Synthesis of a small, cysteine-rich, 29 amino acids long peptide in Mycoplasma pneumoniae.

A 205-210 bases long, small RNA (MP200RNA) of Mycoplasma pneumoniae encodes an open reading frame (ORF pmp200) that has the potential to be translated into a 29 amino acids long peptide with nine cysteines. The expression of this peptide in M. pneumoniae was proven indirectly by constructing a gene fusion between the ORF pmp200 and mrfp1, the gene encoding the monomeric red fluorescent protein. The fusion construct was translated in M. pneumoniae. The corresponding fusion protein, with a molecular mass of approximately 35,000 Da, was isolated and the correct sequence was proven by Edman degradation and by mass spectrometry.

Transcription profiles of the bacterium Mycoplasma pneumoniae grown at different temperatures.

Applying microarray technology, we have investigated the transcriptome of the small bacterium Mycoplasma pneumoniae grown at three different temperature conditions: 32, 37 and 32 degrees C followed by a heat shock for 15 min at 43 degrees C, before isolating the RNA. From 688 proposed open-reading frames, 676 were investigated and 564 were found to be expressed (P < 0.001; 606 with P < 0.01) and at least 33 (P < 0.001; 77 at P < 0.01) regulated. By quantitative real-time PCR of selected mRNA species, the expression data could be linked to absolute molecule numbers. We found M.pneumoniae to be regulated at the transcriptional level. Forty-seven genes were found to be significantly up-regulated after heat shock (P < 0.01). Among those were the conserved heat shock genes like dnaK, lonA and clpB, but also several genes coding for ribosomal proteins and 10 genes of unassigned functions. In addition, 30 genes were found to be down-regulated under the applied heat shock conditions. Further more, we have compared different methods of cDNA synthesis (random hexamer versus gene-specific primers, different RNA concentrations) and various normalization strategies of the raw microarray data.