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Conservation of the opcL gene encoding the peptidoglycan-associated outer-membrane lipoprotein among representatives of the Burkholderia cepacia complex

¹Laboratory of Microbial Interactions, Department of Molecular and Cellular Interactions, Flanders Interuniversity Institute of Biotechnology, Vrije Universiteit Brussel, Building E, Room 6.2, Pleinlaan 2, B-1050 Brussels, Belgium ^2,3Laboratory of Pharmaceutical Microbiology² and Laboratory of Microbiology³, Ghent University, Ghent, Belgium ⁴Department of Medical Microbiology and Genitourinary Medicine, University of Liverpool, Duncan Building, Liverpool L69 3GA, UK

Correspondence Pierre Cornelis pcornel{at}vub.ac.be

Received October 9, 2003
Accepted January 12, 2004

Members of the Burkholderia cepacia complex are Gram-negative ß-proteobacteria that are classified into nine genomic species or genomovars. Some representatives of this group of bacteria, such as Burkholderia multivorans (genomovar II) and Burkholderia cenocepacia (genomovar III), are considered to be dangerous pathogens for cystic fibrosis (CF) patients because of their capacity to colonize CF lungs. The opcL gene, which encodes the peptidoglycan-associated outer-membrane lipoprotein (PAL), was detected in the genome of Burkholderia sp. LB 400 by a similarity search that was based on the sequence of the Pseudomonas aeruginosa PAL, OprL. Primers that could amplify part of opcL from B. multivorans LMG 13010^T were designed. This PCR fragment was used as a probe for screening of a B. multivorans genomic bank, allowing cloning of the complete opcL gene. The complete opcL gene could be PCR-amplified from DNA from all genomovars. The sequences of these opcL genes showed a high degree of conservation (> 95 %) among different species of the B. cepacia complex. OpcL protein that was purified from B. multivorans LMG 13010^T was used to generate mouse polyclonal antisera against OpcL. The OpcL protein could be produced in Escherichia coli and detected in outer-membrane fractions by Western blot. Burkholderia cells were labelled by immunofluorescence staining using antibodies against OpcL, but only after treatment with EDTA and SDS. The opcL gene could be amplified directly from the sputa of 15 CF patients who were known to be colonized by B. cepacia; sequence data derived from the amplicons identified the colonizing strains as B. cenocepacia (genomovar III, n = 14) and B. multivorans (n = 1).

The GenBank/EMBL/DDBJ accession numbers for the sequences described in this article are AY278462–AY278482.

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