Identification of essential operons with a rhamnose-inducible promoter in Burkholderia cenocepacia

Abstract

Scanning of bacterial genomes to identify essential genes is of biological interest, for understanding the basic functions required for life, and of practical interest, for the identification of novel targets for new antimicrobial therapies. In particular, the lack of efficacious antimicrobial treatments for infections caused by the Burkholderia cepacia complex is causing high morbidity and mortality of cystic fibrosis patients and of patients with nosocomial infections. Here, we present a method based on delivery of the tightly regulated rhamnose-inducible promoter P(rhaB) for identifying essential genes and operons in Burkholderia cenocepacia. We demonstrate that different levels of gene expression can be achieved by using two vectors that deliver P(rhaB) at two different distances from the site of insertion. One of these vectors places P(rhaB) at the site of transposon insertion, while the other incorporates the enhanced green fluorescent protein gene (e-gfp) downstream from P(rhaB). This system allows us to identify essential genes and operons in B. cenocepacia and provides a new tool for systematically identifying and functionally characterizing essential genes at the genomic level.

FIG. 1.

Construction of the transposon vectors pSCrhaBout and pSCrhaBoutgfp. The backbone of pCM3gfp was amplified by inverse PCR and ligated to the digested rhaR-rhaS-P_rhaB region to form pSCrhaBoutgfp. pSCrhaBout was obtained by inverse PCR amplification of pSCrhaBoutgfp with divergent primers flanking the e-gfp gene. IR, inverted repeats; oriT, origin of transfer; dhfr, trimethoprim resistance cassette; pMB1 ori, origin of replication for E. coli; rhaR and rhaS, transcription regulator genes of the rhamnose system; P_rhaB, rhamnose-inducible promoter. The numbers represent the primers used in inverse PCR, which are listed in Table 2.

FIG. 2.

Identification of essential B. cenocepacia genes. (a) Western blot analysis of chromosomally located e-gfp expression under inducing and noninducing conditions. The arrow indicates the position of GFP. (b) Identification of rhamnose-dependent mutants by replica plating. (c) Rhamnose-dependent growth phenotype of mutant rhaBoutgfp 3. (d) Identification of the insertion site in rhamnose-dependent mutants by self-cloning. See the text for details.

FIG. 3.

Growth of rhamnose-dependent mutants under inducing and noninducing conditions. Mutants were inoculated with toothpicks onto 96-well microtiter plates containing LB with rhamnose or glucose and incubated for 24 h at 37°C without shaking. (a) Growth was monitored by measuring the OD₅₇₀. The numbers in the x axis correspond to the names of the mutants (Table 3); wt, B. cenocepacia K56-2 parental strain. Results are averages of 12 repetitions. (b) The induction ratio for each mutant was calculated as the OD₅₇₀ under inducing conditions divided by the OD₅₇₀ under repressing conditions, as shown in panel a.

FIG. 4.

Transcriptional analysis of the nrfG-ppiA-ppiB-lpxC cluster. (a) Schematic drawing of the putative operon (top) and location of the primers used in RT-PCR experiments and expected amplified band size for each pair of primers (bottom). (b) RT-PCR amplification of intergenic regions. The arrows indicate the positions of the amplified bands and the observed sizes.

FIG. 5.

Comparative growth and gene expression analysis of rhaBoutgfp 4 and rhaBout12 mutants. (a) Distance of P_rhaB from the start codon (ATG) of nrfG for both mutants. (b) Growth curves in LB medium. Black squares, 0.2% rhamnose; open diamonds, 0.02% rhamnose; open triangles and dotted lines, 0.002% rhamnose. Arrows represent the times at which aliquots were removed for RNA extraction. (c) Relative RT-PCR. Total RNA was extracted from mutants rhaBoutgfp 4 and rhaBout 12 at different levels of rhamnose (rham). The arrows indicate the positions of the internal control band hisD and the intergenic band amplified with primers ppiA2F and ppiB1R. gDNA, genomic DNA. The numbers at the top of the gel represent arbitrary levels of gene expression relative to the internal control hisD.