Isolation and characterization of a Pseudomonas aeruginosa PAO mutant defective in the structural gene for the LIVAT-binding protein

Abstract

A mutant of Pseudomonas aeruginosa PAO which has a defect in the structural gene for a binding protein for leucine, isoleucine, valine, alanine, and threonine (LIVAT-binding protein) was isolated and characterized. DL-4-azaleucine was taken up via the high-affinity branched-chain amino acid transport system (LIV-I), but not via the low affinity system (LIV-II), and then inhibited the growth of P. aeruginosa cells. This finding enabled us to select mutants defective in the LIV-I transport system alone. Among such mutants, strain PAO3530 was found to produce an altered LIVAT-binding protein. The shock fluid of this strain contained a normal level of the protein which corresponded to the wild-type LIVAT-binding protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by an immunological test. However, the shock fluid showed almost no binding activity for branched-chain amino acids, suggesting that strain PAO3530 has a defect in the structural gene for the LIVAT-binding protein. The mutation locus (bra-310) was mapped in a region between cnu-9001 and oru-325 on the chromosome of P. aeruginosa PAO by conjugation mediated by plasmid FP5 or R68.45.

Locus of the Pseudomonas aeruginosa toxin A gene

“…A variant of the R68 plasmid, R68.45, has been described which has chromosome-mobilizing ability from multiple random sites around the chromosome (11). This plasmid can mobilize 10 min of donor chromosome and has facilitated genetic analysis of markers in the late region of the chromosome (13,28). We used the R68.45 plasmid to mediate transfer of chromosome material from strain PAO-PR1 (toxAl) and selected prototrophic recombinants for characterized auxotrophic lesions in recipient strains.…”

Genetic mapping of bra genes affecting branched-chain amino acid transport in Pseudomonas aeruginosa

“…The bacterial strains used in this study were all derivatives of P. aeruginosa PAO (10) and are listed in Table 1. Strains carrying plasmid FP5 (24) or R68.45 (7), except those naturally contain- MT1562 argBJ8 chl-2 rif-8001 A(chr-1061::Tn501-chr-32 1055: :Tn501)c a Symbols for genetic markers are those used in previous papers (16,22,28,33). Markers chr-1055::TnS01 and chr-1061::Tn501 represent the chromosomal sites at which transposon TnS01 is inserted.…”

“…Selection for resistance to azaleucine was used for the preferential isolation of P. aeruginosa PAO mutants defective in the LIV-I transport system, as described previously (16). Five mutants, PA03530 through PA03534, were chosen for detailed characterization.…”

“…Five mutants, PA03530 through PA03534, were chosen for detailed characterization. The properties of PA03530 (braC310) were described previously (16). Assays of transport and binding activities.…”

“…L-Leucine transport activity via the LIV-I or LIV-II system was assayed without Na+ or with 20 mM NaCl and 10 mM L-alanine, respectively (12). The binding activities of shock fluids were measured at 4°C by the equilibrium dialysis method (3,12), with the cap part of an Eppendorf type 3810 micro test tube used as a dialysis capsule (16).…”