Characterization of the mrgRS locus of the opportunistic pathogen Burkholderia pseudomallei: temperature regulates the expression of a two-component signal transduction system

Abstract

Background: Burkholderia pseudomallei is a saprophyte in tropical environments and an opportunistic human pathogen. This versatility requires a sensing mechanism that allows the bacterium to respond rapidly to altered environmental conditions. We characterized a two-component signal transduction locus from B. pseudomallei 204, mrgR and mrgS, encoding products with extensive homology with response regulators and histidine protein kinases of Escherichia coli, Bordetella pertussis, and Vibrio cholerae.

Results: The locus was present and expressed in a variety of B. pseudomallei human and environmental isolates but was absent from other Burkholderia species, B. cepacia, B. cocovenenans, B. plantarii, B. thailandensis, B. vandii, and B. vietnamiensis. A 2128 bp sequence, including the full response regulator mrgR, but not the sensor kinase mrgS, was present in the B. mallei genome. Restriction fragment length polymorphism downstream from mrgRS showed two distinct groups were present among B. pseudomallei isolates. Our analysis of the open reading frames in this region of the genome revealed that transposase and bacteriophage activity may help explain this variation. MrgR and MrgS proteins were expressed in B. pseudomallei 204 cultured at different pH, salinity and temperatures and the expression was substantially reduced at 25 degrees C compared with 37 degrees C or 42 degrees C but was mostly unaffected by pH or salinity, although at 25 degrees C and 0.15% NaCl a small increase in MrgR expression was observed at pH 5. MrgR was recognized by antibodies in convalescent sera pooled from melioidosis patients.

Conclusion: The results suggest that mrgRS regulates an adaptive response to temperature that may be essential for pathogenesis, particularly during the initial phases of infection. B. pseudomallei and B. mallei are very closely related species that differ in their capacity to adapt to changing environmental conditions. Modifications in this region of the genome may assist our understanding of the reasons for this difference.

Figure 1

Map of the mrgRS locus of the B. pseudomallei genome (GenBank accession no. DQ418486). Open reading frames and direction of transcription are marked (arrows). See text for details of mrgR and mrgS, ORFs1-4, and oligonucleotide probes FR1-3. E = EcoRI site, B = BclI site. The positions of the additional EcoRI site that is described in the text and which is present in many B. pseudomallei isolates, and the 450 bp bacteriophage-like insertion and 650 bp transposase-like sequence that accompany this extra restriction site, are indicated by dotted lines.

Figure 2

Southern blot hybridisation of EcoRI-digested genomic DNA from different bacterial species using a digoxigenin-labeled oligonucleotide probe for mrgRS: a) B. pseudomallei isolates, pMRG2 contains a 4.3 kb EcoRI fragment of B. pseudomallei genomic DNA, spanning most of the mrgRS locus, inserted in pUC18 and digested with EcoRI b) B. thailandensis, other Burkholderia species, including B. cocovenenans (B. coco.), B. plantarii (B. plant.), B. vietnamiensis (B. viet.), B. cepacia (B. cep.) and B. vandii (B. van.), Pseudomonas aeruginosa (P. aer.), Pseudomonas fluorescens (P. fluo.) and E. coli. Lane M, λ-HindIII markers with molecular sizes (in kilobases) indicated on the left. See Table 1 for isolate details. All of the samples in the figure were separated on the same single agarose gel, transferred to the same single nylon membrane, and hybridized in the same single hybridization tube using the same labeled probe. The image of the resulting single Southern blot is reproduced in the figure as 2 panels, a) and b), for convenience and clarity and therefore pMRG2 represents a positive control for all samples.

Figure 3

Western blot probed with an affinity purified antibody, anti-MrgR, showing the detection of MrgR in whole cell lysates from 6 isolates of B. pseudomallei grown at 37°C. The isolate number is indicated above each lane. Lane M: molecular weight markers indicated in kilodaltons (kDa). See Table 1 for isolate details. Arrows indicate the expected position of the 24 kDa MrgR protein and slightly larger phosphorylated forms of MrgR. See text for details.

Figure 4

Western blot probed with an affinity purified antibody, anti-MrgS, showing MrgS in cell lysates from 6 isolates of B. pseudomallei grown at 37°C. The isolate number is indicated above each lane. Lane M: molecular weight markers indicated in kilodaltons (kDa). See Table 1 for isolate details. Arrow indicates the expected position of the 118 kDa MrgS protein. Smaller bands may represent processed forms of MrgS. See text for details.

Figure 5

Western blot showing the recognition of MBP-MrgR fusion protein by antibodies in convalescent sera pooled from 6 melioidosis patients. Lane 1: Uninduced cells, lane 2: cells induced for 3 h with IPTG, lane 3: amylose resin purified cell extract, lane 4: crude cell extract, and lane 5: insoluble matter, lane 6: purified maltose binding protein. Lane M: molecular weight markers indicated in kilodaltons (kDa).

Figure 6

Expression of MrgR in B. pseudomallei 204 cultured under different combinations of temperature, pH and NaCl concentration. Western blots of whole cell lysates were probed with anti-MrgR. The identity of each lane is indicated at the top. Lane M: molecular weight markers indicated in kilodaltons (kDa). Arrows indicate the expected position of the 24 kDa MrgR protein and slightly larger phosphorylated forms of MrgR.

Figure 7

Expression of MrgS in B. pseudomallei 204 cultured under different combinations of temperature, pH and NaCl concentration. Western blots of whole cell lysates were probed with anti-MrgS. The identity of each lane is indicated at the top. Lane M: molecular weight markers indicated in kilodaltons (kDa). Arrow indicates the expected position of the 118 kDa MrgS protein.

Figure 8

Southern blot hybridisation of EcoRI-digested genomic DNA from isolates of B. pseudomallei and B. thailandensis using oligonucleotide probe FR3. Lane M, λ-HindIII markers with molecular sizes (in kilobases) indicated on the left. Lane pMRG4 contains an 8.4 kb BclI fragment of the B. pseudomallei 204 genome spanning most of the mrgRS locus and 5 kb downstream, inserted in the phagemid pBK-CMV which was excised from λZAP Express and digested with EcoRI. See Table 1 for isolate details.

Figure 9

Alignment of lipases of B. pseudomallei isolates 112 and 204. Boxes enclose the "lid" motif (residues 112–127) and serine active site (residues 187–191). Asterisks mark the catalytic triad of serine, aspartic acid and histidine. +: conservative amino acid substitutions. Non-conservative amino acid substitutions are underlined. The 6 substitutions that are marked were also present in many of the B. pseudomallei genome sequences of 10 isolates that are available in GenBank: S47N: 10 isolates; I157V: 10 isolates; G180E: 5 isolates; V229A: 9 isolates; N340D: 4 isolates; E346Q: 3 isolates. Other substitutions were also found occasionally: S252F: 1 isolate; D341A: 1 isolate; V249L: 1 isolate.