Simon J. McGowan

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.

N‐acyl homoserine lactone binding to the CarR receptor determines quorum‐sensing specificity in Erwinia

Quorum sensing via an N‐acyl homoserine lactone (HSL) pheromone controls the biosynthesis of a carbapenem antibiotic in Erwinia carotovora. Transcription of the carbapenem biosynthetic genes is dependent on the LuxR‐type activator protein, CarR. Equilibrium binding of a range of HSL molecules, which are thought to activate CarR to bind to its DNA target sequence, was examined using fluorescence quenching, DNA bandshift analysis, limited proteolysis and reporter gene assays. CarR bound the most physiologically relevant ligand, N‐(3‐oxohexanoyl)‐L‐homoserine lactone, with a stoichiometry of two molecules of ligand per dimer of protein and a dissociation constant of 1.8 μM, in good agreement with the concentration of HSL required to activate carbapenem production in vivo. In the presence of HSL, CarR formed a very high molecular weight complex with its target DNA, indicating that the ligand causes the protein to multimerize. Chemical cross‐linking analysis supported this interpretation. Our data show that the ability of a given HSL to facilitate CarR binding to its target DNA sequence is directly proportional to the affinity of the HSL for the protein.

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.

Carbapenem antibiotic biosynthesis in Erwinia carotovora is regulated by physiological and genetic factors modulating the quorum sensing-dependent control pathway

Erwinia carotovora produces the β‐lactam antibiotic, carbapenem, in response to a quorum sensing signalling molecule, N‐(3‐oxohexanoyl)‐ l‐homoserine lactone (OHHL). We have mapped the OHHL‐dependent promoter upstream of the first of the biosynthetic genes, carA. We have also analysed the effect on this promoter of the known genetic regulators of carbapenem expression, carR, carI (encoding homologues of LuxR and LuxI respectively) and hor (encoding a SlyA/MarR‐like transcriptional regulator). We describe a previously unknown promoter located within the carA‐H operon. This promoter does not respond to CarR and is required for quorum sensing‐independent expression of the carbapenem resistance determinants encoded by the carFG genes. We have mapped the carR, carI and hor transcription start points, shown that CarR is positively autoregulated in the presence of OHHL, and have demonstrated negative feedback affecting transcription of carI. In addition, various environmental  and physiological factors were shown to impinge on the transcription of the car biosynthetic genes. The nature of the carbon source and the temperature of growth influence carbapenem production by modulating the level of the OHHL signalling molecule, and thereby physiologically fine‐tune the quorum sensing regulatory system.

Cryptic carbapenem antibiotic production genes are widespread in Erwinia carotovora: facile trans activation by the carR transcriptional regulator.

Few strains of Erwinia carotovora subsp. carotovora (Ecc) make carbapenem antibiotics. Strain GS101 makes the basic carbapenem molecule, 1-carbapen-2-em-3-carboxylic acid (Car). The production of this antibiotic has been shown to be cell density dependent, requiring the accumulation of the small diffusible molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) in the growth medium. When the concentration of this inducer rises above a threshold level, OHHL is proposed to interact with the transcriptional activator of the carbapenem cluster (CarR) and induce carbapenem biosynthesis. The introduction of the GS101 carR gene into an Ecc strain (SCRI 193) which is naturally carbapenem-negative resulted in the production of Car. This suggested that strain SCRI 193 contained functional cryptic carbapenem biosynthetic genes, but lacked a functional carR homologue. The distribution of trans-activatable antibiotic genes was assayed in Erwinia strains from a culture collection and was found to be common in a large proportion of Ecc strains. Significantly, amongst the Ecc strains identified, a larger proportion contained trans-activatable cryptic genes than produced antibiotics constitutively. Southern hybridization of the chromosomal DNA of cryptic Ecc strains confirmed the presence of both the car biosynthetic cluster and the regulatory genes. Identification of homologues of the transcriptional activator carR suggests that the cause of the silencing of the carbapenem biosynthetic cluster in these strains is not the deletion of carR. In an attempt to identify the cause of the silencing in the Ecc strain SCRI 193 the carR homologue from this strain was cloned and sequenced. The SCRI 193 CarR homologue was 94% identical to the GS101 CarR and contained 14 amino acid substitutions. Both homologues could be expressed from their native promoters and ribosome-binding sites using an in vitro prokaryotic transcription and translation assay, and when the SCRI 193 carR homologue was cloned in multicopy plasmids and reintroduced into SCRI 193, antibiotic production was observed. This suggested that the mutation causing the silencing of the biosynthetic cluster in SCRI 193 was leaky and the cryptic Car phenotype could be suppressed by multiple copies of the apparently mutant transcriptional activator.

