DOI: 10.1128/jb.00564-25 ISSN: 0021-9193
High levels of antisense transcription from numerous genes in the obligate intracellular bacterium
Orientia tsutsugamushi
Chitrasak Kullapanich, Regan Hayward, Lars Barquist, Jeanne Salje ABSTRACT
Orientia tsutsugamushi
(Ot) is a cytoplasmic, gram-negative, obligate intracellular bacterium that causes the human disease scrub typhus. It has a highly repetitive genome, whereby approximately 50% is composed of multiple copies of a proliferated integrative and conjugative element, the Rickettsial amplified Genetic Element (RAGE). Previous RNA sequencing analysis revealed a high level of antisense transcription in Ot, particularly from RAGE-encoded genes, with 18% of all genes having a sense:antisense ratio <1 and 5% with a ratio <0.1. In our current study, we have confirmed the earlier RNA sequencing findings via polymerase chain reaction (PCR)-based methods and established that this antisense transcription is a consistent feature across eight distinct Ot strains. Furthermore, we have utilized PCR to monitor differences in sense and antisense expression between intracellular and extracellular bacteria. Our findings lend weight to the hypothesis that antisense transcription in Ot is a regulated mechanism, possibly playing a crucial role in the pathogen’s lifecycle during infection.
IMPORTANCE
Orientia tsutsugamushi
(Ot) is an obligate intracellular bacterium with a highly repetitive genome and an unusual abundance of antisense RNA transcripts. Antisense transcription has been proposed to regulate gene expression, yet its biological significance in intracellular bacteria remains unclear. By confirming high and conserved levels of antisense transcription in specific genes across multiple
O. tsutsugamushi
strains and showing that antisense expression decreases in extracellular forms, this study provides new evidence that antisense transcription is a regulated process rather than transcriptional noise. These findings suggest that antisense RNAs may play a role in controlling gene activity during the bacterial life cycle, offering insights into gene regulation in organisms with reduced and repetitive genomes.