Site-directed mutagenesis studies to probe the role of specific residues in the external loop (L3) of OmpF and OmpC porins in susceptibility of Serratia marcescens to antibiotics.
Serratia marcescens is a nosocomial bacterium with natural resistance to a broad spectrum of antibiotics, making treatment challenging. One factor contributing to this natural antibiotic resistance is reduced outer membrane permeability, controlled in part by OmpF and OmpC porin proteins. To investigate the direct role of these porins in the diffusion of antibiotics across the outer membrane, we have created an ompF-ompC porin-deficient strain of S. marcescens. A considerable similarity between the S. marcescens porins and those from other members of Enterobacteriaceae was detected by sequence alignment, with the exception of a change in a conserved region of the third external loop (L3) of the S. marcescens OmpC protein. Serratia marcescens OmpC has aspartic acid instead of glycine in position 112, methionine instead of aspartic acid in position 114, and glutamine in position 124, while in S. marcescens OmpF this is a glycine at position 124. To investigate the role of amino acid positions 112, 114, and 124 and how the observed changes within OmpC porin may play a part in pore permeability, 2 OmpC sites were altered in the Enterobacteriaceae consensus (D112G and M114D) through site-directed mutagenesis. Also, Q124G in OmpC, G124Q in OmpF, and double mutants of these amino acid residues were constructed. Antibiotic accumulation assays and minimal inhibitory concentrations of the strains harboring the mutated porins were performed, while liposome swelling experiments were performed on purified porins. Our results demonstrate that the amino acid at position 114 is not responsible for either antibiotic size or ionic selection, the amino acid at position 112 is responsible for size selection only, and position 124 is involved in both size and ionic selection.
Begic
Sanela
S
Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
Worobec
Elizabeth A
EA
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D024841:Fluoroquinolones / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005816:Genetic Complementation Test; D008081:Liposomes / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D018272:Porins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012706:Serratia marcescens / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D024841:Fluoroquinolones; D008081:Liposomes; C113724:OmpC protein; C109237:OmpF protein; D018272:Porins