Article Figures & Data
Figures
- FIG. 1.
Genetic organization of the bpsI-bpsR locus of B. pseudomallei KHW (GenBank accession no. AY373337 ). Gray box arrows denote the divergently transcribed bpsI and bpsR genes; short thin arrows denote the PCR primer positions, while short white box arrows denote putative bpsI and bpsR promoters (P). Triangles indicate the locations where kanamycin resistance cassettes were inserted in the null mutants, KHWbpsI::Km and KHWbpsR::Km. The insertion sites were confirmed by PCR and DNA sequencing, while the null phenotypes were confirmed by Northern blotting (data not shown). Black rectangles denote the lux boxes, and their similarities with the palindromic lux box consensus sequence are shown in the insert (10). Consensus sequence abbreviations are as follows: N is A, T, C, or G; R is A or G; S is C or G; Y is T or C; and X is N or a gap in the sequence. The length of the bar represents a distance of 200 bp.
- FIG. 2.
Detection of acyl-HSLs produced by BpsI using HPLC. The culture extract (filled circles) contained acyl-HSL extracted from the supernatant of a stationary-phase culture of E. coli DH5α (pGEM-T-bpsIR) in AB medium (4) supplemented with 0.1 μg of thiamine/ml, 0.3% Casamino Acids, and 20 mM glycerol. Concentrated extract was chromatographed on a C18 reversed-phase HPLC column. Each 2-ml fraction collected was concentrated and assayed for β-galactosidase activity using KHWbpsI::Km (pSYI) as the reporter strain and according to the method described by Miller (15). For the profiles of the synthetic C8HSL (open squares) and C10HSL (open triangles) standards, only fractions 5 to 16 are represented, since the others did not yield any detectable activity.
- FIG. 3.
Cell density-dependent expressions of bpsI and bpsR. Open symbols represent cell densities, while filled symbols represent β-galactosidase activities. Cell densities and β-galactosidase activities of KHW(pSYI) and KHW(pSYR) are represented as open and filled triangles, respectively. Cell density-dependent expression of bpsI was abolished in KHWbpsI::Km(pSYI) (circles, A), and KHWbpsR::Km(pSYI) (circles, B). Addition of exogenous 0.125 nM C8HSL to KHWbpsR::Km(pSYI) did not affect the growth curve (open squares, A) but restored bpsI expression to almost wild-type levels (filled squares, A). KHW did not express any endogenous β-galactosidase activity (data not shown). Cell density-dependent expression of bpsR was also abolished in KHWbpsI::Km(pSYR) (circles, C) and KHWbpsR::Km(pSYR) (circles, D). β-Galactosidase activities were expressed in Miller units. The experiments were performed in triplicate. OD600, optical density at 600 nm.
- FIG. 4.
Effects of bpsI and bpsR mutations on B. pseudomallei virulence. Both KHWbpsI::Km and KHWbspR::Km were attenuated in virulence in the B. pseudomallei-C. elegans coculture assays. After 48 h of coculture, 79% of the worms fed on KHWbpsI::Km survived, compared to 39 and 46% survival rates for those worms fed on KHW and the complemented KHWbpsI::Km strains, respectively (A). KHWbpsR::Km was also attenuated in virulence with 62% of the worms surviving after 48 h of coculture, compared to 31 and 37% survival rates in the wild-type KHW and complemented KHWbpsR::Km strains, respectively (B). E. coli OP50 was used as a negative control in this assay.
- FIG. 5.
(A) Siderophore secretion in B. pseudomallei KHW was negatively regulated by the BpsIR quorum-sensing system. Siderophore activities were assayed in the 24-h-old culture supernatants of B. pseudomallei of KHW, KHWbpsI::Km, KHWbpsI::Km(pUCP28T-bpsI), KHWbpsR::Km, and KHWbpsR::Km(pUCP28T-bpsR) by measuring the differential in readings of optical density at 630 nm (OD630) between the test and the sample blank using the chrome azurol S assay as described in Yang et al. (31). The values shown have been normalized for cell density by expressing them as a ratio of ΔOD630/OD600. Each bar represents the average (± standard deviation) of readings from three independent experiments. (B) PLC secretion by B. pseudomallei KHW is positively regulated by the BpsIR quorum-sensing system. PLC activities were determined in the supernatants of 24-h cultures of B. pseudomallei KHW, KHWbpsI::Km, KHWbpsI::Km(pUCP28T-bpsI), KHWbpsR::Km, and KHWbpsR::Km(pUCP28T-bpsR) by the method described by Kurioka et al. (12). The data presented are the averages (± standard deviations) of the results from three independent experiments.
