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

Abstract

SummaryMembers of two genera of Gram-negative bacteria, Serratia and Erwinia, produce a ␤-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 LuxRtype 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 ␤-lactam resistance genes have evolved in concert with the biosynthetic genes to ensure 'self-resistance' in the Erwinia carbapenem producer.

Exploitation of a β-lactamase reporter gene fusion in the carbapenem antibiotic production operon to study adaptive evolution in Erwinia carotovora

“…The presence of carFG does not confer cross-resistance to related b-lactam antibiotics such as imipenem (McGowan et al, 1997). CarF and CarG both contain predicted signal peptides that direct their export to the periplasm (McGowan et al, 1997). The carH gene product has no known function, but is also exported to the periplasm (McGowan et al, 1997).…”

“…The first three genes, carABC, are essential for Car biosynthesis (McGowan et al, 1997). carDE are also involved in Car biosynthesis, but are not essential, because when they are disrupted by in-frame deletions, Car is still produced, though at a reduced level.…”

“…carFG are involved in Car intrinsic resistance via an uncharacterized, novel mechanism (McGowan et al, 1997). The presence of carFG does not confer cross-resistance to related b-lactam antibiotics such as imipenem (McGowan et al, 1997). CarF and CarG both contain predicted signal peptides that direct their export to the periplasm (McGowan et al, 1997).…”

“…CarF and CarG both contain predicted signal peptides that direct their export to the periplasm (McGowan et al, 1997). The carH gene product has no known function, but is also exported to the periplasm (McGowan et al, 1997).…”

“…carDE are also involved in Car biosynthesis, but are not essential, because when they are disrupted by in-frame deletions, Car is still produced, though at a reduced level. carFG are involved in Car intrinsic resistance via an uncharacterized, novel mechanism (McGowan et al, 1997). The presence of carFG does not confer cross-resistance to related b-lactam antibiotics such as imipenem (McGowan et al, 1997).…”