Analysis of three different repeated DNA elements present in the P1 operon of Mycoplasma pneumoniae: size, number and distribution on the genome.

Mycoplasma pneumoniae, a bacterium pathogenic for humans, has a relatively small genome size of 840 kbp. Even though, several repeated DNA elements have been identified in the genome of this prokaryote, particularly within the P1 gene which codes for a major adhesin protein of M. pneumoniae. These elements were characterized in detail with respect to size, number and distribution on the genome, represented by an ordered cloned library covering the complete chromosome. Three different repetitive elements were detected in and around the P1 gene designated as RepMP2/3, RepMP4 and RepMP5. The length of these elements varies between 1.1-1.5 kbp (RepMP4), 1.8 kbp (RepMP2/3) and 1.9-2.2 kpb (RepMP5). They occur at least 8 to 10 times on the chromosome. Possible functions are discussed and a uniform nomenclature for these repeats is proposed.     

Repetitive DNA sequences in Mycoplasma pneumoniae.

Two types of different repetitive DNA sequences called RepMP1 and RepMP2 were identified in the genome of Mycoplasma pneumoniae. The number of these repeated elements, their nucleotide sequence and their localization on a physical map of the M. pneumoniae genome were determined. The results show that RepMP1 appears at least 10 times and RepMP2 at least 8 times in the genome. The repeated elements are dispersed on the chromosome and, in three cases, linked to each other by a homologous DNA sequence of 400 bp. The elements themselves are 300 bp (for RepMP1) and 150 bp (for RepMP2) long showing a high degree of homology. One copy of RepMP2 is a translated part of the gene for the major cytadhesin protein P1 which is responsible for the adsorption of M. pneumoniae to its host cell.     

Identification of a small RNA within the pdh gene cluster of Mycoplasma pneumoniae and Mycoplasma genitalium

A highly abundant and heterogeneous small RNA about 205 to 210 bases long named MP200 RNA has been identified in Mycoplasma pneumoniae. It was localized on the genome within a 319-bp-long intergenic space of the pyruvate dehydrogenase (pdh) gene cluster. A database search at the DNA level revealed the highest similarity to a sequence located within the pdh gene cluster of Mycoplasma genitalium that was also shown to be transcribed into two abundant, but smaller RNAs than the ones in Mycoplasma pneumoniae. The RNAs from both M. pneumoniae and M. genitalium have the potential to code for cysteine-rich 29- and 23-amino-acid-long peptides, but so far, these peptides have not been identified experimentally in bacterial protein extracts.     

Sequence divergence in the ORF6 gene of Mycoplasma pneumonia.

The ORF6 gene product of Mycoplasma pneumoniae is involved in a yet-unknown manner in the adhesion of the bacterium to its host cell. Part of the ORF6 gene is a repetitive DNA sequence (RepMP5), about 1,900 bp long. Seven additional similar copies of RepMP5 are dispersed on the genome. In the independently isolated strains M. pneumoniae M129 and FH, the RepMP5 copies residing in the ORF6 gene are not identical. Two conserved regions, ranging from nucleotides 1 to 799 and from nucleotide 1795 to the end of the gene, border a variable region, ranging from nucleotides 800 to 1794. This variable region differs in DNA sequence and by 201 bp. Analysis of RepMP5 copies outside the ORF6 gene showed that both M. pneumoniae M129 and M. pneumoniae FH carry a RepMP5 copy on a 6-kbp EcoRI fragment which has the same DNA sequence as the variable region of RepMP5 in the M. pneumoniae FH ORF6 gene. According to these data, a switch from the M. pneumoniae M129 ORF6 gene to the M. pneumoniae FH ORF6 gene could take place by gene conversion.     

Molecular characterization of the P1-like adhesin gene from Mycoplasma pirum.

A DNA fragment has been isolated from the genome of Mycoplasma pirum by use of a genetic probe derived from the conserved region within the genes for the major adhesins of Mycoplasma genitalium and Mycoplasma pneumoniae. A gene encoding an adhesin-like polypeptide was localized, and sequence analysis indicated a G + C content of only 28%, with A- and T-rich codons being preferentially used. A total of 91% of positions 3 were either A or T. The deduced polypeptide is 1,144 amino acids long (126 kDa) and shows 26% identity with the adhesins of M. genitalium and M. pneumoniae. Other features in common with these two membrane proteins include a similar hydropathic profile and a proline-rich C terminus. Antibodies were prepared by using as an immunogen a peptide derived from the C terminus of the M. pirum adhesin-like polypeptide and were found to recognize on immunoblots a 126-kDa polypeptide from an M. pirum cellular extract. The characterization of the adhesin-like gene is a first step toward a better understanding of the mechanisms allowing this human mycoplasma to attach to host cells.     

Complete genome sequence of Mycoplasma pneumoniae type 2a strain 309, isolated in Japan

Mycoplasma pneumoniae strain 309, a type 2a (subtype 2 variant) strain of this bacterium, has variations in the P1 protein, which is responsible for attachment of the bacterium to host cells. Here, we report the complete genome sequence of M. pneumoniae strain 309 isolated from a pneumonia patient in Japan.     

