The Mycoplasma pneumoniae cytadhesin P1 structural gene with flanking regions was labeled by nick translation and used as a probe to analyze gene copy nuimber in M. pneumoniae. Multiple bands of genomic DNA were hybridized by the probe. To establish what part of the P1 gene existed as multiple copies, the P1 gene and regions adjacent to the 3' and 5' ends were divided with restriction enzymes into 14 segments ranging in size from 174 to 651 base pairs. These pieces were purified on agarose gels, subcloned into pUC19, purified, labeled by nick translation, and used to probe the entire M. pneumoniae genome. Several regions near the middle and carboxy end of the P1 structural gene hybridized to single copies. The remaining P1 subclones hybridized to multiple bands under stringent hybridization conditions, indicating extensive homology with other parts of the M. pneumoniae genome. The singleversus multiple-copy nature of P1 structural gene domains is discussed.

The Mycoplasma pneumoniae cytadhesin P1 genes from two groups of clinical isolates that display restriction fragment length polymorphisms were cloned and sequenced. Within each group the nucleotide sequences were identical, but two major differences were detected between the groups. These two stretches of sequence divergence were located in multiple-copy regions of the Pl gene and resulted in considerable amino acid changes. Mycoplasma pneumoniae is a cell wall-less procaryote that colonizes the ciliated respiratory epithelium of humans and causes primary atypical pneumonia (5, 11). Mycoplasma attachment to the respiratory cell surface is a critical step in the infection process, and previous studies have established that a 170-kilodalton surface protein, P1, located at the tiplike structure of virulent M. pneumoniae mediates cytadherence (2, 9, 12). Mutants that lack P1 or cannot cluster P1 at the tip are noncytadhering and avirulent (2, 4, 18). During M. pneumoniae infection both humans and experimental animals mount a strong immunological response against P1 that correlates with resolution of the disease (19, 33). To elucidate structural and functional properties of the P1 cytadhesin, we cloned and sequenced the gene (30). The P1 structural gene was contained within a 5.6-kilobase (kb) EcoRI piece of DNA, and further analysis revealed that two-thirds of the P1 gene was multiple copies (29). Since repeated gene sequences of major surface antigens can be associated with antigenic variation in pathogenic microorganisms (24, 27, 28, 31), P1 subclones generated in our previous study (29) were used to probe a collection of M. pneumoniae strains and clinical isolates. Two distinct hybridization patterns were detected, suggesting variability in their P1 genes (6). To further understand the nature of these observations, the P1 genes from several representative isolates of M. pneumoniae were cloned and sequenced. M. pneumoniae M129-B16 (ATCC 29342 in its sixteenth passage) was isolated in 1968 from a patient with M. pneumoniae disease (20) and served as the standard wild-type strain. M. pneumoniae FH (ATCC 15531) was purchased from the American Type Culture Collection, Rockville, Md. (5). M. pneumoniae TW 7-5 was obtained from J. G. Tully (National Institute of Allergy and Infectious Diseases) and originated from a group of mycoplasma isolates obtained from military recruits in 1974 and 1975 (3, 34). M. pneumo

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 molecular 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.

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The P30 adhesin genes of spontaneous, hemadsorption-negative (HA ؊) class II Mycoplasma pneumoniae mutants that displayed P30 adhesin-deficient protein profiles were analyzed. One subclass of P30-deficient mutants possessed the entire p30 structural gene without alterations (825 nucleotides, encoding 275 amino acids with a predicted molecular mass of 29,743 Da [S. F. Dallo, A. Chavoya, and J. B. Baseman, Infect. Immun. 58:4163-4165, 1990]). However, the second mutant subclass contained a deletion in p30 resulting in the expression of a 25-kDa peptide (681 nucleotides, encoding 227 amino acids with a calculated molecular mass of 24,823 Da). This P25-truncated peptide lacked 8 of the 13 proline-rich amino acid repeat sequences at the carboxy terminus. Whole-cell radioimmunoprecipitation of M. pneumoniae with antibodies directed against the proline-rich repeat sequences located in the carboxy terminus demonstrated their surface accessibility. In contrast, antibodies generated against N-terminal amino acid sequences upstream of the repeats did not bind to intact mycoplasmas. The amino acid sequence homologies exhibited by the P30 adhesin and eucaryotic structural proteins were corroborated by cross-reactive epitopes shared between the P30 adhesin and fibrinogen, keratin, and myosin. These data reinforce the importance of the P30 protein in cytadherence and virulence and provide a molecular basis for postinfectious autoimmunity associated with M. pneumoniaemediated pathologies.