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

Abstract

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 fee 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.

A Novel Plant Ferredoxin-Like Protein and the Regulator Hor Are Quorum-Sensing Targets in the Plant Pathogen Erwinia carotovora

“…In certain strains of E. carotovora subsp. carotovora, Hor controls the expression of biosynthetic genes for the antibiotic carbapenem, which are also regulated by QS via the regulator CarR of the LuxR family (Holden et al 1998;Thomson et al 1997Thomson et al , 2000. In these strains, Hor and QS control of the same genes seem to work in parallel.…”

“…However, E. carotovora subsp. carotovora SCC3193 appears to lack the carbapenem-synthesizing genes (Holden et al 1998). Our results demonstrate that, in SCC3193, expression of hor is controlled in an AHSLdependent manner in contrast to the E. carotovora subsp.…”

Discovery of indolotryptoline antiproliferative agents by homology-guided metagenomic screening

“…However, in the case of the bor cluster, most clone-specific metabolites were produced by S. albus at such low levels that it was not possible to isolate sufficient quantities for structural elucidation. In genome-mining studies with cultured bacteria, it has been possible to increase metabolite production from "silent/cryptic" biosynthetic pathways by overexpressing positive acting transcriptional regulators found within gene clusters of interest (13,14). A putative transcription factor from the bor cluster, borR, was therefore cloned into a Streptomyces expression vector (pIJ10257) under the control of the strong constitutive ermE* promoter and cotransformed into S. albus along with the cosmid AB1091.…”

Quorum sensing, communication and cross-kingdom signalling in the bacterial world

“…The function of CarH is unknown but it is also not essential for antibiotic production (McGowan et al, 1996(McGowan et al, , 1997. Intriguingly, many Erwinia strains are carbapenem-negative but retain the car operon; antibiotic biosynthesis can be restored by introducing a plasmid-borne copy of carR as these strains appear to contain an inactive carR gene (Holden et al, 1998).…”