SOS involvement in stress-inducible biofilm formation.

Bacterial biofilm formation can be induced by antimicrobial and DNA damage agents. These agents trigger the SOS response, in which SOS sensor RecA stimulates auto-cleavage of repressor LexA. These observations lead to a hypothesis of a connection between stress-inducible biofilm formation and the RecA-LexA interplay. To test this hypothesis, three biofilm assays were conducted, viz. the standard 96-well assay, confocal laser scanning microscopy, and the newly developed biofilm-on-paper assay. It was found that biofilm stimulation by the DNA replication inhibitor hydroxyurea was dependent on RecA and appeared repressed by the non-cleavable LexA of Pseudomonas aeruginosa. Surprisingly, deletion of lexA led to reduction of both normal and stress-inducible biofilm formation, suggesting that the wild-type LexA contributes to biofilm formation. The decreases was not the result of poor growth of the mutants. These results suggest SOS involvement in hydroxyurea-inducible biofilm formation. In addition, with the paper biofilm assay, it was found that degradation of the biofilm matrix DNA by DNase I appeared to render the biofilms susceptible to the replication inhibitor. The puzzling questions concerning the roles of LexA in DNA release in the biofilm context are discussed.

Intracellular DNA replication and long-term survival of pathogenic mycoplasmas.

We examined intracellular survival and growth of pathogenic mycoplasmas (Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma genitalium) in cultured human cells. By using the eukaryotic nuclear DNA synthesis inhibitor, aphidicolin, we detected the selective synthesis of mycoplasma (My) and mitochondria (Mt) DNA, which could be further differentiated by restriction enzyme analyses. Also, intracellular M. pneumoniae and M. penetrans infectivity of human cells was detected over 6 months using subfractionation of infected cells and determination of mycoIplasma colony forming units (cfu). For M. genitalium, which we failed to re-grow from infected cells, species-specific PCR primers were used to implicate long-term mycoplasma survivability. Data indicated that pathogenic mycoplasmas reside and replicate intracellularly over extended periods in human cells, consistent with the ability of mycoplasmas to circumvent antibiotic therapy and immune surveillance and establish chronic infections.

Interplay between mycoplasma surface proteins, airway cells, and the protean manifestations of mycoplasma-mediated human infections.

Adherence of mycoplasmas to specific tissue surfaces is a crucial step in the establishment of infection. Several pathogenic mycoplasmas are flask-shaped and possess specialized tips that permit a highly oriented surface parasitism of host target cells. Mycoplasma pneumoniae, which causes primary atypical pneumonia in humans, requires a network of interactive adhesins and accessory proteins to cytadhere. The adhesins must cluster at the mycoplasma tip organelle in close association with cytadherence-related accessory proteins and a naplike structure, which together appear to comprise a primitive cytoskeleton-like system. Proline-rich regions associated with these proteins play critical roles in the maintenance of the structural and functional integrity of the tip. Mycoplasma genitalium, originally isolated from the human urogenital tract of patients with nongonococcal urethritis, also colonizes airway cells along with M. pneumoniae. The molecular basis for cytadherence of these mycoplasmas is discussed in terms of the identification, cloning, and sequencing of the implicated mycoplasma genes, their common DNA and amino acid homologies and structural and functional domains, and the organizational similarities in their cytadherence-related operons. In addition, the multiorgan protean manifestations of mycoplasma infection are discussed in terms of the role that mycoplasma adhesins may play in molecular mimicry, postinfectious autoimmunity, and immune-mediated damage.

Biofunctional domains of the Mycoplasma pneumoniae P30 adhesin.

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 p3O 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 p3O 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. pneumoniae-mediated pathologies.

Mycoplasma pneumoniae and Mycoplasma genitalium mixture in synovial fluid isolate.

A mycoplasma cultured from synovial fluid specimens from a patient with pneumonia and subsequent polyarthritis was identified initially as Mycoplasma pneumoniae. In retrospective studies, the culture was shown also to contain Mycoplasma genitalium. In this paper, the laboratory techniques employed in the identification and separation of the two species are presented, and evidence to implicate postinfectious autoimmunity is provided. An increasing number of reports of M. genitalium in human tissue sites and difficulties in isolation and identification of the organism in the clinical laboratory suggest the need for more extensive application of rapid and specific detection systems for both M. genitalium and M. pneumoniae in the clinical laboratory.

Properties of adhering and nonadhering populations of Mycoplasma genitalium.

The interaction between Mycoplasma genitalium and human lung fibroblasts (HLFs) was studied with the use of wild-type and hemadsorption-negative (HA-) mycoplasmas. [35S]-methionine-labeled M. genitalium adhered to HLFs by first-order kinetics, with maximal interaction occurring at approximately 2 hours. Electron microscopy of chemically fixed cells revealed an almost immediate association of mycoplasmas with the HLF plasma membrane that was mediated by the mycoplasma tip and a nap-like layer, which appeared to extend from the tip around much of the mycoplasmal unit membrane. Following cytadherence, M. genitalium appeared capable of invading the intracellular spaces of targeted HLF cells, possibly by receptor-mediated endocytosis. Spontaneously arising HA- variants of M. genitalium strain G37 failed to adhere to HLF cells and were distinguished on the basis of their protein profiles. SDS-PAGE analysis of the class I (lacking the 140-kd protein but containing a polypeptide doublet at approximately 140-kd) and class II (lacking the 140-kd protein and doublet) variants of M. genitalium revealed that class I variants contain a doublet protein in the 140-kd region, which reacted with a monoclonal antibody generated to the adhesin-implicated 140-kd protein (P140) of wild-type M. genitalium. Class II variants completely lacked the 140-kd protein or immunologically related peptides.

