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

The introduction of extended-spectrum cephalosporins and ß-lactamase inhibitors has driven the evolution of extended-spectrum ß-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. The evolution of TEM ESBLs is facilitated by mutations that act as global suppressors of protein stability defects in that they allow the enzyme to absorb multiple amino acid changes despite incremental losses in stability associated with the substitutions. The best-studied example is the M182T substitution, which corrects protein stability defects and is commonly found in TEM ESBLs or inhibitor-resistant variants from clinical isolates. In this study, a genetic selection for second-site mutations that could partially restore function to a severely destabilized primary mutant enabled the identification of A184V, T265M, R275Q, and N276D, which are known to occur in TEM ESBLs from clinical isolates, as suppressors of TEM-1 protein stability defects. Further characterization demonstrated that these substitutions increased the thermal stability of TEM-1 and were able to correct the stability defects of two different sets of destabilizing mutations. The acquisition of compensatory global suppressors of stability costs associated with active-site mutations may be a common mechanism for the evolution of novel protein function.

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. An L201P substitution distant from the active site that enhanced ampicillin resistance levels and increased protein expression levels of the R244A TEM-1 mutant was identified. The L201P substitution also increases the ampicillin resistance levels and restores expression levels of a poorly expressed TEM-1 mutant with a core-disrupting substitution. In vitro thermal denaturation of purified protein indicated that the L201P mutation increases the T(m) value of the TEM-1 enzyme. The X-ray structure of the L201P TEM-1 mutant was determined to gain insight into the increase in enzyme stability. The proline substitution occurs at the N-terminus of an alpha-helix and may stabilize the enzyme by reducing the helix dipole, as well as by lowering the conformational entropy cost of folding due to the reduced number of conformations available in the unfolded state. Collectively, the data suggest that L201P promotes tolerance of some deleterious TEM-1 mutations by enhancing the protein stability of these mutants.

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). We report efficient detection of single chromosomal DSBs and rates of spontaneous breakage approximately 20- to 100-fold lower than predicted. In addition, we implicate DNA replication in the origin of spontaneous DSBs with the finding of fewer spontaneous DSBs in a mutant with altered DNA polymerase III. The data imply that spontaneous DSBs induce genomic changes and instability 20-100 times more potently than previously appreciated. Finally, FACS demonstrated two main cell fates after spontaneous DNA damage: viability with or without resumption of proliferation.

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. coli RecQ can promote the opposite: the net accumulation of BRIs. We report that cells lacking Ruv and UvrD BRI-resolution and -prevention proteins die and display failed chromosome segregation attributable to accumulation of BRIs. Death and segregation failure require RecA and RecF strand exchange proteins. FISH data show that replication is completed during chromosome-segregation failure/death of ruv uvrD recA(Ts) cells. Surprisingly, RecQ (and RecJ) promotes this death. The data imply that RecQ promotes the net accumulation of BRIs in vivo, indicating a second paradigm for the in vivo effect of RecQ-like proteins. The E. coli RecQ paradigm may provide a useful model for some human RecQ homologs.

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. Either recQ or recA null mutations, but not helD null or lexA3, partially rescued the synthetic lethality of the double topoisomerase III/IV mutant, indicating a role for topoisomerase III in recombination. We find a bias against deleting the gene encoding topoisomerase III in ruvC53 or DeltaruvABC backgrounds compared with the isogenic wild-type strains. The topoisomerase III RuvC double mutants that can be constructed are five- to 10-fold more sensitive to UV irradiation and mitomycin C treatment and are twofold less efficient in transduction efficiency than ruvC53 mutants. The overexpression of ruvABC allows the construction of the topoisomerase III/IV double mutant. These data are consistent with a role for topoisomerase III in disentangling recombination intermediates as an alternative to RuvABC to maintain the stability of the genome.