Abstract
Mycoplasmas are bacteria without a cell wall that evolved by drastically reducing the size of the genome. Whole-genome analysis of Mycoplasma pneumoniae revealed the presence of numerous copies of four distinct large repetitive elements (RepMPs) from M. pneumonia. One copy of RepMP2/3, RepMP4, and RepMP5 are located within the P1 operon (MPN140 to MPN142 loci), and their involvement in sequence variation in P1 adhesin and adhesion-related protein B/C has been documented.
Here we analyze a clinical strain of Mycoplasma pneumonia Recombinants designated S1 isolated from a 1993 outbreak of respiratory infections in San Antonio, TX. Based on the type of RepMP within the P1 operon, we classified clinical isolate S1 as type 2 with unique minor sequence variations. Hybridization with oligonucleotide arrays revealed sequence divergence in two previously unsuspected hypothetical genes (loci MPN137 and MPN138). Closer inspection of this region revealed that loci MPN137 and MPN138 harboured previously unrecognized unique RepMP1 sequences found only in M. pneumoniae.
Sequence and PCR analyses revealed a recombination event involving three RepMP1-containing genes that resulted in a fusion of the MPN137 and MPN138 reading frames and loss of all but a short fragment of another RepMP1-containing locus, MPN130. The multiple copies of unique RepMP1 elements spread throughout the chromosome could allow for a large number of sequence variations in clinical strains. Amino acid sequence comparisons showed the presence of leucine zipper motifs in the MPN130 and MPN138 proteins in the reference strain M129 and the absence of these motifs in the S1 fused protein. The presence of tandem leucine and other repeats points to possible regulatory functions of proteins encoded by genes containing RepMP1.
M. pneumonia strains and DNA isolation.
M. pneumonia reference strain M129 and clinical strain S1, which was isolated from an acute outbreak of M. pneumonia respiratory infections in San Antonio in 1993, were grown in SP-4 medium at 37°C for 72 to 96 hours. h in tissue culture flasks as previously described. Chromosomal DNA was isolated using an Easy DNA kit as recommended by the manufacturer (Invitrogen).
Microarray and hybridization.
Qiagen Operon, Inc. designed and synthesized 70 mers representing 689 open reading frames from strain M129, and Microarrays Inc. printed slides. For hybridization, 4 μg portions of purified chromosomal DNA from M129 and S1 were labelled using random hexamers and the aminoallyl method recommended by the Institute for Genomics Research (TIGR). The generated probes, Cy3-labeled M129 DNA and Cy5-labeled S1 DNA were hybridized to the same slide. Following post-hybridization washes, images were collected with a GenePix 4000B microarray scanner and analyzed using Equity Rev4.
PCR amplification of chromosomal regions of M. pneumoniae.
Specific primers for selected loci were designed based on the available genomic sequence of the reference strain M129. All PCR amplifications were performed using Platinum Taq High-Fidelity DNA Polymerase (Invitrogen) at appropriate annealing temperatures and extension times. The amplified products were separated on 1% agarose and their sizes and specificities were evaluated.
Restriction fragment length polymorphism analysis by PCR of the S1-P1 gene sequence of the strain. M. pneumonia strains M129 and S1 were typed by performing PCR restriction fragment length polymorphism analysis based on sequence divergence of the gene encoding the P1 adhesin. The primers ADH1 (5′-CTGCCTTGTCCAAGTCCACT-3′), ADH2 (5′-AACCTTGTCGGGAAGAGCTG-3′), ADH3 (5′-CGAGTTTGCTGCTAACGAGT-3′), and ADH4 (5′-CTTGACTG ATACCTGTGCGG-3′) were used to amplify the ADH1-2 (~2280 bp) and ADH3-4 (~2580 bp) regions, respectively.
Amplification reactions were performed using 50 μl mixtures containing 100 ng of chromosomal DNA and Platinum Taq high fidelity DNA polymerase (Invitrogen). Regions of the gene encoding P1 were amplified using 30 cycles consisting of 94 °C for 30 s, 55 °C for 30 s and 68 °C for 3 min and visualized in 1% agarose. The amplified products were then subjected to complete restriction digestion with HaeIII, HhaI, HpaII, and RsaI, and the generated restriction patterns were resolved in 2% agarose for comparison and analysis.
Sequencing.
The PCR products that were selected for sequencing were cloned into the PCR vector (Invitrogen) and plasmid DNA isolated using a QIAprep Spin kit (Qiagen Sciences). Sequencing was performed at the Nucleic Acid Center of the Department of Microbiology and Immunology (University of Texas Health Sciences Center at San Antonio). Sequences were analyzed using the BLAST program available in the NCBI database.