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. 2004 Jul;72(7):4172-87.
doi: 10.1128/IAI.72.7.4172-4187.2004.

Contribution of Gene Loss to the Pathogenic Evolution of Burkholderia Pseudomallei and Burkholderia Mallei

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Free PMC article

Contribution of Gene Loss to the Pathogenic Evolution of Burkholderia Pseudomallei and Burkholderia Mallei

Richard A Moore et al. Infect Immun. .
Free PMC article

Abstract

Burkholderia pseudomallei is the causative agent of melioidosis. Burkholderia thailandensis is a closely related species that can readily utilize l-arabinose as a sole carbon source, whereas B. pseudomallei cannot. We used Tn5-OT182 mutagenesis to isolate an arabinose-negative mutant of B. thailandensis. Sequence analysis of regions flanking the transposon insertion revealed the presence of an arabinose assimilation operon consisting of nine genes. Analysis of the B. pseudomallei chromosome showed a deletion of the operon from this organism. This deletion was detected in all B. pseudomallei and Burkholderia mallei strains investigated. We cloned the B. thailandensis E264 arabinose assimilation operon and introduced the entire operon into the chromosome of B. pseudomallei 406e via homologous recombination. The resultant strain, B. pseudomallei SZ5028, was able to utilize l-arabinose as a sole carbon source. Strain SZ5028 had a significantly higher 50% lethal dose for Syrian hamsters compared to the parent strain 406e. Microarray analysis revealed that a number of genes in a type III secretion system were down-regulated in strain SZ5028 when cells were grown in l-arabinose, suggesting a regulatory role for l-arabinose or a metabolite of l-arabinose. These results suggest that the ability to metabolize l-arabinose reduces the virulence of B. pseudomallei and that the genes encoding arabinose assimilation may be considered antivirulence genes. The increase in virulence associated with the loss of these genes may have provided a selective advantage for B. pseudomallei as these organisms adapted to survival in animal hosts.

