A cyclic AMP receptor protein-regulated cell-cell communication system mediates expression of a FecA homologue in Stenotrophomonas maltophilia

Abstract

Stenotrophomonas maltophilia WR-C possesses an rpf/diffusible signal factor (DSF) cell-cell communication system. It produces cis-Delta2-11-methyl-dodecenoic acid, a DSF, and seven structural derivatives, which require rpfF and rpfB for synthesis. Acquisition of iron from the environment is important for bacterial growth as well as the expression of virulence genes. We identified a gene homologous to fecA, which encodes a ferric citrate receptor that transports exogenous siderophore ferric citrate from the environment into the bacterial periplasm. Western blot analysis with anti-FecA-His(6) antibody showed that the FecA homologue was induced in the iron-depleted medium supplemented with a low concentration of ferric citrate. Deletion of rpfF or rpfB resulted in reduced FecA expression compared to the wild type. Synthetic DSF restored FecA expression by the DeltarpfF mutant to the wild-type level. Reverse transcription-PCR showed that the fecA transcript was decreased in the DeltarpfF mutant compared to the wild type. These data suggest that DSF affected the level of fecA mRNA. Transposon inactivation of crp, which encodes cyclic AMP (cAMP) receptor protein (CRP) resulted in reduced FecA expression and rpfF transcript level. Putative CRP binding sites were located upstream of the rpfF promoter, indicating that the effect of CRP on FecA is through the rpf/DSF pathway and by directly controlling rpfF. We propose that CRP may serve as a checkpoint for iron uptake, protease activity, and hemolysis in response to environmental changes such as changes in concentrations of glucose, cAMP, iron, or DSF.

FIG. 1.

Genetic organization and expression of FecA. (A) fecA and flanking genes in Escherichia coli K-12 (E. coli) (7) and S. maltophilia WR-C (GenBank accession no. EF011626). Genes depicted are not proportional to their respective sequence lengths. (B) SDS-PAGE of cell lysates from E. coli BL21(DE3) harboring pET22b(+) (lane 1) or pfecA::ET22b(+) (lane 2). M, SDS-PAGE standards, high range (Bio-Rad). (C) Anti-His tag Western blot of cell lysate from E. coli BL21(DE3) harboring pET22b(+) (lane 1) or pfecA::ET22b(+) (lane 2). (D) Anti-FecA-His₆ Western blot of cell lysates from E. coli BL21(DE3) harboring pfecA::ET22b(+) and S. maltophilia WR-C grown under different concentrations (0, 0.5, 2.5, 25, 50, and 100 mM) of ferric citrate in fecA medium at 30°C and 175 rpm for 3 days.

FIG. 2.

Anti-FecA-His₆ Western blot of cell lysates from S. maltophilia WT and ΔrpfB, ΔrpfF, and crp::EZTN mutants and the respective complemented strains. Strains were cultured in fecA medium at 30°C and 175 rpm for 3 days.

FIG. 3.

Effect of synthetic cis-Δ2-11-methyl-dodecenoic acid (DSF) on FecA expression. Cell lysates from S. maltophilia WR-C WT, the ΔrpfF mutant, and the ΔrpfF mutant harboring prpfF (ΔrpfF/prpfF) were analyzed by Western blotting using anti-FecA-His₆ antibody. Strains were cultured at 30°C and 175 rpm for 3 days in fecA medium without DSF (−) and with different concentrations of DSF (0.25, 2.5, 25, and 50 μg ml⁻¹).

FIG. 4.

Steady-state levels of fecA and rmlA mRNA in S. maltophilia WT and the ΔrpfF mutant. RNA was isolated from cultures of S. maltophilia WT and the ΔrpfF mutant in fecA medium after 6 h and 18 h at 30°C and 175 rpm. RT-PCR was performed with primers specific for fecA (994 bp) and rmlA (613 bp). The experiment was repeated four times with reproducible results. Data from one representative experiment are presented.

FIG. 5.

Levels of rpfF transcript determined by RT-PCR in the WT, crp mutant, and its complemented strain (A) and predicted CRP binding sites upstream of the rpfF promoter (B). rpfF mRNA level was determined by RT-PCR. RNA was isolated from cultures of S. maltophilia WT, crp mutant (crp::EZTN), and crp-complemented (crp::EZTN/pcrp) strains grown in fecA medium without glucose at 30°C and 175 rpm for 2 days. RT-PCR was performed with primers specific for rpfF (845 bp). The experiment was repeated four times with reproducible results. Data from one representative experiment are presented. The partial rpfF sequence and upstream regions flanking rpfF were extracted from GenBank accession no. EF011627 nucleotides 4380 to 4600. Boxes, predicted CRP binding sites; solid underline, rpfF start codon; dashed underline, predicted rpfF promoter; +1, rpfF transcriptional start site.

FIG. 6.

A proposed model for the roles of CRP and DSFs in the regulation of various physiological processes in S. maltophilia WR-C. The organism produces DSFs (triangles), which require rpfF and rpfB for synthesis and are secreted outside the cell. The DSF system is involved in iron uptake mediated by FecA, ultimately affecting growth. It does not play a role in biofilm formation, lipopolysaccharide (LPS) biosynthesis, protease production, and hemolysis of rabbit blood cells. The divergent regulation of bacterial growth controlled by the DSF system and virulence factors in response to environmental stimuli may be mediated by CRP. CRP responds to environmental changes, such as iron and glucose levels, and binds to the predicted CRP binding site upstream of rpfF, activating the rpf system. OM, outer membrane; CM, cytoplasmic membrane.