Ralstonia solanacearum iron scavenging by the siderophore staphyloferrin B is controlled by PhcA, the global virulence regulator

Abstract

PhcA is a transcriptional regulator that activates expression of multiple virulence genes in the plant pathogen Ralstonia solanacearum. Relative to their wild-type parents, phcA mutants overproduced iron-scavenging activity detected with chrome azurol S siderophore detection medium. Transposon mutagenesis of strain AW1-PC (phcA1) generated strain GB6, which was siderophore negative but retained weak iron-scavenging activity. The ssd gene inactivated in GB6 encodes a protein similar to group IV amino acid decarboxylases, and its transcription was repressed by iron(III) and PhcA. ssd is the terminal gene in a putative operon that also appears to encode three siderophore synthetase subunits, a integral membrane exporter, and three genes with no obvious role in siderophore production. A homologous operon was found in the genomes of Ralstonia metallidurans and Staphylococcus aureus, both of which produce the polycarboxylate siderophore staphyloferrin B. Comparison of the siderophores present in culture supernatants of R. solanacearum, R. metallidurans, and Bacillus megaterium using chemical tests, a siderophore utilization bioassay, thin-layer chromatography, and mass spectroscopy indicated that R. solanacearum produces staphyloferrin B rather than schizokinen as was reported previously. Inactivation of ssd in a wild-type AW1 background resulted in a mutant almost incapable of scavenging iron but normally virulent on tomato plants. AW1 did not produce siderophore activity when cultured in tomato xylem sap, suggesting that the main location in tomato for R. solanacearum during pathogenesis is iron replete.

FIG. 1.

Iron scavenging by wild-type and mutant R. solanacearum strains on CAS indicator plates. The light halos surrounding the bacterial patches are where siderophore has removed iron from CAS, changing its color from blue to orange. (A) AW1-PC (phcA1) overproduced siderophore compared to AW1, its wild-type parent (photographed 48 h after stab inoculation). (B) The phcA mutants of eight R. solanacearum strains overproduced siderophores (photographed 48 h after droplets with 10⁴ CFU were deposited on the agar; wild types are to the left and phcA mutants are to the right of each pair of columns). (C) Inactivation of ssd in AW1-GB6 and GB6 greatly reduced the iron-scavenging ability of these strains compared to their parents. The plate was incubated for 4 days after droplets with 10⁴ CFU were deposited on the agar and was then photographed before and after washing off the bacteria. This image shows the plate after bacteria were removed, but with the colony margins indicated by the dotted lines. Pictures were taken with a Canon PowerShot A40 digital camera. Image contrast and brightness were adjusted with Adobe Photoshop, and the figure was assembled with Microsoft PowerPoint.

FIG. 2.

Putative locus for scavenging of iron(III) in R. solanacearum GMI1000 and related loci in R. metallidurans and S. aureus, two bacteria that produce the polycarboxylate staphyloferrin B. A related locus in the R. eutropha H16 megaplasmid (AY305387) that might encode iron(III) acquisition is also shown. The upper panel shows the size and arrangement of the ORFs. Open and patterned arrows denote genes potentially required for regulation, biosynthesis, and export of staphyloferrin B or uptake of ferri-staphyloferrin B. Filled arrows denote genes not involved in these processes. The R. eutropha putative siderophore receptor (gene b) has low homology to R. solanacearum gene B, and this is indicated by its having a lowercase letter. An asterisk designates that the R. eutropha putative MFS transporter gene is not homologous to R. solanacearum gene C4. The number of bases between the ORFs is indicated below each line (negative numbers indicate overlap of the ORFs). The lower panel gives selected details for the ORFs in GMI1000 and how similar the encoded proteins are to orthologs in three other bacteria. COG, cluster of orthologous group number; Score, BLASTp score; % ID, percentage of identical amino acids; % Tot, percentage of total amino acids aligned; sid. synth., siderophore synthesis.

FIG. 3.

Mass spectra of culture supernatants to detect staphyloferrin B. Samples were partially purified and concentrated about 200-fold prior to mass spectroscopy. (A) R. metallidurans CH34, which produces staphyloferrin B, has a peak at 449 m/z that is consistent with the molecular ion [M + H]⁺ of this siderophore. (B) R. solanacearum AW1-PC (phcA1), which is siderophore positive, also has a peak at 449 m/z. (C) R. solanacearum GB6 (phcA1 ssd), which is siderophore negative, lacks the peak at 449 m/z. See the text for further analysis of the spectra. The spectra for both AW1-PC and GB6 were recorded about 6.7 min after sample injection. The spectra shown had the background electronically subtracted, and the signal from the GB6 sample was amplified slightly compared to the other two samples.