Figures

FIG. 1.
FIG. 1.
Arabinose assimilation pathway in B. thailandensis.
FIG. 2.
FIG. 2.
Arrangement of arabinose assimilation genes on chromosome 2 (Chr2) in B. thailandensis (Bt). The corresponding regions on B. pseudomallei (Bp) Chr2 and B. mallei (Bm) Chr2 are also shown, including the location of the araA-araH deletion. The percent amino acid (AA) identity was determined using tBlastn, and the results are color coded. The figure also depicts the duplication and movement of araF, araG, and araH to chromosome 1 in both B. pseudomallei and B. mallei. The gene order is conserved, but the identity has decayed to lower than 60%. The exact chromosomal locations of all genes are shown in parentheses.
FIG. 3.
FIG. 3.
PCR amplification of ara genes. (A) PCR survey for the presence of ara genes in B. pseudomallei and B. thailandensis isolates. PCR primers ARA1 and ARA2 were used to amplify a 638-bp product spanning the araA-araB intergenic region of the arabinose utilization locus. Lanes: 1, K96243; 2, 1026b; 3, 576; 4, E8; 5, E12; 6, E13; 7, 112c; 8, 238; 9, 295; 10, 296; 11, 713; 12, 730; 13, E27; 14, E30; 15, E32; 16, E96; 17, E100; 18, E105; 19, E111; 20, E120; 21, E125; 22, E132; 23, E135; 24, E264. (B) All B. pseudomallei and B. mallei strains examined contained the Δ(araA-araH) mutation. The PCR primer pair ARA1 and ARA3 was used to amplify a 454-bp product flanking the Δ(araA-araH) mutation in B. pseudomallei and B. mallei strains. Lanes: 1, K96243; 2, 1026b; 3, 576; 4, E8; 5, E12; 6, E13; 7, 112c; 8, 238; 9, 295; 10, 296; 11, 713; 12, 730; 13, 423; 14, 439a; 15, 465a; 16, 487; 17, 503; 18, 644; 19, NCTC 10248; 20, NCTC 10229; 21, NCTC 10260; 22, NCTC 10247; 23, ATCC 23344; 24, NCTC 120.
FIG. 4.
FIG. 4.
Induction of arabinose assimilation genes in B. thailandensis RM601 (DW503 derivative; araC::pGSV3-lux Gmr)and RM602 (DW503 derivative; araE::pGSV3-lux Gmr). ▾, RM601 in glucose; □, RM602 in glucose; ⋄, RM601 in arabinose; ★, RM602 in arabinose; ▴, RM601 in glucose plus arabinose; •, RM602 in glucose plus arabinose. RLU, relative light units (luminescence).
FIG. 5.
FIG. 5.
Construction of the arabinose-utilizing B. pseudomallei strain SZ5028. The transcriptional regulator gene (araA) from the B. thailandensis arabinose assimilation operon was amplified using PCR. A 1,133-bp PCR product was generated with primers ARA6 and ARA2 and E264 chromosomal DNA. This PCR product was cloned into the suicide vector pSKM11 to produce the plasmid pDD157. The B. thailandensis strain DD5026 was generated by integrating pDD157 into the E264 chromosome. The plasmid pDD5026H was obtained by self-cloning with the restriction enzyme HindIII. This plasmid contains pSKM11 and the entire arabinose assimilation operon from B. thailandensis. pDD5026H was transformed and conjugated to B. pseudomallei 406e to produce an arabinose-utilizing strain of B. pseudomallei. The resulting strain, B. pseudomallei SZ5028, was obtained by integrating pDD5026H into the 406e chromosome. The locations and names of the arabinose genes from B. thailandensis are indicated, and the direction of transcription is represented by arrows. The region of homology between B. thailandensis and B. pseudomallei is indicated. The plasmids pDD157 and pDD5026H are illustrated. The locations of relevant restriction endonuclease recognition sites (Bg, BglII; H, HindIII) are shown.
FIG. 6.
FIG. 6.
PCR amplification of the araA gene and the arabinose assimilation operon from B. thailandensis and B. pseudomallei chromosomal DNA. Lanes 1 to 5, PCR amplification of the araA gene using primers ARA6 and ARA2. Lanes 6 to 10, PCR amplification of the arabinose assimilation operon using primers ARA-F and ARA-R2. Lane M, 1-kb Plus DNA ladder (Invitrogen Life Technologies); lanes 1 and 6, B. pseudomallei 1026b; lanes 2 and 7, B. thailandensis E264; lanes 3 and 8, B. pseudomallei 406e; lanes 4 and 9, B. pseudomallei SZ5026; lanes 5 and 10, B. pseudomallei SZ5028.
FIG. 7.
FIG. 7.
Gene expression profiles of the arabinose-assimilating B. pseudomallei strain SZ5028 grown in M9 medium containing 1% arabinose versus that with 1% glucose. (a) Microarray image showing up-regulated (red) and down-regulated (green) genes. (b) Scatter plot of the normalized data, showing genes involved in TTSS3 that were significantly down-regulated. LEX.R, intensities of the reference sample (cells grown in 1% glucose); LEX.E, intensities of the evaluated sample (cells grown in 1% arabinose). The plotted data were normalized using the LOWESS smoothing (subgrid) method based on the GeneTraffic software, and the regression line shows the 1:1 ratio of 100% LOWESS smoothing factor.
FIG. 8.
FIG. 8.
Northern blot analysis of gene expression. Lanes 1, 3, 5, 8, 11, 14, and 17, total RNA from strain SZ5028 grown in M9 medium supplemented with 1% glucose; lanes 2, 4, 6, 9, 12, 15, and 18, total RNA from strain SZ5028 grown in M9 medium supplemented with 1% arabinose; lanes 7, 10, 13, and 16, total RNA from strain 406e grown in M9 medium supplemented with 1% glucose. The RNA blots were probed with the genes bsaN, bsaP, araC, bpss0766, bpss0769, bpss0780, and bpss0782.

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