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S M Rosenberg

Publications and source records attributed to S M Rosenberg.

At least 19 recordsLinked to original sources
PubMed2001

The TGV transgenic vectors for single-copy gene expression from the Escherichia coli chromosome.

Plasmid-based cloning and expression of genes in Escherichia coli can have several problems: plasmid destabilization; toxicity of gene products; inability to achieve complete repression of gene expression; non-physiological overexpression of the cloned gene; titration of regulatory proteins; and the requirement for antibiotic selection. We describe a simple system for cloning and expression of genes in single copy in the E. coli chromosome, using a non-antibiotic selection for transgene insertion. The transgene is inserted into a vector containing homology to the chromosomal region flanking the attachment site for phage lambda. This vector is then linearized and introduced into a recombination-proficient E. coli strain carrying a temperature-sensitive lambda prophage. Selection for replacement of the prophage with the transgene is performed at high temperature. Once in the chromosome, transgenes can be moved into other lysogenic E. coli strains using standard phage-mediated transduction techniques, selecting against a resident prophage. Additional vector constructs provide an arabinose-inducible promoter (P(BAD)), P(BAD) plus a translation-initiation sequence, and optional chloramphenicol-, tetracycline-, or kanamycin-resistance cassettes. These Transgenic E. coli Vectors (TGV) allow drug-free, single-copy expression of genes from the E. coli chromosome, and are useful for genetic studies of gene function.

Attachment Sites, Microbiological↗
PubMed2001

Stationary-phase mutation in the bacterial chromosome: recombination protein and DNA polymerase IV dependence.

Several microbial systems have been shown to yield advantageous mutations in slowly growing or nongrowing cultures. In one assay system, the stationary-phase mutation mechanism differs from growth-dependent mutation, demonstrating that the two are different processes. This system assays reversion of a lac frameshift allele on an F' plasmid in Escherichia coli. The stationary-phase mutation mechanism at lac requires recombination proteins of the RecBCD double-strand-break repair system and the inducible error-prone DNA polymerase IV, and the mutations are mostly -1 deletions in small mononucleotide repeats. This mutation mechanism is proposed to occur by DNA polymerase errors made during replication primed by recombinational double-strand-break repair. It has been suggested that this mechanism is confined to the F plasmid. However, the cells that acquire the adaptive mutations show hypermutation of unrelated chromosomal genes, suggesting that chromosomal sites also might experience recombination protein-dependent stationary-phase mutation. Here we test directly whether the stationary-phase mutations in the bacterial chromosome also occur via a recombination protein- and pol IV-dependent mechanism. We describe an assay for chromosomal mutation in cells carrying the F' lac. We show that the chromosomal mutation is recombination protein- and pol IV-dependent and also is associated with general hypermutation. The data indicate that, at least in these male cells, recombination protein-dependent stationary-phase mutation is a mechanism of general inducible genetic change capable of affecting genes in the bacterial chromosome.

Alleles↗
PubMed2001

SOS mutator DNA polymerase IV functions in adaptive mutation and not adaptive amplification.

Adaptive point mutation and amplification are induced responses to environmental stress, promoting genetic changes that can enhance survival. A specialized adaptive mutation mechanism has been documented in one Escherichia coli assay, but its enzymatic basis remained unclear. We report that the SOS-inducible, error-prone DNA polymerase (pol) IV, encoded by dinB, is required for adaptive point mutation in the E. coli lac operon. A nonpolar dinB mutation reduces adaptive mutation frequencies by 85% but does not affect adaptive amplification, growth-dependent mutation, or survival after oxidative or UV damage. We show that pol IV, together with the major replicase, pol III, can account for all adaptive point mutations at lac. The results identify a role for pol IV in inducible genetic change.

Adaptation, Physiological↗
PubMed2001

Adaptive mutations, mutator DNA polymerases and genetic change strategies of pathogens.

"Adaptive" or "stationary-phase" mutation is a collection of stress responses promoting mutations, some of which are advantageous. In 2000 and 2001, in Escherichia coli, adaptive gene amplification was documented, and a parallel adaptive point-mutation mechanism was linked to the error-prone DNA polymerase, DinB (pol IV). We suggest that DinB homologues may contribute to adaptive strategies of pathogens, including antigenic variation.

Adaptation, Physiological↗
PubMed2001

Evolving responsively: adaptive mutation.

A basic principle of genetics is that the likelihood that a particular mutation occurs is independent of its phenotypic consequences. The concept of adaptive mutation seemed to challenge this principle with the discoveries of mutations stimulated by stress, some of which allow adaptation to the stress. The emerging mechanisms of adaptive genetic change cast evolution, development and heredity into a new perspective, indicating new models for the genetic changes that fuel these processes.

