Abstract
The avian bacterial pathogen Mycoplasma gallisepticum is a good model for systems studies due to small genome and simplicity of regulatory pathways. In this study, we used RNA-Seq and MS-based proteomics to accurately map coding sequences, transcription start sites (TSSs) and transcript 3′-ends (T3Es). We used obtained data to investigate roles of TSSs and T3Es in stress-induced transcriptional responses. We identified 1061 TSSs at a false discovery rate of 10% and showed that almost all transcription in M. gallisepticum is initiated from classic TATAAT promoters surrounded by A/T-rich sequences. Our analysis revealed the pronounced operon structure complexity: on average, each coding operon has one internal TSS and T3Es in addition to the primary ones. Our transcriptomic approach based on the intervals between the two nearest transcript ends allowed us to identify two classes of T3Es: strong, unregulated, hairpin-containing T3Es and weak, heat shock-regulated, hairpinless T3Es. Comparing gene expression levels under different conditions revealed widespread and divergent transcription regulation in M. gallisepticum. Modeling suggested that the core promoter structure plays an important role in gene expression regulation. We have shown that the heat stress activation of cryptic promoters combined with the hairpinless T3Es suppression leads to widespread, seemingly non-functional transcription.
Highlights
Mycoplasma gallisepticum belongs to the Mollicutes class––a specialized branch of microorganisms related to Gram-positive bacteria [1]
The only transcription factors (TFs) whose mechanism is known in M. gallisepticum is a heat-shock repressor (HrcA) that binds a palindromic sequence known as controlling inverted repeat of chaperone expression (CIRCE) in the promoters of several chaperone genes [5], whereas other common bacterial TFs, such as the LexA repressor of the SOS response [6], are lacking
We strand- sequenced two sets of samples (16 samples in each data set) of the total RNA extracted from M. gallisepticum that was cultured under different conditions in at least two biological replicates using a SOLiD 4 sequencer (Life Technologies)
Summary
Mycoplasma gallisepticum belongs to the Mollicutes class––a specialized branch of microorganisms related to Gram-positive bacteria [1]. Mollicutes feature reduced genomes with an average size of 1 Mb, and they lack a cell wall [3]. Their cell physiology is considerably simplified compared to that of most bacteria, making Mollicutes a good model for systemic studies and, in particular, for studying the complex response to stress. M. gallisepticum, along with most Mollicutes, shows a reduced repertoire of transcription factors (TFs) compared to that of related bacteria, such as Bacillus subtilis [4]. Experimental identification of transcription start sites (TSSs) and transcription terminators (TTs) could help to resolve this ‘regulation without regulators’ puzzle
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