The Burkholderia cepacia epidemic strain marker is part of a novel genomic island encoding both virulence and metabolism-associated genes in Burkholderia cenocepacia

Abstract

The Burkholderia cepacia epidemic strain marker (BCESM) is a useful epidemiological marker for virulent B. cenocepacia strains that infect patients with cystic fibrosis. However, there was no evidence that the original marker, identified by random amplified polymorphic DNA fingerprinting, contributed to pathogenicity. Here we demonstrate that the BCESM is part of a novel genomic island encoding genes linked to both virulence and metabolism. The BCESM was present on a 31.7-kb low-GC-content island that encoded 35 predicted coding sequences (CDSs): an N-acyl homoserine lactone (AHL) synthase gene (cciI) and corresponding transcriptional regulator (cciR), representing the first time cell signaling genes have been found on a genomic island; fatty acid biosynthesis genes; an IS66 family transposase; transcriptional regulator CDSs; amino acid metabolism genes; and a group of hypothetical genes. Mutagenesis of the AHL synthase, amidase (amiI), and porin (opcI) genes on the island was carried out. Testing of the isogenic mutants in a rat model of chronic lung infection demonstrated that the amidase played a role in persistence, while the AHL synthase and porin were both involved in virulence. The island, designated the B. cenocepacia island (cci), is the first genomic island to be defined in the B. cepacia complex and its discovery validates the original epidemiological correlation of the BCESM with virulent CF strains. The features of the cci, which overlap both pathogenicity and metabolism, expand the concept of bacterial pathogenicity islands and illustrate the diversity of accessory functions that can be acquired by lateral gene transfer in bacteria.

FIG. 1.

PFGE mapping of the BCESM in CF strains from patients with chronic infection. PFGE macrorestriction and Southern hybridization results of B. cenocepacia CF strains are shown. Lanes are as follows: M, molecular size markers with relevant fragment sizes (in kilobases) indicated on the left of the figure; 1 and 2, CEP54A and CEP54B, respectively, digested with SpeI; 3 and 4, CEP54A and CEP54B, respectively, digested with SwaI; 5, 6, and 7, strain type 2 sequential isolates recovered from patient A, after 1, 2, and 3 years of chronic infection (SpeI digests); 8, 9, and 10, corresponding Southern blot of lanes 5 to 7 hybridized to the BCESM; 11, 12, and 13, strain type 4 isolates recovered from patient B, after 1, 3, and 5 years of infection (SpeI digests); 14, 15, and 16, Southern blot of lanes 11 to 13 hybridized to the BCESM. The arrow indicates the conserved 503-kb SpeI fragment to which the BCESM hybridized in all strains.

FIG. 2.

Bioinformatic analysis of the B. cenocepacia island. (A and B) GC content plots and GC frame plots are shown, respectively; GC percentage was calculated over a window size of 500 bases, and the mean GC content of the second chromosome, 67.3%, is indicated by the line across each panel. (C) The gene content of the DNA in the BCESM region is shown, with each predicted CDS designated by its BCAM number or designated name. Genes mapped by Southern hybridization are shown as black arrows above the sequence bar (size in kilobases indicated on bar); other CDSs are indicated below the sequence bar as grey arrows. The predicted functions of each gene cluster are shown below the brackets. The position of the 13-bp direct imperfect repeats which flank the cci are shown by the arrows linked to the sequence bar.

FIG. 3.

Southern hybridization to BCC genomic DNA arrays. (A) Control hybridization to pooled 16S rRNA gene probes. (B) Specific hybridization of the amiI gene probe to strains of B. cenocepacia encoding the BCESM DNA.

FIG. 4.

Confirmation of the B. cenocepacia K56-2cciI, K56-2amiI, and K56-2opcI mutants. (A and B) Conventional SalI RFLP mapping of the amidase gene mutation. (A) Lanes are as follows: M, 1-kb molecular size marker; 1, K56-2; 2, K56-2cciI; 3, K56-2amiI; and 4, K56-2opcI. (B) Corresponding Southern hybridization probed with the amidase gene probe (Table 2). (C) PFGE SpeI analysis of the following strains: lane 1, K56-2 parent; lane 2, K56-2cciI; lane 3, K56-2amiI; and lane 4, K56-2opcI. PFGE molecular size markers were run in lane M, and the sizes of relevant bands are indicated.

FIG. 5.

Scans of representative lung sections from animals infected with K56-2 (A), K56-2cciI (B), or K56-2opcI (C). Regions of darker staining are indicative of areas of inflammation. The inflammation calculated for the sections in panels A to C are 28, 13, and 11%, respectively.