The 40- and 90-kDa membrane proteins (ORF6 gene product) of Mycoplasma pneumoniae are responsible for the tip structure formation and P1 (adhesin) association with the Triton shell

After Triton X-100 treatment of Mycoplasma pneumoniae cells, a portion of the adhesin P1 (transmembrane protein) proved to remain tightly associated with the Triton insoluble material (Triton shell) as shown previously by several authors. However, the spontaneous loss of two cytadherence-associated membrane proteins of 90 and 40 kDa (gene product of the open reading frame 6 of the P1 operon) in a hemadsorption-negative mutant, designated M5, resulted in a 100% release of the P1 protein into the Triton phase and in the lack of the characteristic tip-like attachment organelle of M. pneumoniae indicating an essential role of the open reading frame 6 gene product in tip structure formation.     

Complete nucleotide sequence of the mycoplasma virus P1 genome

Mycoplasma virus P1 is one of only four viruses isolated from the genus Mycoplasma. The host for P1, Mycoplasma pulmonis, possesses complex, phase-variable restriction and modification enzymes and the Vsa family of phase-variable surface proteins. The ability of P1 virus to infect host cells is influenced by these phase-variable systems, rendering P1 a valuable tool for assessing host properties. The double-stranded P1 DNA genome was sequenced (11,660 bp) and 11 ORFs were identified. The predicted P1 DNA polymerase is similar to that of phages that are known to have terminal protein (TP) attached to the 5' end of their genome, consistent with previous studies indicating that P1 DNA has covalently attached TP. Most of the other predicted P1 proteins have little sequence similarity to known proteins, and P1 virus is unrelated to the other mycoplasma virus, MAV1, for which the genome sequence is known. One of the predicted P1 proteins, the ORF 8 gene product, contains a repetitive collagen-like motif characteristic of some bacteriophage tail fiber proteins and is a candidate for interacting with the Vsa proteins.     

Genomic organization of the Papaver rhoeas self-incompatibility S(1) locus

The self-incompatibility (SI) response in Papaver rhoeas depends upon the cognate interaction between a pollen-expressed receptor and a stigmatically expressed ligand. The genes encoding these components are situated within the S-locus. In order for SI to be maintained, the genes encoded by the S-locus must be co-inherited with no recombination between them. Several hypotheses, including sequence heterogeneity and chromosomal position, have been put forward to explain the maintenance of the S-locus in the SI systems of the Brassicaceae and the Solanaceae. A region of the Papaver rhoeas genome encompassing part of the self-incompatibility S(1) locus has been cloned and sequenced. The clone contains the gene encoding the stigmatic component of the response, but does not contain a putative pollen S-gene. The sequence surrounding the S(1) gene contains several diverse repetitive DNA elements. As such, the P. rhoeas S-locus bears similarities to the S-loci of other SI systems. An attempt to localize the P. rhoeas S-locus using fluorescence in situ hybridization (FISH) has also been made. The potential relevance of the findings to mechanisms of recombination suppression is discussed.     

Sequence analysis of 56 kb from the genome of the bacterium Mycoplasma pneumoniae comprising the dnaA region, the atp operon and a cluster of ribosomal protein genes.

To sequence the entire 800 kilobase pair genome of the bacterium Mycoplasma pneumoniae, a plasmid library was established with contained the majority of the EcoR1 fragments from M.pneumoniae. The EcoR1 fragments were subcloned from an ordered cosmid library comprising the complete M.pneumoniae genome. Individual plasmid clones were sequenced in an ordered fashion mainly by primer walking. We report here the initial results from the sequence analysis of -56 kb comprising the dnaA region as a potential origin of replication, the ATPase operon and a region coding for a cluster of ribosomal protein genes. The data were compared with the corresponding genes/operons from Bacillus subtilis, Escherichia coli, Mycoplasma capricolum and Mycoplasma gallisepticum.     

Expression of UGA-containing Mycoplasma genes in Bacillus subtilis

We used Bacillus subtilis to express UGA-containing Mycoplasma genes encoding the P30 adhesin (one UGA) of Mycoplasma pneumoniae and methionine sulfoxide reductase (two UGAs) of Mycoplasma genitalium. Due to natural UGA suppression, these Mycoplasma genes were expressed as full-length protein products, but at relatively low efficiency, in recombinant wild-type Bacillus. The B. subtilis-expressed Mycoplasma proteins appeared as single bands and not as multiple bands compared to expression in recombinant Escherichia coli. Bacillus mutants carrying mutations in the structural gene (prfB) for release factor 2 markedly enhanced the level of readthrough of UGA-containing Mycoplasma genes.     