Expression in Spiroplasma citri of an epitope carried on the G fragment of the cytadhesin P1 gene from Mycoplasma pneumoniae.

We have previously described the use of the replicative form (RF) of Spiroplasma citri virus SpV1 as a vector for cloning and expressing foreign genes in S. citri, an organism which reads UGA as a tryptophan codon (C. Stamburski, J. Renaudin, and J.M. Bové, J. Bacteriol. 173:2225-2230, 1991). We now report cloning and expression in S. citri of the G fragment of cytadhesin P1 gene from Mycoplasma pneumoniae. The G fragment was inserted in the SpV1 RF downstream of a synthetic ribosome binding site and introduced into S. citri by electroporation. Northern (RNA) blot analyses showed that in S. citri, the G fragment was transcribed from an SpV1 RF promoter as a 1.2-kb mRNA. The translation product was detected by Western blotting (immunoblotting) with a rabbit antiserum raised against total proteins from M. pneumoniae (strain FH) and was proved to be P1 specific by using monoclonal antibodies specific for the G region of the P1 protein. The apparent molecular mass of the polypeptide (24.5 kDa) indicates that in S. citri, the G fragment was fully translated in spite of the seven UGA codons present in the reading frame.

Possible origin of sequence divergence in the P1 cytadhesin gene of Mycoplasma pneumoniae.

Specific regions of the P1 adhesin structural gene of Mycoplasma pneumoniae hybridize to various parts of the mycoplasma genome, indicating their multiple-copy nature. In addition, restriction fragment length polymorphisms and sequence divergence have been observed in the P1 gene, permitting the classification of clinical isolates of M. pneumoniae into two groups, I and II. These data suggest that the observed P1 gene diversity may be explained by homologous recombination between similar but not identical multicopy P1-related sequences and the P1 structural gene. We used oligonucleotide probes specific to the diverged regions of the group I and group II P1 structural genes to clone and sequence multicopy P1-related DNA segments. We detected sequences in group I M. pneumoniae isolates that were homologous not only to the group I P1 structural gene but also to the diverged regions of the group II P1 structural gene. Likewise, sequences in group II clinical isolates that were homologous both to the group II P1 structural gene and the diverged regions of the group I P1 structural gene were detected.

Distinctions in DNA and protein profiles among clinical isolates of Mycoplasma pneumoniae.

Clinical isolates of Mycoplasma pneumoniae previously shown to exhibit significant sequence divergency in a major 170 kDa adhesin, designated P1, were further characterized using restriction enzyme fingerprinting of genomic DNA and two-dimensional gel electrophoresis of total proteins. Numerous differences in DNA restriction patterns and protein profiles were found, possibly reflecting various degrees of virulence and antigenic potential.

Adhesin gene of Mycoplasma genitalium exists as multiple copies.

The structural gene of the 140 kDa adhesin of Mycoplasma genitalium was used to probe M. genitalium genomic DNA for gene copy number. Since multiple banding patterns were observed, the 140 kDa structural gene was subdivided into 10 contiguous fragments in size from 165 to 657 base pairs in order to determine which parts of the adhesin gene existed as multiple copies. Each fragment was labeled by nick translation and used to probe the entire M. genitalium genomic DNA. Approximately half the gene was present in single copy while the remaining sequences were multiple copied. Both single and multiple copy regions were interspersed throughout the structural gene.

Characterization of the gene for a 30-kilodalton adhesion-related protein of Mycoplasma pneumoniae.

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.

Sequence divergency of the cytadhesin gene of Mycoplasma pneumoniae.

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 P1 gene and resulted in considerable amino acid changes.

Molecular distinctions among clinical isolates of Mycoplasma pneumoniae.

Restriction enzyme fingerprinting of genomic DNA and Southern blots probed with subclones of the Mycoplasma pneumoniae cytadhesin P1 gene were used to characterize clinical isolates of M. pneumoniae. On the basis of the examination of 29 individual M. pneumoniae isolates, two distinct groups were established. Group 1, which displayed a 12-kilobase band following DNA digestion with HindIII, consisted of strain M129-B16 and three others obtained in the state of Washington during the 1960s. The remaining M. pneumoniae strains belonged to group 2, which lacked the 12-kilobase band and included samples from the 1940s, 1970s, and 1980s. This category also included the only M. pneumoniae strain isolated from the synovial fluid of an arthritic patient.

Restriction fragment length polymorphism in the cytadhesin P1 gene of human clinical isolates of Mycoplasma pneumoniae.

