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The Mycoplasma pneumoniae MPN490 and Mycoplasma genitalium MG339 genes encode reca homologs that promote homologous DNA strand exchange.

ABSTRACT: The P1, P40, and P90 proteins of Mycoplasma pneumoniae and the MgPa and P110 proteins of Mycoplasma genitalium are immunogenic adhesion proteins that display sequence variation. Consequently, these proteins are thought to play eminent roles in immune evasive strategies. For each of the five proteins, a similar underlying molecular mechanism for sequence variation was hypothesized, i.e., modification of the DNA sequences of their respective genes. This modification is thought to result from homologous recombination of parts of these genes with repeat elements (RepMp and MgPar elements in M. pneumoniae and M. genitalium, respectively) that are dispersed throughout the bacterial genome. Proteins that are potentially involved in homologous DNA recombination have been suggested to be implicated in recombination between these repeat elements and thereby in antigenic variation. To investigate this notion, we set out to study the function of the RecA homologs that are encoded by the M. pneumoniae MPN490 and M. genitalium MG339 genes. Both proteins, which are 79% identical on the amino acid level, were found to promote recombination between homologous DNA substrates in an ATP-dependent fashion. The recombinational activities of both proteins were Mg2+ and pH dependent and were strongly supported by the presence of single-stranded DNA binding protein, either from M. pneumoniae or from Escherichia coli. We conclude that the MPN490- and MG339-encoded proteins are RecA homologs that have the capacity to recombine homologous DNA substrates. Thus, they may play a central role in recombination between repetitive elements in both M. pneumoniae and M. genitalium.

SUBMITTER: Sluijter M

PROVIDER: S-EPMC2772507 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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The Mycoplasma pneumoniae MPN490 and Mycoplasma genitalium MG339 genes encode reca homologs that promote homologous DNA strand exchange.

Sluijter Marcel M Spuesens Emiel B M EB Hartwig Nico G NG van Rossum Annemarie M C AM Vink Cornelis C

Infection and immunity 20090908 11

The P1, P40, and P90 proteins of Mycoplasma pneumoniae and the MgPa and P110 proteins of Mycoplasma genitalium are immunogenic adhesion proteins that display sequence variation. Consequently, these proteins are thought to play eminent roles in immune evasive strategies. For each of the five proteins, a similar underlying molecular mechanism for sequence variation was hypothesized, i.e., modification of the DNA sequences of their respective genes. This modification is thought to result from homol ...[more]

PMID: 19737894

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Project description:The discovery of small open reading frames (smORFs) encoding for polypeptides (SEPs; <100aa) highlights that the coding capacity of the genomes has been underestimated. Most ORF-finding algorithms have historically set a minimum threshold length of 100 aa. As consequence, some transcripts encoding for SEPs, were erroneously discarded or classified as non-coding RNAs (ncRNAs). With this experiments we try to experimentally assess the existence and complexity of these small proteins. The experimental design includes: After growing each Mycoplasma for 6h at 37°C, cells were washed twice with PBS and lysed with 700 µl of Qiazol buffer. Then, samples were lysed with 700 µl of Qiazol buffer. RNA extractions were performed by using the miRNeasy mini Kit (Qiagen) following the instructions of the manufacturer. Libraries for RNA-seq were prepared following directional RNA-seq library preparation and sequencing. Briefly, 1 µg of total RNA was fragmented to ~100-150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). Treatments with Antarctic phosphatase (ref. M0289S, NEB) and PNK (ref. M0201S, NEB) were performed in order to make the 5’ and 3’ ends of the RNA available for adapter ligation. Samples were further processed using the TruSeq small RNA Sample Prep Kit (ref. RS-200-0012, Illumina) according to the manufacturer's protocol. In summary, 3’ adapters and subsequently 5’ adapters were ligated to the RNA. cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064-014, Invitrogen) and a specific primer (RNA RT Primer) complementary to the 3’ RNA adapter. cDNA was further amplified by PCR using indexed adapters supplied in the kit. Finally, size selection of the libraries was performed using 6% Novex® TBE Gels (ref. EC6265BOX, Life Technologies). Fragments with insert sizes of 100 to 130 bp were cut from the gel, and cDNA was precipitated and eluted in 10 µl of elution buffer. Double-stranded templates were cluster amplified and sequenced on an Illumina HiSeq 2000.

Dataset Information

The Mycoplasma pneumoniae MPN490 and Mycoplasma genitalium MG339 genes encode reca homologs that promote homologous DNA strand exchange.

Publications

The Mycoplasma pneumoniae MPN490 and Mycoplasma genitalium MG339 genes encode reca homologs that promote homologous DNA strand exchange.

Similar Datasets

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