The sbiTRS Operon Contributes to Stenobactin-Mediated Iron Utilization in Stenotrophomonas maltophilia

Abstract

Iron is an essential micronutrient for various bacterial cellular processes. Fur is a global transcriptional regulator participating in iron homeostasis. Stenotrophomonas maltophilia is a ubiquitous environmental bacterium that has emerged as an opportunistic pathogen. To elucidate the novel regulatory mechanism behind iron homeostasis in S. maltophilia, wild-type KJ and KJΔFur, a fur mutant, were subjected to transcriptome assay. A five-gene cluster, sbiBA-sbiTRS, was significantly upregulated in KJΔFur. SbiAB is an ATP type efflux pump, SbiT is an inner membrane protein, and SbiSR is a two-component regulatory system (TCS). The sbiTRS operon organization was verified by reverse transcription-PCR (RT-PCR). Localization prediction and bacterial two-hybrid studies revealed that SbiT resided in the inner membrane and had an intramembrane interaction with SbiS. In iron-replete conditions, SbiT interacted with SbiS and maintained SbiSR TCS in a resting state. In response to iron depletion stress, SbiT no longer interacted with SbiS, leading to SbiSR TCS activation. The iron source utilization assay demonstrated the contribution of SbiSR TCS to stenobactin-mediated ferric iron utilization but notto the utilization of hemin and ferric citrate. Furthermore, SmeDEF and SbiAB pumps, known stenobactin secretion outlets, were members of the SbiSR regulon. Collectively, in an iron-depleted condition, SbiSR activation is regulated by Fur at the transcriptional level and by SbiT at the posttranslational level. Activated SbiSR contributes to stenobactin-mediated ferric iron utilization by upregulating the smeDEF and sbiAB operons. SbiSR is the first TCS found to be involved in iron homeostasis in S. maltophilia. IMPORTANCE Therapeutic options for Stenotrophomonas maltophilia infections are limited because S. maltophilia is intrinsically resistant to several antibiotics. Iron is an essential element for viability, but iron overload is a lethal threat to bacteria. Therefore, disruption of iron homeostasis can be an alternative strategy to cope with S. maltophilia infection. The intricate regulatory networks involved in iron hemostasis have been reported in various pathogens; however, little is known about S. maltophilia. Herein, a novel sbiTRS operon, a member of Fur regulon, was characterized. SbiT, an inner membrane protein, negatively modulated the SbiSR two-component regulatory system by intramembrane protein-protein interaction with SbiS. In response to iron-depleted stress, SbiSR was activated via the regulation of Fur and SbiT. Activated SbiSR upregulated smeDEF and sbiAB, which contributed to stenobactin-mediated ferric iron utilization. A novel fur-sbiT-sbiSR-smeDEF/sbiAB regulatory circuit in S. maltophilia was revealed.

Keywords: ABC-type efflux pump; Stenotrophomonas maltophilia; iron homeostasis; stenobactin; two-component regulatory system.

FIG 1

Gene ontology classification of KJ and KJΔFur transcriptomes. Gene enrichment analysis of the differentially expressed genes (DEGs) was carried out using topGO package with Fisher exact test and weighted 01 algorithm. The GO terms with P < 0.01 were selected as significantly enriched functional groups.

FIG 2

SbiT, sbiR, and sbiS form an operon. (A) The genetic organization of the sbiB-sbiA-sbiT-sbiR-sbiS genes cluster of S. maltophilia. Arrows represent open reading frames (ORFs) and the direction of transcription. Small black arrow indicates the position of primer SbiR-C used for reverse transcription. The solid lines represent the PCR amplicons amplified using the SbiTQ102-F/R and SbiRQ94-F/R primer sets. (B) Agarose gel electrophoresis of the PCR products. DNA-free RNA was purified from logarithmically-grown KJΔFur cells, and cDNAs were obtained by reverse transcription using the primer SbiR-C. The cDNA was used as template for PCR with the primers indicated. Lane 1, primers SbiTQ102-F and SbiTQ102-R; Lane 2, primers SbiRQ94-F/R. The SbiRQ94-F/R primer sets were used as a control for a check of DNA contamination during cDNA preparation.