Genomic DNA obtained from Mycoplasma pneumoniae clinical isolates spanning a 30-year period was analyzed for the presence of polymorphism in their P1 cytadhesin genes. All clinical isolates expressed a 170-kilodalton P1 protein that reacted with anti-P1 monoclonal antibodies. However, Southern blot analysis of specific M. pneumoniae isolates with subclones of the P1 structural gene revealed the presence of restriction fragment length polymorphism, permitting the classification of their P1 genes into two distinct categories.

Cross-hybridization between the cytadhesin genes of Mycoplasma pneumoniae and Mycoplasma genitalium and genomic DNA of Mycoplasma gallisepticum.

Immunological cross-reactivity was observed between the cytadhesin proteins of Mycoplasma pneumoniae and Mycoplasma genitalium and a 155 kDa protein of Mycoplasma gallisepticum. Furthermore, the cytadhesin genes of M. pneumoniae and M. genitalium were used to demonstrate homology with M. gallisepticum genomic DNA under low stringency conditions suggesting that a family of adhesin-related genes exists among these pathogenic mycoplasmas.

DNA and protein sequence homologies between the adhesins of Mycoplasma genitalium and Mycoplasma pneumoniae.

Mycoplasma genitalium and Mycoplasma pneumoniae are morphologically and serologically related pathogens that colonize the human host. Their successful parasitism appears to be dependent on the product, an adhesin protein, of a gene that is carried by each of these mycoplasmas. Here we describe the cloning and determine the sequence of the structural gene for the putative adhesin of M. genitalium and compare its sequence to the counterpart P1 gene of M. pneumoniae. Regions of homology that were consistent with the observed serological cross-reactivity between these adhesins were detected at both DNA and protein levels. However, the degree of homology between these two genes and their products was much higher than anticipated. Interestingly, the A + T content of the M. genitalium adhesin gene was calculated as 60.1%, which is substantially higher tham that of the P1 gene (46.5%). Comparisons of codon usage between the two organisms revealed that M. genitalium preferentially used A- and T-rich codons. A total of 65% of positions 3 and 56% of positions 1 in M. genitalium codons were either A or T, whereas M. pneumoniae utilized A or T for positions 3 and 1 at a frequency of 40 and 47%, respectively. The biased choice of the A- and T-rich codons in M. genitalium could also account for the preferential use of A- and T-rich codons in conservative amino acid substitutions found in the M. genitalium adhesin. These facts suggest that M. genitalium might have evolved independently of other human mycoplasma species, including M. pneumoniae.

Homologous regions shared by adhesin genes of Mycoplasma pneumoniae and Mycoplasma genitalium.

A lambda gt11 library of Mycoplasma genitalium genomic DNA was generated, and clones were identified using a pool of monoclonal antibodies directed against different epitopes of the 140 kDa adhesin protein. Because the 140 kDa protein of M. genitalium and the 170 kDa P1 adhesin of M. pneumoniae share biological properties such as a tip-associated location, cytadherence function and immunologic crossreactivity, we performed Southern blot analysis using these cloned partial 140 kDa gene fragments and 14 subclones that span the P1 structural gene of M. pneumoniae. Homologous regions of the two genes were identified.

Isolation and characterization of Mycoplasma genitalium strains from the human respiratory tract.

Mycoplasma genitalium, an organism first isolated from the urethras of two men with nongonococcal urethritis, has been found in throat specimens from military recruits participating in an inactivated Mycoplasma pneumoniae vaccine field trial in 1974-1975. Four of 16 preserved throat isolates, previously identified as strains of M. pneumoniae, have now been shown to be mixtures of M. pneumoniae and M. genitalium. Purification of these mixed mycoplasmas by selection of single colonies confirmed the presence of M. genitalium. Identification of M. genitalium was based upon the occurrence of a species-specific 140-kilodalton protein adhesin in these isolates and their serologic reactivity to an M. genitalium antiserum. The frequent occurrence of both M. pneumoniae and M. genitalium in a number of these throat specimens, in combination with their shared antigenic cross-reactivities, suggests the likelihood that M. genitalium strains are easily missed in the usual laboratory identification procedures. What role M. genitalium may play in human respiratory disease remains to be determined.

Identification of P1 gene domain containing epitope(s) mediating Mycoplasma pneumoniae cytoadherence.

A genomic library of Mycoplasma pneumoniae was constructed by cloning sheared genomic DNA into the expression vector lambda gt11. Recombinant clones were screened using anti-M. pneumoniae mAbs reactive with adhesin P1 epitopes that mediate cytadherence. 10 clones with different size inserts were isolated. These clones possessed P1 sequences localized to the COOH terminus of the P1 gene. All clones produced fusion proteins that reacted with acute and convalescent sera of patients infected with M. pneumoniae. Interestingly, one clone, P1-7, contained an epitope that was confined to a region of 13 amino acids present in the M. pneumoniae genome as a single copy. The identification of this cytadherence-related epitope permits the production of a synthetic peptide that can be used as a rational vaccine candidate and serodiagnostic probe.