RpoS-dependent stress tolerance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is able to persist during feast and famine in many different environments including soil, water, plants, animals and humans. The alternative sigma factor encoded by the rpoS gene is known to be important for survival under stressful conditions in several other bacterial species. To determine if the P. aeruginosa RpoS protein plays a similar role in stationary-phase-mediated resistance, an rpoS mutant was constructed and survival during exposure to hydrogen peroxide, high temperature, hyperosmolarity, low pH and ethanol was investigated. Disruption of the rpoS gene resulted in a two- to threefold increase in the rate of kill of stationary-phase cells. The rpoS mutant also survived less well than the parental strain during the initial phase of carbon or phosphate-carbon starvation. However, after 25 d starvation the remaining population of culturable cells was not significantly different. Stationary-phase cells of the RpoS-negative strain were much more stress resistant than exponentially growing RpoS-positive cells, suggesting that factors other than the RpoS protein must be associated with stationary-phase stress tolerance in P. aeruginosa. Comparison of two-dimensional PAGE of the rpoS mutant and the parental strain showed four major modifications of protein patterns associated with the rpoS mutation.

A pheromone-independent CarR protein controls carbapenem antibiotic synthesis in the opportunistic human pathogen Serratia marcescens.

Strain ATCC 39006 of Serratia marcescens makes the same carbapenem, (5R)-carbapen-2-em-3-carboxylic acid (Car), as the Erwinia carotovora strain GS101. Unlike E. carotovora, where the onset of production occurs in the late-exponential phase of growth in response to the accumulation of the small diffusible pheromone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), in S. marcescens carbapenem is produced throughout the growth phase and does not appear to involve any diffusible pheromone molecule. Two cosmids capable of restoring antibiotic production in E. carotovora group I carbapenem mutants were isolated from an S. marcescens gene library. These cosmids were shown to contain a homologue of the E. carotovora carR gene, encoding a CarR protein with homology to the LuxR family of transcriptional regulators. The S. marcescens carR was subcloned and shown to be capable of complementing in trans, in the absence of OHHL, an E. carotovora carR carI double mutant, releasing the heterologous E. carotovora host from pheromone dependence for carbapenem production. The apparent OHHL-independence of the S. marcescens CarR explains the constitutive nature of carbapenem production in this strain of S. marcescens.

Analysis of bacterial carbapenem antibiotic production genes reveals a novel ß-lactam biosynthesis pathway.

Carbapenems are ß-lactam antibiotics which have an increasing utility in chemotherapy, particularly for nosocomial, multidrug-resistant infections. Strain GS101 of the bacterial phytopathogen, Erwinia carotovora, makes the simple ß-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid. We have mapped and sequenced the Erwinia genes encoding carbapenem production and have cloned these genes into Escherichia coli where we have reconstituted, for the first time, functional expression of the ß-lactam in a heterologous host. The carbapenem synthesis gene products are unrelated to enzymes involved in the synthesis of the so-called sulphur-containing ß-lactams, namely penicillins, cephamycins and cephalosporins. However, two of the carbapenem biosynthesis genes, carA and carC, encode proteins which show significant homology with proteins encoded by the Streptomycesclavuligerus gene cluster responsible for the production of the ß-lactamase inhibitor, clavulanic acid. These homologies, and some similarities in genetic organization between the clusters, suggest an evolutionary relatedness between some of the genes encoding production of the antibiotic and the ß-lactamase inhibitor. Our observations are consistent with the evolution of a second major biosynthetic route to the production of ß-lactam-ring-containing antibiotics.