Inhibition of the multiple antibiotic resistance (mar) operon in Escherichia coli by antisense DNA analogs.

The multiple antibiotic resistance operon (marORAB) in Escherichia coli controls intrinsic susceptibility and resistance to multiple, structurally different antibiotics and other noxious agents. A plasmid construct with marA cloned in the antisense direction reduced LacZ expression from a constitutively expressed marA::lacZ translational fusion and inhibited the induced expression of LacZ in cells bearing the wild-type repressed fusion. The marA antisense construction also decreased the multiple antibiotic resistance of a Mar mutant.

Construction of derivatives of the F plasmid pOX-tra715: characterization of traY and traD mutants that can be complemented in trans.

A derivative of the F plasmid, pOX38-tra715, expresses the entire F tra operon from a foreign promoter (PT7) derived from phage T7. A series of plasmids related to pOX38-tra715 were constructed which carry either deletion mutations or point mutations in traY. When the PT7 promoter was induced, these plasmids expressed the F pilus but were transfer deficient unless TraY was supplied In trans from compatible plasmids.

Identification for mar mutants among quinolone-resistant clinical isolates of Escherichia coli.

Quinolone-resistant clinical Escherichia coli isolates were examined for mutations in the marRAB operon of the multiple antibiotic resistance (mar) locus. Among 23 strains evaluated, 8 were chosen for further study: 3 that showed relatively high levels of uninduced, i.e.

Characterization of traX, the F plasmid locus required for acetylation of F-pilin subunits.

Acetylation of F-pilin subunits has previously been shown to depend upon expression of the F plasmid transfer operon gene traX. To assess the requirement for pilin acetylation in conjugative transfer of F, we constructed traX::kan insertion mutations and crossed them onto the transmissible F derivative pOX38. Under standard conditions, the function of traX seemed to be dispensable.

The pKSM710 vector cassette provides tightly regulated lac and T7lac promoters and strategies for manipulating N-terminal protein sequences.

We describe a set of plasmid vectors that are very useful for cloning, expressing, mutagenizing, deleting, and sequencing DNA fragments. A strategy for using one (pKSM717) to obtain mutant protein products that contain deletions of N-terminal amino acids is also presented. Desirable sequences were first combined in plasmid pKSM710 in a manner that facilitates construction of similar vectors carrying alternative selectable markers or replication origins: a cassette that includes LacI-regulated T7 (T7lac) and lacUV5 promoters, a multiple cloning site (MCS)/lacZ alpha sequence, a set of transcription terminators (T phi, rrnBT1, rrnBT2, and Tfd), and an fd origin of replication can be moved as a single unit.