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.

Cross-complementation between the products of the genes P1 and ORF6 of Mycoplasma pneumoniae subtypes 1 and 2

“…The expression unit was defined as the DNA segment beginning at the first nucleotide in front of the start codon of MPN531 and ending 313 nucleotides further upstream (genome position 653950-654263). The transcriptional start point of this gene (Weiner et al, 2000) and its relative transcription rate (Weiner et al, 2003) were determined experimentally. We selected this expression unit, because clpB is well transcribed and translated (Ueberle et al, 2002) and can be up-and downregulated by changes in temperature (Weiner et al, 2003).…”

“…The transcriptional start point of this gene (Weiner et al, 2000) and its relative transcription rate (Weiner et al, 2003) were determined experimentally. We selected this expression unit, because clpB is well transcribed and translated (Ueberle et al, 2002) and can be up-and downregulated by changes in temperature (Weiner et al, 2003). This promoter fusion was necessary, because the ORF6 gene, as part of an operon, does not have its own promoter (Fig.…”

Detection and quantification of intergenic transcription in Mycoplasma hyopneumoniae

“…DNA replication, transcription and translation, are thought to be constitutively expressed, and in constant environments, the organism supposedly has little need for sophisticated genetic control mechanisms (Muto & Ushida, 2002). Our knowledge of gene expression in mycoplasmas is rudimentary, with few studies that actually address the mechanisms involved (Benders et al, 2005;Chang et al, 2008;Güell et al, 2009;Lluch-Senar et al, 2007;Weiner et al, 2000Weiner et al, , 2003. Transcription is essential to the basic process of gene expression, but surprisingly, little information has been obtained experimentally defining the transcription process in mycoplasmas (Muto & Ushida, 2002).…”

“…Transcription is essential to the basic process of gene expression, but surprisingly, little information has been obtained experimentally defining the transcription process in mycoplasmas (Muto & Ushida, 2002). The heat-shock response has been studied in a limited number of mycoplasma species (Madsen et al, 2006b;Musatovova et al, 2006;Weiner et al, 2003), and it is clear that a transcriptional activator, HrcA, is involved in regulating transcription of two heat-shock genes (Chang et al, 2008). In addition, transcriptional start sites have been mapped for a limited number of genes (Hyman et al, 1988;Taschke & Herrmann, 1988;Taschke et al, 1986;Weiner et al, 2000), and promoter-probe vectors have been used in several different species (Janis et al, 2005;Knudtson & Minion, 1993, 1994Lluch-Senar et al, 2007).…”

“…The limited genome capacity suggests simple regulatory mechanisms, and experimental evidence indicates that mycoplasmas have few regulatory systems for several reasons: (1) only a single sigma factor has been identified in any of the complete mycoplasma genome sequences, (2) transcriptional activators have rarely been identified (Chang et al, 2008), and (3) the genes for a few repressor-like proteins have been identified in genome sequence studies (Fraser et al, 1995;Glass et al, 2000;Minion et al, 2004). The global assessment of transcription has been reported using arrays (Madsen et al, 2006a(Madsen et al, , b, 2008Oneal et al, 2008;Schafer et al, 2007;Weiner et al, 2003), but these tools are limited because they only assess transcriptional changes in annotated gene sequences and do not address mechanisms of transcription control.…”