Antibiotic resistance is a threat to our modern society, and new strategies leading to the identification of new molecules or targets to combat multidrug-resistant pathogens are needed. Species of the genus
Burkholderia, including the Burkholderia cepaciacomplex (Bcc), Burkholderia pseudomallei, and Burkholderia mallei, can be highly pathogenic and are intrinsically resistant to multiple classes of antibiotics. Bcc species are nonetheless sensitive to extracellular products released by Pseudomonas aeruginosain interspecies competition. We screened for Burkholderiatransposon mutants with increased sensitivity to P. aeruginosaspent medium and identified multiple mutants in genes sharing homology with the Mla pathway. Insertional mutants in representative genes of the Bcc Mla pathway had a compromised cell membrane and were more sensitive to various extracellular stresses, including antibiotics and human serum. More precisely, mlamutants in the Bcc species Burkholderia cenocepaciaand Burkholderia dolosawere more susceptible to Gram-positive antibiotics (i.e., macrolides and rifampin), fluoroquinolones, tetracyclines, and chloramphenicol. Genetic complementation of mlaCinsertional mutants restored cell permeability and resistance to Gram-positive antibiotics. Importantly, Bcc mlamutants were not universally weaker strains since their susceptibilities to other classes of antibiotics were unaffected. Although cell permeability of homologous mlamutants in Escherichia colior P. aeruginosawas also impaired, they were not more sensitive to Gram-positive antibiotics or other antimicrobials as was observed in Bcc mlamutants. Together, the data suggest that the Mla pathway in Burkholderiamay play a different biological role, which could potentially represent a Burkholderia-specific drug target in combination therapy with antibiotic adjuvants. IMPORTANCEThe outer membrane of Gram-negative bacteria acts as an effective barrier against toxic compounds, and therefore compromising this structure could increase sensitivity to currently available antibiotics. In this study, we show that the Mla pathway, a system involved in maintaining the integrity of the outer membrane, is genetically and functionally different in Burkholderia cepaciacomplex species compared to that in other proteobacteria. Mutants in mlagenes of Burkholderia cenocepaciaor Burkholderia dolosawere sensitive to Gram-positive antibiotics, while this effect was not observed in Escherichia colior Pseudomonas aeruginosa. The Mla pathway in Burkholderiaspecies may represent an ideal genus-specific target to address their intrinsic antimicrobial resistances.