George P. C. Salmond

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.

Membrance traffic wardens and protein secretion in Gram-negative bacteria

Abstract Recent progress in the genetic analysis of protein secretion in diverse Gram-negative bacteria has revealed three major, highly conserved but Protein secretion via the Type I pathway is signal sequence-independent with no free periplasmic intermediate. Secretion by the Type II pathway is signal sequence-dependent and via the periplasm. Recent results also suggest that a third (Type III) secretory pathway exists in which protein secretion is signal sequence-independent.

A general role for the lux autoinducer in bacterial cell signalling: control of antibiotic biosynthesis in Erwinia

Micro-organisms have evolved complex and diverse mechanisms to sense environmental changes. Activation of a sensory mechanism typically leads to alterations in gene expression facilitating an adaptive response. This may take several forms, but many are mediated by response-regulator proteins. The luxR-encoded protein (LuxR) has previously been characterised as a member of the response-regulator superfamily and is known to respond to the small diffusible autoinducer signal molecule N-(beta-ketocaproyl) homoserine lactone (KHL). Observed previously in only a few marine bacteria, we now report that KHL is in fact produced by a diverse group of terrestrial bacteria. In one of these (Erwinia carotovora), we show that it acts as a molecular control signal for the expression of genes controlling carbapenem antibiotic biosynthesis. This represents the first substantive evidence to support the previous postulate that the lux autoinducer, KHL, is widely involved in bacterial signalling.

Biosynthesis of carbapenem antibiotic and prodigiosin pigment in Serratia is under quorum sensing control.

Serratia sp. ATCC 39006 produces the carbapenem antibiotic, carbapen‐2‐em‐3‐carboxylic acid and the red pigment, prodigiosin. We have previously reported the characterization of a gene, carR, controlling production of carbapenem in this strain. We now describe further characterization of the carR locus to locate the genes encoding carbapenem biosynthetic and resistance functions. A novel family of diverse proteins showing sequence similarity to the C‐terminal domain of CarF (required for carbapenem resistance) is described. We also report the isolation of the locus involved in the biosynthesis of the red pigment, prodigiosin. A cosmid containing ≈ 35 kb of the Serratia chromosome encodes synthesis of the pigment in the heterologous host, Erwinia carotovora, demonstrating, for the first time, that the complete prodigiosin biosynthetic gene cluster had been cloned and functionally expressed. We report the isolation of a third locus in Serratia, containing convergently transcribed genes, smaI and smaR, encoding LuxI and LuxR homologues respectively. SmaI directs the synthesis of N‐acyl homoserine lactones involved in the quorum sensing process. We demonstrate that biosynthesis of the two secondary metabolites, carbapenem antibiotic and prodigiosin pigment, is under pheromone‐mediated transcriptional regulation in this bacterium. Finally, we describe a new prodigiosin‐based bioassay for detection of some N‐acyl homoserine lactones.

A novel strategy for the isolation of luxl homologues: evidence for the widespread distribution of a LuxR:Luxl superfamily in enteric bacteria

The pheromone N‐(3‐oxohexanoyl)‐L‐homoserine lactone (OHHL) regulates expression of bioluminescence in the marine bacterium Vibrio fischeri, the production of carbapenem antibiotic in Erwinia carotovora and exoenzymes in both E. carotovora and Pseudomonas aeruginosa. A characteristic feature of this regulatory mechanism in V. fischeri is that it is cell density‐dependent, reflecting the need to accumulate sufficient pheromone to trigger the induction of gene expression. Using a lux plasmid‐based bioluminescent sensor for OHHL, pheromone production by E. carotovora, Enterobacter agglomerans, Hafnia alvei, Rahnella aquatilis and Serratia marcescens has been demonstrated and shown also to be cell density‐dependent. Production of OHHL implies the presence in these bacteria of a gene equivalent to luxl. Chromosomal banks from all five enteric bacteria have yielded clones capable of eliciting OHHL production when expressed in Escherichia coli. The luxl homologue from both E. carotovora (carl) and E. agglomerans (eagl) were characterized at the DNA sequence level and the deduced protein sequences have only 25% identity with the V. fischeri Luxl. Despite this, carl, eagl and luxl are shown to be biologically equivalent. An insertion mutant of eagl demonstrates that this gene is essential for OHHL production in E. agglomerans.

Molecular analysis of the methane monooxygenase (MMO) gene cluster of Methylosinus trichosporium OB3b

The oxidation of methane to methanol in methano‐trophic bacteria is catalysed by the enzyme methane monooxygenase (MMO). This multicomponent enzyme catalyses a range of oxidations including that of aliphatic and aromatic compounds and therefore has potential for commercial exploitation. This study details the molecular characterization of the soluble MMO (sMMO) genes from the Type II methanotroph Methylosinus trichosporium OB3b. The structural genes encoding the α, β and γ subunits of sMMO protein A and the structural gene encoding component B have been isolated and sequenced. These genes have been expressed and their products identified using an in vitro system. A comparative analysis of sMMO predicted sequences of M. trichosporium OB3b and the taxonomically related M. capsulatus (Bath) is also presented.

