Multiple global suppressors of protein stability defects facilitate the evolution of extended-spectrum TEM β-lactamases.

The introduction of extended-spectrum cephalosporins and β-lactamase inhibitors has driven the evolution of extended-spectrum β-lactamases (ESBLs) that possess the ability to hydrolyze these drugs. The evolved TEM ESBLs from clinical isolates of bacteria often contain substitutions that occur in the active site and alter the catalytic properties of the enzyme to provide an increased hydrolysis of extended-spectrum cephalosporins or an increased resistance to inhibitors. These active-site substitutions often result in a cost in the form of reduced enzyme stability.

Genetic and structural characterization of an L201P global suppressor substitution in TEM-1 beta-lactamase.

TEM-1 beta-lactamase confers bacterial resistance to penicillin antibiotics and has acquired mutations that permit the enzyme to hydrolyze extended-spectrum cephalosporins or to avoid inactivation by beta-lactamase inhibitors. However, many of these substitutions have been shown to reduce activity against penicillin antibiotics and/or result in loss of stability for the enzyme. In order to gain more information concerning the tradeoffs associated with active site substitutions, a genetic selection was used to find second site mutations that partially restore ampicillin resistance levels conferred by an R244A active site TEM-1 beta-lactamase mutant.

Spontaneous DNA breakage in single living Escherichia coli cells.

Spontaneous DNA breakage is predicted to be a frequent, inevitable consequence of DNA replication and is thought to underlie much of the genomic change that fuels cancer and evolution. Despite its importance, there has been little direct measurement of the amounts, types, sources and fates of spontaneous DNA lesions in living cells. We present a direct, sensitive flow cytometric assay in single living Escherichia coli cells for DNA lesions capable of inducing the SOS DNA damage response, and we report its use in quantification of spontaneous DNA double-strand breaks (DSBs).

RecQ promotes toxic recombination in cells lacking recombination intermediate-removal proteins.

The RecQ-helicase family is widespread, is highly conserved, and includes human orthologs that suppress genomic instability and cancer. In vivo, some RecQ homologs promote reduction of steady-state levels of bimolecular recombination intermediates (BRIs), which block chromosome segregation if not resolved. We find that, in vivo, E.

A role for topoisomerase III in a recombination pathway alternative to RuvABC.

The physiological role of topoisomerase III is unclear for any organism. We show here that the removal of topoisomerase III in temperature sensitive topoisomerase IV mutants in Escherichia coli results in inviability at the permissive temperature. The removal of topoisomerase III has no effect on the accumulation of catenated intermediates of DNA replication, even when topoisomerase IV activity is removed.