A putative gene cluster for aminoarabinose biosynthesis is essential for Burkholderia cenocepacia viability

Abstract

Using a conditional mutagenesis strategy we demonstrate here that a gene cluster encoding putative aminoarabinose (Ara4N) biosynthesis enzymes is essential for the viability of Burkholderia cenocepacia. Loss of viability is associated with dramatic changes in bacterial cell morphology and ultrastructure, increased permeability to propidium iodide, and sensitivity to sodium dodecyl sulfate, suggesting a general cell envelope defect caused by the lack of Ara4N.

FIG. 1.

(A) Genetic organization of the putative arn gene cluster and flanking regions on chromosome 1 of B. cenocepacia strains J2315 and K56-2. Solid boxes around gene and locus names indicate the genes for which polar mutations failed; dotted boxes indicate genes for which the creation of polar mutations was successful. Mutagenesis of the remaining genes was not attempted. The dotted arrows indicate the locations of the inserted rhamnose-inducible promoter in strains XOA10, XOA11, and XOA12. Black and gray arrows denote the genes within the two transcriptional units of the putative arn cluster that were identified by RT-PCR. (B) RT-PCR analysis of the arn cluster, performed on strain J2315 in the absence of polymyxin B challenge.

FIG. 2.

(A) Conditional lethal phenotype of strains XOA11 and XOA12 on M9 agar plates supplemented with 0.5% (wt/vol) rhamnose or 0.5% (wt/vol) glucose. XOA11 and XOA12 grow only in the presence of rhamnose, while XOA10 and SAL10 grow equally well in either media. STC280 is shown as a control mutant for a known essential gene. (B) Depletion experiments using strains K56-2(pSCrhaB2), XOA10, XOA11, and XOA12 in nonpermissive conditions. Growth was monitored every hour using a Klett-Summerson colorimeter. The figure is representative of two independent experiments with similar results.

FIG. 3.

Microscopy and live-dead staining of strains K56-2, XOA10, XOA11, and XOA12 at 8 h of growth during the depletion experiments. Live bacteria appear fluorescent green, while dead bacteria and bacteria with compromised membranes appear fluorescent red. These experiments were carried out twice with similar results.

FIG. 4.

Transmission electron microscopy of strains K56-2, XOA11, and XOA12 after 8 h in nonpermissive conditions. The inserts in each panel are digital magnifications of selected regions of the electron micrographs (indicated by the dotted-line squares) showing the accumulation of membranous material, the presence of empty cells, and cell division abnormalities in more detail. Bars, 0.5 μm.