Summary auto-generated
This study investigates carbapenem antibiotic production in Erwinia carotovora, revealing that cryptic (silent) carbapenem biosynthetic genes are widespread among strains. While only a few natural isolates produce the carbapenem antibiotic 1-carbapen-2-em-3-carboxylic acid (Car), introduction of the transcriptional regulator carR from the producing strain GS101 activates antibiotic production in non-producing strains. The researchers surveyed over 80 Erwinia strains and found that 16% of E. carotovora subsp. carotovora strains contain trans-activatable cryptic carbapenem genes. Analysis revealed these strains possess functional biosynthetic gene clusters but harbor defective carR homologues. Carbapenem production is controlled by quorum sensing through the signaling molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), which activates CarR-mediated transcription when threshold concentrations accumulate. Southern blot hybridization confirmed that cryptic strains contain both biosynthetic and regulatory genes. The carR homologue in non-producing strain SCRI 193 showed 94% identity to GS101's carR but contained 14 amino acid substitutions. Multicopy expression of the SCRI 193 carR variant restored antibiotic production, suggesting the regulatory defect involves a leaky mutation in carR rather than complete gene deletion.
Key findings
- Carbapenem biosynthetic genes are cryptic and widespread in E. carotovora strains; only 16% of surveyed Ecc strains produce antibiotic constitutively, but 16% contain trans-activatable genes that are activated by introduction of functional carR.
- Carbapenem production is controlled by quorum sensing through OHHL accumulation and the transcriptional activator CarR; the CarR-OHHL complex activates biosynthetic gene transcription above threshold inducer concentrations.
- Defects in carR homologues, rather than deletions of biosynthetic genes, cause the cryptic phenotype; the SCRI 193 carR variant contains 14 amino acid substitutions and produces carbapenems when supplied in multiple copies.
- All E. carotovora strains tested produce OHHL and contain carR and car biosynthetic gene homologues detected by Southern blotting, indicating carbapenem production capacity is a species-wide trait.
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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 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.