass of 29,743 Da. By analogy to the 170-kDa adhesin P1 of M. pneumoniae (26) and the 140-kDa adhesin of Mycoplasma genitalium (4), this protein may be processed. For example, the hydropathicity plot of the 4163