Aminoglycoside-Resistant Mutation of Pseudomonas aeruginosa Defective in Cytochrome c ₅₅₂ and Nitrate Reductase

Abstract

A gentamicin-resistant mutant of Pseudomonas aeruginosa PA0503 was selected after ethyl methane sulfonate mutagenesis. The strain, P. aeruginosa PA02401 had increased resistance to all aminoglycosides tested but exhibited no change for other antibiotics. The mutation designated aglA (aminoglycoside resistance) was 50% cotransducible with the 8-min ilvB,C marker on the P. aeruginosa chromosome. It showed a marked reduction in cytochrome c52 and nitrate reductase (Nar) and a change in terminal oxidase activity. Cytochrome c552 is a component of the P. aeruginosa Nar. No changes in succinate and reduced nicotinamide adenine dinucleotide dehydrogenases, ubiquinone content, Mg2"/Ca2" membrane adenosine triphosphatase, and energy coupling of electron transport to adenosine 5'-triphosphate synthesis were detected. Transport of gentamicin and dihydrostreptomycin was impaired in PA02401, but transport of proline, arginine, glutamine, glucose or the polyamine spermidine was not reduced. Ribosomes of PA02401, and PA0503 bound dihydrostreptomycin equally well, and cell extracts did not inactivate gentamicin or dihydrostreptomycin. Strain PA02401 is resistant to gentamicin and dihydrostreptomycin because of impaired transport of these compounds. The transport studies indicate a selective coupling of dihydrostreptomycin and gentamicin transport with terminal electron transport. This conclusion was supported by results from another mutant (PA0417-T2) with increased Nar activity, enhanced dihydrostreptomycin and gentamicin transport and a reduction in resistance to these drugs. These results are discussed in relation to a refined model for aminoglycoside transport and briefly relative to plasmid-mediated aminoglycoside resistance.

Peripheral inactivation of gentamicin

“…The correlation of increased intracellular accumulation of gentamicin with high sensitivity of the bacteria to the drug is good (Bryan et al, 1976(Bryan et al, , 1980Bryan & Van den Elzen, 1975, 1977. Experimental data show further that neither strictly anaerobic bacteria (Bryan et al, 1979) nor facultative bacteria growing anaerobically (Bryan & Van den Elzen, 1975) can accumulate bactericidal levels of gentamicin.…”

“…In contrast to these results, aerobic bacteria or facultative bacteria grown in aerobic conditions (Bryant et al, 1976(Bryant et al, , 1980Bryan & Van den Elzen, 1975, 1977 rapidly accumulated toxic levels of gentamicin, which thereafter could interact with ribosomes and which could lead eventually to cell death. With the use of metabolic inhibitors and of metabolic bacterial mutants, Bryan and his colleagues (Bryan et al, 1976(Bryan et al, , 1980Bryan & Van den Elzen, 1975, 1977 could demonstrate an absolute requirement for energy-dependent transport for gentamicin accumulation. This energy had to be generated by oxidative phosphorylation and could not be substituted by ATP produced under anaerobic conditions.…”

“…A better understanding of the specific effect of reduced pO 2 on the susceptibility of target bacteria to the drug, stems from detailed transport studies of this aminoglycoside across the cell wall into the bacteria (Bryan, Haraphongse & Van den Elzen, 1976;Bryan et al, 1980;Bryan & Van den Elzen, 1975, 1977. The correlation of increased intracellular accumulation of gentamicin with high sensitivity of the bacteria to the drug is good (Bryan et al, 1976(Bryan et al, , 1980Bryan & Van den Elzen, 1975, 1977.…”

Cysteine Catabolism and Cysteine Desulfhydrase (CdsH/STM0458) in Salmonella enterica Serovar Typhimurium

“…A previous study revealed that increasing exogenous cysteine decreases reduced periplasmic cytochrome c levels (49). Since aminoglycoside antibiotic uptake relies on anionic transporters energized mainly by the respiratory chain, impaired electron transport could explain the observed increase in aminoglycoside resistance with cysteine (12,59). Defects in cysteine catabolism may have little effect on exogenous cysteine under these conditions, and this could explain the lack of effect on antibiotic susceptibility.…”

Precise excision and instability of the transposon Tn5 in Pseudomonas aeruginosa

“…As in strain FRD, PAOl (pJG 1 : : Tn5-34) transconjugant colonies were small compared to PAOl(pJGl), and > 30% of the PAOl (pJG 1 : : Tn5-34) transconjugants appeared Kms. Kmr strains of PAOl(pJG1 : :Tn5-34) were taken from Tc-medium, rather than from Km-medium, to avoid the possibility of spontaneous Kmr [which is common in P. aeruginosa (Bryan et al, 1980)], and were grown in L broth with Tc (50 pg ml-l) overnight. Following subculture, 60 to 100% of the colonies obtained again appeared Kms or showed poor growth on Km.…”