A new promoterless reporter vector reveals antisense transcription in Mycoplasma genitalium

Abstract

The mechanisms that promote and regulate transcription in mycoplasmas are poorly understood. Here, a promoter-probe vector based on the pMTnTetM438 minitransposon and containing a promoterless lacZ reporter gene was constructed to analyse Mycoplasma genitalium transcription in vivo. Recovered transposon insertions were in monocopy, with 16 % expressing enough b-galactosidase (b-Gal) to yield colonies exhibiting a detectable blue colour. A sample of 52 blue colonies was propagated and selected for further analyses. The b-Gal activity of the corresponding cultures was measured to quantify, in a reproducible way, the transcription levels of the interrupted ORFs. Several insertions were found in sense with the interrupted ORF, but surprisingly there was also a number of insertions in non-coding regions, many of them in repetitive DNA regions known as MgPa islands. Moreover, 30 % of the analysed transposon insertions had the lacZ gene in the opposite orientation to the coding frame, suggesting the existence of antisense transcripts that may be involved in the control of gene expression in M. genitalium.

Characterization of the Operon Encoding the Holliday Junction Helicase RuvAB from Mycoplasma genitalium and Its Role in mgpB and mgpC Gene Variation

“…To further characterize the transcriptional organization of the ruvAB operon, we mapped the transcriptional start sites (TSSs) using a fluorescence-based primer extension protocol (34,35). As shown in Fig.…”

“…Primer extension analyses were performed using a previously described nonradioactive protocol (34,35). Briefly, 10 g of total RNA was combined with 2 pmol of a 5=-6-carboxyfluorescein (FAM)-labeled primer (Table 1) in reverse transcription reactions using the SuperScript III first-strand synthesis kit (Invitrogen) according to the manufacturer's instructions.…”

Detection and quantification of intergenic transcription in Mycoplasma hyopneumoniae

“…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). These studies show that the 210 box of the Pribnow sequence is relatively conserved, but not the 235 sequence.…”

“…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).…”

Natural Antisense Transcripts in Bacteria

“…Antisense transcription occurs throughout the bacterial kingdom. Bacteria for which at least a single antisense RNA was reported are plotted along a phylogenetic tree based on 16S rRNA sequences (Albrecht et al 2010;Andre et al 2008;Beaume et al 2010;Berghoff et al 2009;Csiszar et al 1987;D'Alia et al 2010;Dühring et al 2006;Eiamphungporn and Helmann 2009;Filiatrault et al 2010;Fozo et al 2008;Georg et al 2009;Giangrossi et al 2010;Guell et al 2009;Hernandez et al 2005;Kawano et al 2005;Landt et al 2008;Lee et al 2001;Lee and Groisman 2010;Liu et al 2009;Lluch-Senar et al 2007;Mitschke et al 2011;Nelson et al 2008;Opdyke et al 2004;Qiu et al 2010;Rasmussen et al 2009;Schluter et al 2010;Selinger et al 2000;Sharma et al 2010;Silby and Levy 2008;Silvaggi et al 2005Silvaggi et al , 2006Sittka et al 2008;Steglich et al 2008;Stork et al 2007;Swiercz et al 2008;Toledo-Arana et al 2009;Vogel et al 2003;Xiao et al 2009;Yachie et al 2006;Zemanova et al 2008).…”