Roles of the rpoEc-chrR-chrA operon in superoxide tolerance and β-lactam susceptibility of Stenotrophomonas maltophilia

Ren-Hsuan Ku ¹ Hsu-Feng Lu ² Li-Hua Li ³ Ting-Yu Yeh ¹ Yi-Tsung Lin ³ Tsuey-Ching Yang ^1*

• ¹ National Yang Ming Chiao Tung University, Taipei City, Taiwan
• ² Asia University, Taichung, Taiwan
• ³ Taipei Veterans General Hospital, Taipei, Taipei County, Taiwan

The rpoE-chrR pair is a regulatory system used by photosynthetic microorganisms to overcome singlet oxygen stress. rpoE and chrR encode the sigma factor σ E and anti-sigma factor ChrR, respectively. Stenotrophomonas maltophilia, an opportunistic pathogen, is a multidrug-resistant gram-negative bacterium. Although it is not a photosynthetic microorganism, a rpoE-chrR homolog (smlt2377-smlt2378) was found in the S. maltophilia genome. In this study, we aimed to assess the significance of σ Εc -ChrR pair in oxidative stress alleviation and antibiotic susceptibility of S. maltophilia KJ. Reverse transcription-polymerase chain reaction revealed that the rpoEc-chrR pair and downstream chrA formed an operon. Inactivation of chrR upregulated the expression of rpoEc-chrR-chrA operon in an σ Ec -and ChrA-dependent manner. The contribution of rpoEc-chrR-chrA operon to oxidative stress alleviation and antibiotic susceptibility was further evaluated using mutant constructs and stresstolerance assays. σ Ec activation contributed to superoxide tolerance and increased β-lactam susceptibility but did not affect the tolerance to singlet oxygen and hydrogen peroxide. To elucidate the underlying mechanism responsible for ∆chrR-mediated increase in superoxide tolerance and βlactam susceptibility, RNA-seq transcriptome assay of wild-type KJ, KJ∆ChrR (chrR mutant), and KJ∆ChrR∆RpoEc (chrR/rpoEc double mutant) was performed to reveal the σ Ec regulon. Expression of the nine-gene cluster, smlt2375-smlt2367, was significantly upregulated in KJ∆ChrR and reverted to the wild-type level in KJ∆ChrR∆RpoEc. smlt2375-smlt2367 cluster was located upstream of the rpoEc-chrR-chrA operon and divergently transcribed, seeming to be involved in membrane lipid modification. Deletion of smlt2375-smlt2367 cluster from the chromosome of KJ∆ChrR reverted the superoxide tolerance and β-lactam susceptibility to the wild-type level. Collectively, the rpoEc-chrR pair of S. maltophilia was involved in superoxide tolerance and β-lactam susceptibility. Notably, a novel regulatory circuit involving rpoEc-chrR-chrA operon and smlt2375-smlt2367 cluster was revealed.