Transcriptome analysis reveals novel regulatory mechanisms in a genome-reduced bacterium

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.

Global Changes in Mycoplasma gallisepticum Phase-Variable Lipoprotein Gene vlhA Expression during In Vivo Infection of the Natural Chicken Host

“…However, M. gallisepticum exhibits differential levels of gene expression in response to coincubation with MRC-5 human lung fibroblasts (7) and in response to various stress conditions (8,9). These data suggest that changes in transcription are indeed occurring as mycoplasmas respond to external stimuli and that the regulation is, in fact, more dynamic than originally hypothesized, with genes potentially affected by multiple regulators (10).…”

Data on translatome analysis of Mycoplasma gallisepticum

“…Mycoplasma gallisepticum S6 cells were cultivated and subjected to stress as described previously [2].…”

“…RNA-seq libraries were prepared as described previously [2]. Briefly, RNA was sheared by zinc sulfate buffer, ligated to adapters and amplified.…”

“…Briefly, RNA was sheared by zinc sulfate buffer, ligated to adapters and amplified. After that libraries were depleted of ribosomal RNA cDNA using duplex-specific nuclease as described earlier [2]. Samples were prepared in three biological replicates with one technical replicate per one biological replicate.…”

“…Reads were mapped and the coverage data was analyzed as described previously [2]. CP006916.3 genome assembly was used.…”