G. S. A. B. Stewart

The bacterial ‘enigma’: cracking the code of cell–cell communication

In recent years it has become clear that the production of N‐acyl homoserine lactones (N‐AHLs) is widespread in Gram‐negative bacteria. These molecules act as diffusible chemical communication signals (bacterial pheromones) which regulate diverse physiological processes including bioluminescence, antibiotic production, piasmid conjugal transfer and synthesis of exoenzyme virulence factors in plant and animal pathogens. The paradigm for N‐AHL production is in the bioluminescence (lux) phenotype of Photobacterium fischeri (formerly classified as Vibrio fischeri) where the signalling molecule N‐(3‐oxohexanoyl)‐L‐homoserine lactone (OHHL) is synthesized by the action of the Luxl protein. OHHL is thought to bind to the LuxR protein, allowing it to act as a positive transcriptional activator in an autoinduction process that physiologically couples cell density (and growth phase) to the expression of the bioluminescence genes. Based on the growing information on Luxl and LuxR homologues in other N‐AHL‐producing bacterial species such as Erwinia carotovora, Pseudomonas aeruginosa, Yersinia enterocolitica, Agrobacterium tumefaciens and Rhizobium legumino‐sarum, it seems that analogues of the P. fischeri lux autoinducer sensing system are widely distributed in bacteria. The general physiological function of these simple chemical signalling systems appears to be the modulation of discrete and diverse metabolic processes in concert with cell density. In an evolutionary sense, the elaboration and action of these bacterial pheromones can be viewed as an example of multi‐cellularity in prokaryotic populations.

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.

Analysis of bacterial carbapenem antibiotic production genes reveals a novel β‐lactam biosynthesis pathway

Carbapenems are β‐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 β‐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 β‐lactam in a heterologous host. The carbapenem synthesis gene products are unrelated to enzymes involved in the synthesis of the so‐called sulphur‐containing β‐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 β‐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 β‐lactamase inhibitor. Our observations are consistent with the evolution of a second major biosynthetic route to the production of β‐lactam‐ring‐containing antibiotics.

Analysis of the carbapenem gene cluster of Erwinia carotovora: definition of the antibiotic biosynthetic genes and evidence for a novel β‐lactam resistance mechanism

Members of two genera of Gram‐negative bacteria, Serratia and Erwinia, produce a β‐lactam antibiotic, 1‐carbapen‐2‐em‐3‐carboxylic acid. We have reported previously the cloning and sequencing of the genes responsible for production of this carbapenem in Erwinia carotovora. These genes are organized as an operon, carA–H, and are controlled by a LuxR‐type transcriptional activator, encoded by the linked carR gene. We report in this paper the genetic dissection of this putative operon to determine the function of each of the genes. We demonstrate by mutational analysis that the products of the first five genes of the operon are involved in the synthesis of the carbapenem molecule. Three of these, carABC, are absolutely required. In addition, we provide evidence for the existence of a novel carbapenem resistance mechanism, encoded by the carF and carG genes. Both products of these overlapping and potentially translationally coupled genes have functional, N‐terminal signal peptides. Removal of these genes from the Erwinia chromosome results in a carbapenem‐sensitive phenotype. We assume that these novel β‐lactam resistance genes have evolved in concert with the biosynthetic genes to ensure ‘self‐resistance’ in the Erwinia carbapenem producer.

Molecular characterization of the proU loci of Salmonella typhimurium and Escherichia coli encoding osmoregulated glycine betaine transport systems

The proU loci of Salmonella typhimurium and Escherichia coli encode high‐affinity glycine betaine transport systems which play an important role in survival under osmotic stress. Transcription of the proU locus is tightly regulated by osmolarity and this regulation appears to be mediated by osmotically induced changes in DNA supercoiling. In order to study the regulatory mechanisms involved we have cloned and characterized the proU locus of S. typhimurium by an in vivo transductional procedure. The locus is shown to consist of at least three genes, designated proVWX, cotranscribed as a single operon. The first gene in the operon encodes a protein sharing considerable sequence identity with ATP‐binding proteins from other periplasmic transport systems. Unexpectedly, the highly expressed periplasmic glycine betaine binding protein was found to be encoded by a distal gene, proX, in the operon. The operon has no significant internal promoters but is expressed from a single osmoregulated promoter whose transcription start site has been mapped. The proU promoter of E. coli has also been sequenced and the transcription start site shown to be similar to that of S. typhimurium. Evidence is presented which suggests that, besides de novo glycine betaine uptake, an important function of ProU may be the recapture and recycling of other osmolytes that leak from the cell.

A novel, non‐invasive promoter probe vector: cloning of the osmoregulated proU promoter of Escherichia coli K12

We have constructed a novel promoter probe plasmid pSB40, containing a unique lac‐α‐tetracycline marker gene tandem, which allows for both positive and negative selection of active promoters. Promoters cloned in pSB40 can be readily mobilized as EcoR1 cassettes. Using this vector we have performed a non‐invasive analysis of the E. coli chromosome for promoters regulated by osmotic upshift. Only one such promoter, subsequently identified as part of the proU operon, was isolated. A sequence of 253bp, sufficient to mediate osmotic regulation of the proU promoter, was defined. This E coli promoter was normally regulated in Salmonella typhimurium, Klebsiella and Citrobacter but not in Shigella. A proU‐luxAB fusion plasmid was constructed and used to monitor in vivo real‐time kinetics of proU induction following osmotic upshock.