FIG 3

The expression of sbiBA-sbiTRS cluster. Data are represented as the means of values from three independent experiments. Bars represent the means of values from three independent experiments. Error bars represent the standard deviation for triplicates. *, P < 0.01; significance calculated by Student's t test. (A) Regulation of sbiBA-sbiTRS cluster expression. The overnight-cultured bacterial cells as indicated were inoculated into fresh LB broth at an initial OD₄₅₀ of 0.15. The DIP was added to a final concentration of 30 μg/mL. After 15 h culture, DNA-free RNA was isolated. The levels of transcripts as indicated were determined by qRT-PCR. The primers used were listed in Table S3 in the supplemental material. Relative transcript levels were calculated using the 2^−ΔΔCT method with wild-type KJ transcript level set as 1. (B) The expression of sbiTRS and sbiAB operons in logarithmically grown KJ cells. The overnight-cultured bacterial cells as indicated were inoculated into fresh LB broth at an initial OD₄₅₀ of 0.15. After 5 h culture, the C23O activities were determined.

FIG 4

Bacterial adenylate cyclase‐based two‐hybrid analysis of interactions between SbiS and SbiT. (A) Schematic representation of the interaction between T18-SbiT and T25-SbiS₁₋₈₇. Full-length SbiS (1 to 147 aa) and truncated SbiS (1 to 87 aa) were fused in frame to T18 and T25 fragments, respectively. (B) β-galactosidase activity of E. coli DHM1 strain harboring both pUT18- and pKT25-derived plasmids. The plasmids as indicated were cotransformed into E. coli DHM1, and the transformants were grown in LB broth with ampicillin, kanamycin, and isopropyl-β-d-thiogalactopyranoside (IPTG) for 16 h. Beta-galactosidase activity was determined and expressed as Miller units. Each bar represents the mean of values obtained from three independent experiments. Error bars represent the standard error of the mean. *, P < 0.01; significance calculated by Student's t test. Black bars, void, pUT18 and pKT25; SbiT control, pUT18-SbiT and pKT25; SbiS₁₋₈₇ control, pUT18 and pKT25-SbiS₁₋₈₇; SbiT-SbiS₁₋₈₇, pUT18-SbiT and pKT25-SbiS₁₋₈₇. Gray bars, SbiT-SbiS₁₋₈₇, pUT18-SbiT and pKT25-SbiS₁₋₈₇; DIP concentrations as indicated.

FIG 5

Contribution of SbiSR TCS to the utilization of different iron sources in iron-depleted condition. The logarithmic-phase bacterial cells (2 × 10⁵ CFU/μL) were 10-fold serially diluted. Five microliters of bacterial suspension were spotted onto LB agar plates with different additives as indicated. After a 24-h incubation at 37°C, the viabilities were imaged. The concentrations of additives are as follows: FeCl₃, 35 μM; hemin, 150 μM; ferric citrate, 110 μM. The numbers in the parentheses indicate the DIP concentration (μg/mL). (A) Role of SbiSR in iron depletion tolerance. (B) Role of SbiSR in ferric iron utilization. (C) Role of stenobactin in SbiSR-mediated ferric iron utilization. (D) Role of SbiSR in hemin utilization. (E) Role of SbiSR in ferric citrate utilization.

FIG 6

The expression of stenobactin-mediated ferric iron acquisition-related genes in KJ, KJΔSbiT, KJΔSbiTRS, KJΔSbiS, KJΔSbiR, and KJΔSbiSR strains. Overnight culture of the S. maltophilia strains was inoculated into fresh LB at an initial OD₄₅₀ of 0.15. After a 15-h culture, the transcripts as indicated were quantified by qRT-PCR. The relative transcript levels were calculated with the transcript level of KJ cells set as 1. Black dots represent the results of three independent experiments. Bars represent the mean of the values from three independent experiments. *, P < 0.01; significance calculated by Student's t test.

FIG 7

Model of Fur-SbiT-SbiSR-SmeDEF/SbiAB regulatory circuit in S. maltophilia. (A) In iron-replete condition, Fur-ferrous iron complex partially represses sbiTRS operon expression. The expressed SbiT and SbiS show intramembrane protein-protein interaction, which blocks SbiSR TCS activation. (B) In iron-depleted condition, the Fur regulon is derepressed and SbiT-SbiS interaction disappears. Ceasing of repression by Fur regulon leads to stenobactin synthesis and upregulation of the sbiTRS operon. Meanwhile, the loss of the SbiT-SbiS interaction results in SbiSR TCS activation. Phosphorylated SbiR upregulates the expression of sbiAB and smeDEF operons. SmeDEF and SbiAB pumps are the known stenobactin secretion outlets.