Tables
- TABLE 1.
Bacterial strains and plasmids used in this study
Strain or plasmid Relevant genotype or characteristics Reference or source B. pseudomallei strains KHW Wild-type, virulent clinical isolate 3 KHWbpsI::Km Isogenic to KHW containing bpsI::Km This study KHWbpsR::Km Isogenic to KHW containing bpsR::Km This study KHWbpsI::Km (pUCP28T-bpsI) KHWbpsI::Km mutant complemented in trans with pUCP28T carrying full-length bpsI gene This study KHW bpsR::Km (pUCP28T-bpsR) KHWbpsR::Km mutant complemented in trans with pUCP28T carrying full-length bpsR gene This study KHW(pSYI) KHW carrying the plasmid pSYI; Tcr This study KHW(pSYR) KHW carrying the plasmid pSYR; Tcr This study E. coli strains DH5αλpir DH5α with a λ prophage carrying the gene encoding the p protein; Kms Tps Gms 17 HB101(pRK600) Helper strain; containing pRK600 for triparental mating, supE44 hsdS20(rBmB) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1; Cmr 7, 23 SM10 Mobilizing strain; RP4 tra genes integrated in chromosome; Kmr 24 Plasmids pGEM-T Vector for PCR cloning; Apr Promega pUTKm Source of kanamycin resistance cassette; oriR6K mobRP4; Kmr Apr 6 pUCP28T Broad-host-range vector; IncP OriT; pRO1600 ori; Tpr 30 pJQ200mp18 Mobilizable allelic exchange vector; traJ sacB Gmr 22 Mini-CTX1 Mobilizable, broad-host-range plasmid for engineering of reporter strains; oriT Tcr 11 pMC1403 pBR322 derivative carrying a 6.2-kb promoterless lacZYA fragment for the cloning of translational control signals and 5′ coding sequences of exogenously derived genes; Apr 1 pCYY Mini-CTX1 containing a 6.2-kb promoterless lacZYA fragment inserted into EcoRI-SalI site in the multiple cloning sites of pMC1403 This study pSYI pCYY containing a 1.2-kb bpsI′-lacZ+ gene fusion; Tcr This study pSYR pCYY containing a 1.2-kb bpsR′-lacZ+ gene fusion; Tcr This study pGEM-T-bpsI::Km pGEM-T carrying 710-bp bpsI with a 2.3-kb kanamycin resistance cassette inserted at a blunt-ended BamHI site 330 bp from the start of the bpsI coding sequence; Apr Kmr This study pGEM-T-bpsIR pGEM-T carrying the full-length 2,271-bp bpsIR fragment for heterologous expression in E. coli This study pJQbpsI::Km pJQ200mp18 with a 3.0-kb PstI fragment from pGEMT-bpsI::Km containing bpsI::Km; Gmr Kmr This study pJQbpsR::Km pJQ200mp18 carrying bpsR::Km; Gmr Kmr. The 2.3-kb kanamycin resistance cassette from pUTKm was made blunt ended and inserted into the blunt-ended AatII site, 410 bp from the start of the coding sequence of bpsR This study
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- Article
- ABSTRACT
- Autoinducer synthesis by BpsIR in B. pseudomallei KHW.
- Cell density-dependent expression and transcriptional regulation of bpsI and bpsR.
- bpsI and bpsR mutants are partially attenuated in virulence in the Caenorhabditis elegans model.
- BpsIR is involved in the secretion of some exoproducts.
- FOOTNOTES
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