William A. Rosche

Isolation and Characterization of a Generalized Transducing Phage for Pseudomonas aeruginosa Strains PAO1 and PA14

A temperate, type IV pilus-dependent, double-stranded DNA bacteriophage named DMS3 was isolated from a clinical strain of Pseudomonas aeruginosa. A clear-plaque variant of this bacteriophage was isolated. DMS3 is capable of mediating generalized transduction within and between P. aeruginosa strains PA14 and PAO1, thus providing a useful tool for the genetic analysis of P. aeruginosa.

DNA polymerase III proofreading mutants enhance the expansion and deletion of triplet repeat sequences in Escherichia coli.

The influence of mutations in the 3′ to 5′ exonucleolytic proofreading ε-subunit of Escherichia coliDNA polymerase III on the genetic instabilities of the CGG·CCG and the CTG·CAG repeats that cause human hereditary neurological diseases was investigated. The dnaQ49 ts and themutD5 mutations destabilize the CGG·CCG repeats. The distributions of the deletion products indicate that slipped structures containing a small number of repeats in the loop mediate the deletion process. The CTG·CAG repeats were destabilized by thednaQ49 ts mutation by a process mediated by long hairpin loop structures (≥5 repeats). The mutD5 mutator strain stabilized the (CTG·CAG)175 tract, which contained two interruptions. Since the mutD5 mutator strain has a saturated mismatch repair system, the stabilization is probably an indirect effect of the nonfunctional mismatch repair system in these strains. Shorter uninterrupted tracts expand readily in themutD5 strain, presumably due to the greater stability of long CTG·CAG tracts (>100 repeats) in this strain. When parallel studies were conducted in minimal medium, where the mutD5strain is defective in exonucleolytic proofreading but has a functional MMR system, both CTG·CAG and CGG·CCG repeats were destabilized, showing that the proofreading activity is essential for maintaining the integrity of TRS tracts. Thus, we conclude that the expansion and deletion of triplet repeats are enhanced by mutations that reduce the fidelity of replication.

Genetic assays for measuring rates of (CAG) (CTG) repeat instability in Escherichia coli

Genetic selection assays were developed to measure rates of deletion of one or more (CAG).(CTG) repeats, or an entire repeat tract, in Escherichia coli. In-frame insertions of >or=25 repeats in the chloramphenicol acetyltransferase (CAT) gene of pBR325 resulted in a chloramphenicol-sensitive (Cm(s)) phenotype. When (CAG)25 comprised the leading template strand, deletion of one or more repeats resulted in a chloramphenicol resistant (Cm(r)) phenotype at a rate of 4 x 10(-2) revertants per cell per generation. The mutation rates for plasmids containing (CAG)43 or (CAG)79 decreased significantly. When (CTG)n comprised the leading template strand the Cm(r) mutation rates were 100-1000 lower than for the opposite orientation. As an initial application of this assay, the effects of mutations influencing mismatch repair and recombination were examined. The methyl directed mismatch repair system increased repeat stability only when (CTG)n comprised the leading template strand. Replication errors made with the opposite repeat orientation were apparently not recognized. For the (CAG)n leading strand orientation, mutation rates were reduced as much as 3000-fold in a recA- strain. In a second assay, out-of-frame mutation inserts underwent complete deletion at rates ranging from about 5 x 10(-9) to 1 x 10(-7) per cell per generation. These assays allow careful quantitation of triplet repeat instability in E. coli and provide a way to examine the effects of mutations in replication, repair, and recombination on repeat instability.

Strand misalignments lead to quasipalindrome correction

Quasipalindromes, or imperfect inverted repeats, undergo spontaneous mutation to more-perfect inverted repeats. These mutations have been observed in many organisms, ranging from bacteria to humans, where they are associated with mutations leading to disease. We determined the relative frequency of quasipalindromes and perfect palindromes in more than 100 sequenced prokaryotic genomes. In nearly all cases, perfect palindromes were relatively more frequent than quasipalindromes, suggesting that quasipalindrome correction is a general mechanism for mutation in prokaryotes.

DNA-directed mutations. Leading and lagging strand specificity.

ABSTRACT: The fidelity of replication has evolved to reproduce B‐form DNA accurately, while allowing a low frequency of mutation. The fidelity of replication can be compromised, however, by defined order sequence DNA (dosDNA) that can adopt unusual or non B‐DNA conformations. These alternative DNA conformations, including hairpins, cruciforms, triplex DNAs, and slipped‐strand structures, may affect enzyme‐template interactions that potentially lead to mutations. To analyze the effect of dosDNA elements on spontaneous mutagenesis, various mutational inserts containing inverted repeats or direct repeats were cloned in a plasmid containing a unidirectional origin of replication and a selectable marker for the mutation. This system allows for analysis of mutational events that are specific for the leading or lagging strands during DNA replication in Escherichia coli. Deletions between direct repeats, involving misalignment stabilized by DNA secondary structure, occurred preferentially on the lagging strand. Intermolecular strand switch events, correcting quasipalindromes to perfect inverted repeats, occurred preferentially during replication of the leading strand.