Complementation of deletion mutations in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species‐specific secretion of proteins via the type II pathway

The type II or Sec‐dependent secretion system is used by diverse Gram‐negative bacteria for secretion of extracellular proteins. Of the 12–15 proteins involved in secretion, the requirement for many has not been demonstrated and little is known about their functions in the secretion process. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete extracellular pectate lyases (Pels) using the type II or Out pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed genes from the other species, suggesting the presence of species‐specific recognition factors in the Out systems of the two Erwinia species. We previously reported the isolation of a cosmid clone, pCPP2006, from E. chrysanthemi EC16, which enables Escherichia coli to secrete heterologously expressed E. chrysanthemi Pels. Sequencing in a region required for secretion revealed the presence of 12 genes, outC‐M and outO. We report here the construction of functionally non‐polar mutations in each gene in the outC‐M operon and outS and outB using a polAts strain of E. coli to facilitate homologous recombination between out genes carrying deletions and their wild‐type copies on pCPP2006. By testing for complementation of each deletion with wild‐type out genes from E. chrysanthemi EC16 and E. carotovora SCRI193 we have demonstrated that: (i) each out gene is required for secretion of E. chrysanthemi PelE from E. coli with the exception of outH; (ii) each mutation can be complemented by its homologue from E. carotovora, except for outC and outD; (iii) outC and outD from E. carotovora do not confer secretion of Pel1 on the E. chrysanthemi Out system; and (iv) Pel1 secretion can be conferred on the E. chrysanthemi Out system by the presence of outC‐M, S and B from E. carotovora. The data suggest that OutC and OutD are gatekeepers of the Out system involved in recognition of Pels targeted for secretion but that OutC and OutD from E. carotovora cannot be successfully assembled into the E. chrysanthemi Out system.

A new cell division operon in Escherichia coli.

SummaryAt 76 min on theE. coli genetic map there is a cluster of genes affecting essential cellular functions, including the heat shock response and cell division. A combination ofin-vivo andin-vitro genetic analysis of cell division mutants suggests that the cell division genefts E is the second gene in a 3 gene operon. A cold-sensitive mutant, defective in the third gene, is also unable to divide at the restrictive temperature, and we designate this new cell division genefts X. Another cell division gene,fts S, is very close to, but distinct from, the 3 genes of the operon. Thefts E product is a 24.5 Kd polypeptide which shows strong homology with a small group of proteins involved in transport. Both thefts E product and the protein coded by the first gene (fts Y) in the operon have a sequence motif found in a wide range of heterogeneous proteins, including the Ras proteins of yeast. This common domain is indicative of a nucleotide-binding site.

Molecular cloning and characterization of 13 out genes from Erwinia carotovora subspecies carotovora: genes encoding members of a general secretion pathway (GSP) widespread in Gram‐negative bacteria

The chemical mutagen ethylmethanesulphonate (EMS) has been used to generate mutants of Erwinia carotovora subspecies carotovora which are defective in the secretion of pectinases (Pel) and cellulases (Cel) but unaltered for protease (Prt) secretion. Such mutants, called Out− still synthesize Pel and Cel but these enzymes accumulate within the periplasm. Cosmid clones carrying wild‐type E. carotovra ssp. carotovora DNA, identified by their ability to restore the Out+ phenotype when transferred to some Out− mutants, were classified into six complementation groups using cosmids and cosmid derivatives. Analysis of the nucleotide sequence of a 12.7 kb DNA fragment, encompassing complementing cosmid inserts, revealed a coding capacity for 13 potential open reading frames (ORFs), and these were designated outC‐outO. Some of the out gene products were visualized using a T7 gene 10 expression system. The predicted Out proteins are highly similar to components of extracellular enzyme secretion systems from a diverse range of eubacteria including Erwinia chrysanthemi, Klebsiella oxytoca, Aeromonas hydrophila, Pseudomonas aeruginosa and Xanthomonas campestris. Lower levels of similarity exist between Ecc Out proteins and components of macromolecular trafficking systems from Bacillus subtilis, Haemophilus influenzae, Agrobacterium tumefaciens, Yersinia pestis and a protein involved in the morphogenesis of filamentous bacteriophages such as M13.

EXTRACELLULAR AND PERIPLASMIC ISOENZYMES OF PECTATE LYASE FROM ERWINIA-CAROTOVORA SUBSPECIES CAROTOVORA BELONG TO DIFFERENT GENE FAMILIES

Pectate lyase (Pel) plays a crucial role in the maceration of vegetables by soft rot Erwinia spp. We have characterized the four Pel isoenzymes of Erwinia carotovora subspecies carotovora strain SCRI193. In this paper we concentrate on two isoenzymes which have different locations in SCRI193: PLb is periplasmic and PLc is extracellular. Comparison of the gene products and nucleotide sequences of pelB and pelC allowed us to assign them to different gene families. In addition, we have identified a number of conserved amino acid residues that are common to all extracellular Pel isoenzymes.