The gene mpn310 (hmw2) from Mycoplasma pneumoniae encodes two proteins, HMW2 and HMW2-s, which differ in size but use the same reading frame

The gene mpn310 from Mycoplasma pneumoniae encodes the proteins HMW2 with a molecular weight of 215 621 and the smaller P28, here called HMW2-s. Because HMW2-s is not well defined, it was isolated from protein extracts of M. pneumoniae cells and its N-terminal end was determined by MS. HMW2-s starts with the methionine at the amino acid position 1620 of HMW2 and its residual sequence is identical to the last 198 amino acids of HMW2, predicting a molecular weight of 23 204. These results were confirmed by the comparative MS analysis of HMW2-s that had been synthesized in Escherichia coli . A precursor–product relationship between HMW2 and HMW2-s could be excluded, because HMW2-s can be translated from a specific mRNA starting within mpn310 . The conservation of an HMW2-s like protein in M. pneumoniae and Mycoplasma genitalium emphasizes its possible functional importance.     

A Mycoplasma genetic element resembling prokaryotic insertion sequences

Nucleotide sequence analysis of two Mycoplasma hyopneumoniae-derived copies of a repetitive genetic element revealed structural similarities to typical prokaryotic insertion sequences. This is the first such sequence identified in the class Mollicutes. The element spans approximately 1550bp, with 28bp inverted terminal repeats. Two open reading frames occur within the sequence, one potentially encoding a protein with a size-variant alpha-helical domain containing heptameric leucine periodicity. Hybridization data with several strains from each of two mycoplasma species showed that the repetitive sequence is variably distributed within the M. hyopneumoniae and Mycoplasma hyorhinis chromosomes and indicated that in some cases the repeated sequence is contained within a larger genetic element which may be the result of phage or plasmid insertion.     

Homologue of macrophage-activating lipoprotein in Mycoplasma gallisepticum is not essential for growth and pathogenicity in tracheal organ cultures

While the genomes of a number of Mycoplasma species have been fully determined, there has been limited characterization of which genes are essential. The surface protein (p47) identified by monoclonal antibody B3 is the basis for an enzyme-linked immunosorbent assay for serological detection of Mycoplasma gallisepticum infection and appears to be constitutively expressed. Its gene was cloned, and the DNA sequence was determined. Subsequent analysis of the p47 amino acid sequence and searches of DNA databases found homologous gene sequences in the genomes of M. pneumoniae and M. genitalium and identity with a gene family in Ureaplasma urealyticum and genes in M. agalactiae and M. fermentans. The proteins encoded by these genes were found to belong to a family of basic membrane proteins (BMP) that are found in a wide range of bacteria, including a number of pathogens. Several of the BMP family members, including p47, contain selective lipoprotein-associated motifs that are found in macrophage-activating lipoprotein 404 of M. fermentans and lipoprotein P48 of M. agalactiae. The p47 gene was predicted to encode a 59-kDa peptide, but affinity-purified p47 had a molecular mass of approximately 47 kDa, as determined by polyacrylamide gel analysis. Analysis of native and recombinant p47 by mass peptide fingerprinting revealed the absence of the carboxyl end of the protein encoded by the p47 gene in native p47, which would account for the difference seen in the predicted and measured molecular weights and indicated posttranslational cleavage of the lipoprotein at its carboxyl end. A DNA construct containing the p47 gene interrupted by the gene encoding tetracycline resistance was used to transform M. gallisepticum cells. A tetracycline-resistant mycoplasma clone, P2, contained the construct inserted within the genomic p47 gene, with crossovers occurring between 73 bp upstream and 304 bp downstream of the inserted tetracycline resistance gene. The absence of p47 protein in clone P2 was determined by the lack of reactivity with rabbit anti-p47 sera or monoclonal antibody B3 in Western blots of whole-cell proteins. There was no difference between the p47(-) mutant and wild-type M. gallisepticum in pathogenicity in chicken tracheal organ cultures. Thus, p47, although homologous to genes that occur in many prokaryotes, is not essential for growth in vitro or for attachment and the initial stages of pathogenesis in chickens.     

Cytoskeletal protein P41 is required to anchor the terminal organelle of the wall-less prokaryote Mycoplasma pneumoniae

The cell wall-less prokaryote Mycoplasma pneumoniae approaches the minimal requirements for a cell yet produces a complex terminal organelle that mediates cytadherence and gliding motility. Here we explored the molecular nature of the M. pneumoniae gliding machinery, utilizing fluorescent protein fusions and digital microcinematography to characterize gliding-altered mutants having transposon insertions in MPN311, encoding the cytoskeletal protein P41. Disruption of MPN311 resulted in loss of P41 and P24, the downstream gene product. Gliding ceases in wild-type M. pneumoniae during terminal organelle development, which occurs at the cell poles adjacent to an existing structure. In contrast, terminal organelle development in MPN311 mutants did not necessarily coincide with gliding cessation, and new terminal organelles frequently formed at lateral sites. Furthermore, new terminal organelles exhibited gliding capacity quickly, unlike wild-type M. pneumoniae. P41 and P24 localize at the base of the terminal organelle; in their absence this structure detached from the cell body of motile and dividing cells but retained gliding capacity and thus constitutes the gliding apparatus. Recombinant wild-type P41 restored cell integrity, establishing a role for this protein in anchoring the terminal organelle to the cell body.