Instability of repeated DNAs during transformation in Escherichia coli

Escherichia coli has provided an important model system for understanding the molecular basis for genetic instabilities associated with repeated DNA. Changes in triplet repeat length during growth following transformation in E. coli have been used as a measure of repeat instability. However, very little is known about the molecular and biological changes that may occur on transformation. Since only a small proportion of viable cells become competent, uncertainty exists regarding the nature of these transformed cells. To establish whether the process of transformation can be inherently mutagenic for certain DNA sequences, we used a genetic assay in E. coli to compare the frequency of genetic instabilities associated with transformation with those occurring in plasmid maintained in E. coli. Our results indicate that, for certain DNA sequences, bacterial transformation can be highly mutagenic. The deletion frequency of a 106 bp perfect inverted repeat is increased by as much as a factor of 2 x 10(5) following transformation. The high frequency of instability was not observed when cells stably harboring plasmid were rendered competent. Thus, the process of transformation was required to observe the instability. Instabilities of (CAG).(CTG) repeats are also dramatically elevated upon transformation. The magnitude of the instability is dependent on the nature and length of the repeat. Differences in the methylation status of plasmid used for transformation and the methylation and restriction/modification systems present in the bacterial strain used must also be considered in repeat instability measurements. Moreover, different E. coli genetic backgrounds show different levels of instability during transformation.

Mechanisms of Mutation in Nondividing Cells: Insights from the Study of Adaptive Mutation in Escherichia colia

ABSTRACT: When populations of cells are subjected to nonlethal selection, mutations arise in the absence of cell division, a phenomenon that has been called “adaptive mutation.” In a strain of Escherichia coli that cannot metabolize lactose (Lac−) but that reverts to lactose utilization (Lac+) when lactose is its sole energy and carbon source, the mutational process consists of two components. (1) A highly efficient, recombination‐dependent mechanism giving rise to mutations on the F′ episome that carries the Lac− allele; and (2) a less efficient, unknown mechanism giving rise to mutations elsewhere in the genome. Both selected and nonselected mutations arise in the Lac− population, but nonselected mutations are enriched in Lac+ mutants, suggesting that some Lac+ cells have passed though a transient period of increased mutation. These results have several evolutionary implications. (1) DNA synthesis initiated by recombination could be an important source of spontaneous mutation, particularly in cells that are not undergoing genomic replication. (2) The highly active mutational mechanism on the episome could be important in the horizontal transfer of variant alleles among species that carry and exchange conjugal plasmids. (3) A subpopulation of cells in a state of transient mutation could be a source of multiple variant alleles and could provide a mechanism for rapid adaptive evolution under adverse conditions.

(CAG)*(CTG) repeats associated with neurodegenerative diseases are stable in the Escherichia coli chromosome.

(CAG)n·(CTG)n expansion is associated with many neurodegenerative diseases. Repeat instability has been extensively studied in bacterial plasmids, where repeats undergo deletion at high rates. We report an assay for (CAG)n·(CTG)n deletion from the chloramphenicol acetyltransferase gene integrated into the Escherichia coli chromosome. In strain AB1157, deletion rates for 25–60 (CAG)·(CTG) repeats integrated in the chromosome ranged from 6.88 × 10–9 to 1.33 × 10–10, or ∼6,300 to 660,000-fold lower than in plasmid pBR325. In contrast to the situation in plasmids, deletions occur at a higher rate when (CTG)43, rather than (CAG)43, comprised the leading template strand, and complete rather than partial deletions were the predominant mutation observed. Repeats were also stable on long term growth following multiple passages through exponential and stationary phase. Mutations in priA and recG increased or decreased deletion rates, but repeats were still greatly stabilized in the chromosome. The remarkable stability of (CAG)n·(CTG)n repeats in the E. coli chromosome may result from the differences in the mechanisms for replication or the probability for recombination afforded by a high plasmid copy number. The integration of (CAG)n·(CTG)n repeats into the chromosome provides a model system in which the inherent stability of these repeats reflects that in the human genome more closely.

Adaptive mutation inEscherichia coli strain FC40

Mutations can arise in static populations of cells that are subjected to nonlethal selective pressure, a phenomenon that has been called ‘adaptive mutation’. This phenomenon has been extensively studied in FC40, a strain ofEscherichia coli that cannot metabolize lactose (Lac−) but that reverts to lactose utilization (Lac+) when lactose is its sole energy and carbon source. The adaptive Lac+ mutations arise by two mutational processes: a recombination-dependent process that is highly active on the episome carrying the Lac− allele, and an unknown process that affects the whole genome. Most of the Lac+ mutations are due to the first process, which also produces nonselected mutations on the F′ episome. However, about 10% of the Lac+ mutations arise in a subpopulation of cells that experience a period of transient hypermutation. Although minor contributors to any one type of mutation, the hypermutators account for nearly all cases of multiple mutations. The evolutionary implications of these results are: (i) DNA synthesis associated with recombination may be an important source of spontaneous mutation, particularly in cells that are not actively growing; (ii) the efficient mutational mechanism that occurs on the episome could result in the horizontal transfer of new alleles among species that carry and exchange conjugal plasmids; and (iii) a subpopulation of transient hypermutators could be a source of multiple mutations that would allow for rapid adaptive evolution under adverse conditions.