Transcription profiles of the bacterium Mycoplasma pneumoniae grown at different temperatures

Abstract

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.

Figure 1

DNA array of the entire set of 677 postulated ORFs of M.pneumoniae hybridized with radioactively labeled cDNA probes generated from RNA which was extracted from cells grown at 37°C for 96 h. Each ORF was spotted in doublets.

Figure 2

A series of 25 ORFs (MPN507–MPN531) and their hybridization signals. The signals were derived from three temperature experiments where M.pneumoniae was incubated for 96 h at 37°C, 144 h at 32°C or exposed to a heat shock of 43°C for 15 min after incubation for 144 h at 32°C, respectively. Each signal represents a percentage of the sum of all 677 ORF signals. A whole number of 15 experiments were included in the evaluation (four for incubation at 37°C, six for incubation at 32°C and five for heat shock). For each profile, the sum of all signals was calculated and each ORF signal was divided by the sum. An average of all corresponding ORF data was calculated and is presented in the histogram. Error bars represent the standard error within the selected experiments.

Figure 3

Correlation between expression at 32°C and expression in heat shock. Gray circles, genes that are not significantly expressed in either of the tested experimental conditions; black circles, genes that are significantly expressed, but do not show significant differences between the two profiles as tested with the U-test. Genes up-regulated during heat shock: light-red circles (P < 0.01), dark-red circles (P < 0.001); genes significantly down-regulated during heat shock: light-blue circles (P < 0.01), dark-blue circles (P < 0.001).

Figure 4

Expression map of M.pneumoniae. Color scale represents the normalized gene expression. Black boxes represent non-coding RNA genes (tRNAs, ribosomal RNAs, etc.). Genes that were not subject to the microarray analysis are white. White squares above the genome position scale represent the repetitive regions.

Figure 5

RT–PCR results. Total RNA from M.pneumoniae grown at 32°C and from heat shock-treated cells was reverse transcribed with a mix of 688 gene-specific primers. The cDNA was used in a 24 cycle PCR. The synthesized DNA was loaded onto an agarose gel and stained with ethidium bromide. PCR results are shown in pairs (left, 32°C; right, heat shock) for each selected gene. The results were divided into three categories based on human judgment of the ethidium bromide staining intensity: (a) genes which showed a heat shock-induced up-regulation in the RT–PCR, (b) genes which showed no regulation in the RT–PCR and (c) genes which showed a heat shock-induced down-regulation in the RT–PCR. The results were compared with the statistically evaluated microarray signals. The microarray data are represented as: ns, genes which showed no statistically significant regulation in the array analysis; –, genes which showed statistically significant down-regulation in the array analysis.

Figure 6

Results of Q-PCR analysis of nine different M.pneumoniae genes. (a) Correlation between the absolute copy number per 1 µg of a total RNA isolation, and the microarray signal (percentage). Different symbols correspond to the three experimental conditions used. (b) Correlation of the differences in copy number and differences in micorarray signal strength between the three different conditions. Correlation coefficients are given in the legend. Different symbols correspond to the three comparisons possible among three different experimental conditions, as explained in the box.