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Background: Stenotrophomonas maltophilia, an opportunistic pathogen, is ubiquitously distributed in the environment. In response to iron-depletion stress, S. maltophilia synthesizes the sole catecholate-type siderophore, stenobactin, for ferric iron acquisition. FepAsm, a TonB-dependent transporter (TBDT), is the sole known outer membrane receptor responsible for ferri-stenobactin uptake in S. maltophilia K279a. However, S. maltophilia KJ and its isogenic fepA mutant displayed comparable ability to utilize FeCl3 as the sole iron source for growth in iron-depleted conditions, suggesting the involvement of additional TBDT in ferri-stenobactin uptake in the KJ strain. Here, we aimed to determine additional TBDT required for ferri-stenobactin uptake and the post-TBDT ferri-stenobactin transport system in the KJ strain. Methods and results: Twelve TBDTs, whose expression were significantly upregulated in 2,2′-dipyridyl-treated KJ strain, were selected as candidates for ferri-stenobactin uptake. The involvement of these selected candidates in ferri-stenobactin acquisition was investigated using deletion mutant construction and FeCl3 utilization assay. Among the 12 TBDTs tested, FepAsm, FciA, and SbaA were the TBDTs for ferri-stenobactin uptake in KJ strain. Because fciA is a member of fciTABC operon, the involvement of fciTABC operon in ferri-stenobactin uptake was also investigated. Of the fciTABC operon, fciA, fciB and fciC, but not fciT, contributed to ferri-stenobatin acquisition. SbaE is the homolog of FepD/FepG, the inner membrane transporters for ferri-enterobactin in E. coli; therefore, sbaBCDEF operon was selected as a candidate for the post-TBDT transport system of ferri-stenobactin. All proteins encoded by sbaBCDEF operon participated in ferri-stenobactin acquisition. Due to the contribution of the putative periplasmic esterase SbaB to ferri-stenobactin acquisition, FeoB, a ferrous iron inner membrane transporter, was included as a candidate and proved to be involved in ferri-stenobactin acquisition. Accordingly, contributions of feoB and sbaE to ferri-stenobactin acquisition illustrated that ferric and ferrous iron could be transported across the inner membrane via SbaE and FeoB, respectively. Conclusions: FepAsm, fciABC, sbaA, sbaBCDEF, and feoB contribute to ferri-stenobatin acquisition in Stenotrophomonas maltophilia KJ. [ABSTRACT FROM AUTHOR]

Introduction: The rpoE-chrR pair is a regulatory system used by photosynthetic microorganisms to overcome singlet oxygen stress. rpoE and chrR encode the sigma factor σE 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 σEc-ChrR pair in oxidative stress alleviation and antibiotic susceptibility of S. maltophilia KJ. Methods: Reverse transcription-polymerase chain reaction was used to validate the presence of operon. The contribution of rpoEc-chrR-chrA operon to oxidative stress alleviation and antibiotic susceptibility was evaluated using mutant constructs and stress-tolerance assays. RNA-seq transcriptome assay of wild-type KJ, KJΔChrR (chrR mutant), and KJΔChrRΔRpoEc (chrR/rpoEc double mutant) was performed to reveal the σEc regulon. Results: The rpoEc-chrR pair and downstream chrA formed an operon. Inactivation of chrR upregulated the expression of rpoEc-chrR-chrA operon in an σEc- and ChrA-dependent manner. σEc activation contributed to superoxide tolerance and increased β-lactam susceptibility but did not affect the tolerance to singlet oxygen and hydrogen peroxide. Transcriptome analysis revealed that expression of the nine-gene cluster, smlt2375-smlt2367 , was significantly upregulated in KJΔChrR and reverted to the wild-type level in KJΔChrRΔRpoEc. 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 KJΔChrR reverted the superoxide tolerance and β-lactam susceptibility to the wild-type level. Discussion: 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. [ABSTRACT FROM AUTHOR]

Background SmeYZ is a constitutively expressed efflux pump in Stenotrophomonas maltophilia. Previous studies demonstrated that: (i) smeYZ inactivation causes compromised swimming, oxidative stress tolerance and aminoglycoside resistance; and (ii) the ΔsmeYZ -mediated pleiotropic defects, except aminoglycoside susceptibility, result from up-regulation of entSCEBB′FA and sbiAB operons, and decreased intracellular iron level. Objectives To elucidate the modulatory role of SmeQ, a novel cytoplasmic protein, in ΔsmeYZ -mediated pleiotropic defects. Methods The presence of operons was verified using RT–PCR. The role of SmeQ in ΔsmeYZ -mediated pleiotropic defects was assessed using in-frame deletion mutants and functional assays. A bacterial adenylate cyclase two-hybrid assay was used to investigate the protein–protein interactions. Gene expression was quantified using quantitative RT–PCR (RT–qPCR). Results SmeYZ and the downstream smeQ formed an operon. SmeQ inactivation in the WT KJ decreased aminoglycoside resistance but did not affect swimming and tolerance to oxidative stress or iron depletion. However, smeQ inactivation in the smeYZ mutant rescued the ΔsmeYZ -mediated pleiotropic defects, except for aminoglycoside susceptibility. In the WT KJ, SmeQ positively modulated SmeYZ pump function by transcriptionally up-regulating the smeYZQ operon. Nevertheless, in the smeYZ mutant, SmeQ exerted its modulatory role by up-regulating entSCEBB′FA and sbiAB operons, decreasing intracellular iron levels, and causing ΔsmeYZ -mediated pleiotropic defects, except for aminoglycoside susceptibility. Conclusions SmeQ is the first small protein identified to be involved in efflux pump function in S. maltophilia. It exerts modulatory effect by transcriptionally altering the expression of target genes, which are the smeYZQ operon in the WT KJ, and smeYZQ , entSCEBB′FA and sbiAB operons in smeYZ mutants. [ABSTRACT FROM AUTHOR]