Bacterial N-acyl-homoserine-lactone-dependent signalling and its potential biotechnological applications.

N-acyl homoserine lactones are bacterial signalling molecules involved in regulating diverse metabolic functions, particularly those relating to virulence, in concert with cell density. Each aspect of the signalling pathway, from production and recognition of the signal to expression of the target genes, offers a potential opportunity for exploitation. Attention is now focusing on the development of novel methods for bacterial enumeration, modulation of bacterial virulence and flexible, coordinated expression of heterologous genes through the use of N-acyl-homoserine-lactone-based systems.

Erwinia carotovora has two KdgR-like proteins belonging to the IcIR family of transcriptional regulators: identification and characterization of the RexZ activator and the KdgR repressor of pathogenesis

A novel Erwinia carotovora subsp. carotovora mutant designated RexZ, (regulator of exoenzymes) showed reduced production of the degradative exoenzymes. The rexZ gene product shows similarity of the KdgR regulatory protein from Erwinia chrysanthemi, described as the major repressor of the pectin catabolism pathway genes in the latter species. In vitro DNA-protein interaction experiments demonstrated that the synthesis of the RexZ protein is controlled by the cAMP-CRP (cAMP-receptor protein) complex. Western blot analysis also revealed the presence of a second KdgR homologue (distinct from RexZ) which, like RexZ, was present in all species of the genus Erwinia tested. The corresponding KdgR proteins from both E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica share a high level of sequence identity with the KdgR homologues from E. chrysanthemi and Escherichia coli. Although the E. carotovora subsp. carotovora rexZ regulatory region displayed specific interactions with both the purified E. chrysanthemi KdgR repressor and the partially purified E. carotovora subsp. carotovora KdgR, in vivo quantification revealed that the cellular level of RexZ protein was unaffected by the presence of pectic compounds. This study shows that the complex regulatory network governing virulence in the erwinias involves two totally distinct, but highly conserved, members of the IcIR class of DNA binding proteins: RexZ and KdgR.

Molecular genetics of carbapenem antibiotic biosynthesis

Carbapenems are potent β-lactam antibiotics with a broad spectrum of activity against both Gram positive and Gram negative bacteria. As naturally produced metabolites, they have been isolated from species of Streptomyces, Erwinia and Serratia. The latter two members of the Enterobacteriaceae have proved to be genetically amenable and a growing body of research on these organisms now exists concerning the genes responsible for carbapenem biosynthesis and the regulatory mechanisms controlling their expression. A cluster of nine carbapenem (car) genes has been identified on the chromosome of Erwinia carotovora. These genes encode the enzymes required for construction of carbapenem and the proteins responsible for a novel β-lactam resistance mechanism, conferring carbapenem immunity in the producing host. Although sharing no homology with the well known enzymes of penicillin biosynthesis, two of the encoded proteins are apparently similar to enzymes of the clavulanic acid biosynthetic pathway implying a common mechanism for construction of the β-lactam ring. In addition, a transcriptional activator is encoded as the first gene of the carbapenem cluster and this allows positive expression of the remaining downstream genes in response to a quorum sensing, N-acyl homoserine lactone, signalling molecule.

Bacterial production of carbapenems and clavams: evolution of β-lactam antibiotic pathways

Research into two of the four classes of naturally produced beta-lactams--the clavams and carbapenems--has started to throw light upon their biochemical pathways and underlying genetics. Interesting similarities between these two classes, from their joint discovery to an apparently common beta-lactam ring-forming enzyme, are now being revealed.