Adaptation, Physiological↗
PubMed2000

Adaptive amplification: an inducible chromosomal instability mechanism.

Adaptive mutation is an induced response to environmental stress in which mutation rates rise, producing permanent genetic changes that can adapt cells to stress. This contrasts with neo-Darwinian views of genetic change rates blind to environmental conditions. DNA amplification is a flexible, reversible genomic change that has long been postulated to be adaptive. We report the discovery of adaptive amplification at the lac operon in Escherichia coli. Additionally, we find that adaptive amplification is separate from, and does not lead to, adaptive point mutation. This contradicts a prevailing alternative hypothesis whereby adaptive mutation is normal mutability in amplified DNA. Instead, adaptive mutation and amplification are parallel routes of inducible genetic instability allowing rapid evolution under stress, and escape from growth inhibition.

Base Pair Mismatch↗
PubMed2000

The SOS response regulates adaptive mutation.

Upon starvation some Escherichia coli cells undergo a transient, genome-wide hypermutation (called adaptive mutation) that is recombination-dependent and appears to be a response to a stressful environment. Adaptive mutation may reflect an inducible mechanism that generates genetic variability in times of stress. Previously, however, the regulatory components and signal transduction pathways controlling adaptive mutation were unknown. Here we show that adaptive mutation is regulated by the SOS response, a complex, graded response to DNA damage that includes induction of gene products blocking cell division and promoting mutation, recombination, and DNA repair. We find that SOS-induced levels of proteins other than RecA are needed for adaptive mutation. We report a requirement of RecF for efficient adaptive mutation and provide evidence that the role of RecF in mutation is to allow SOS induction. We also report the discovery of an SOS-controlled inhibitor of adaptive mutation, PsiB. These results indicate that adaptive mutation is a tightly regulated response, controlled both positively and negatively by the SOS system.

Adaptation, Physiological↗
PubMed2000

Evidence that stationary-phase hypermutation in the Escherichia coli chromosome is promoted by recombination.

Adaptive (or stationary-phase) mutation is a group of phenomena in which mutations appear to occur more often when selected than when not. They may represent cellular responses to the environment in which the genome is altered to allow survival. The best-characterized assay system and mechanism is reversion of a lac allele on an F' sex plasmid in Escherichia coli, in which the stationary-phase mutability requires homologous recombination functions. A key issue has concerned whether the recombination-dependent mutation mechanism is F' specific or is general. Hypermutation of chromosomal genes occurs in association with adaptive Lac(+) mutation. Here we present evidence that the chromosomal hypermutation is promoted by recombination. Hyperrecombinagenic recD cells show elevated chromosomal hypermutation. Further, recG mutation, which promotes accumulation of recombination intermediates proposed to prime replication and mutation, also stimulates chromosomal hypermutation. The coincident mutations at lac (on the F') and chromosomal genes behave as independent events, whereas coincident mutations at lac and other F-linked sites do not. This implies that transient covalent linkage of F' and chromosomal DNA (Hfr formation) does not underlie chromosomal mutation. The data suggest that recombinational stationary-phase mutation occurs in the bacterial chromosome and thus can be a general strategy for programmed genetic change.

Bacterial Proteins↗
PubMed2000

radC102 of Escherichia coli is an allele of recG.

The radC102 mutation causes mild UV and X-ray sensitivity and was mapped previously to near pyrE and recG at 82 min on the Escherichia coli chromosome (I. Felzenszwalb, N. J. Sargentini, and K. C. Smith, Radiat. Res. 97:615-625, 1984). We report that radC102 has two striking phenotypes characteristic of recG mutations. First, it causes dramatically increased RecA-dependent mutation in a stationary-phase mutation assay. Second, it causes extreme UV sensitivity in combination with ruv mutations affecting the RuvABC Holliday junction resolution system. DNA sequencing of the radC and recG genes in radC102 strains revealed that the radC102 mutation creates a stop codon in recG that is predicted to truncate the RecG protein at 410 of 603 amino acids. A low-copy-number plasmid carrying the radC(+) gene did not affect the UV sensitivity of a wild-type strain, a radC102 strain, or a recG258::Tn10mini-kan strain. We conclude that radC102 is an allele of recG and that the function of the RadC protein remains to be determined.

Alleles↗
PubMed1999

Double-strand-break repair recombination in Escherichia coli: physical evidence for a DNA replication mechanism in vivo.

DNA double-strand-break repair (DSBR) is, in many organisms, accomplished by homologous recombination. In Escherichia coli DSBR was thought to result from breakage and reunion of parental DNA molecules, assisted by known endonucleases, the Holliday junction resolvases. Under special circumstances, for example, SOS induction, recombination forks were proposed to initiate replication. We provide physical evidence that this is a major alternative mechanism in which replication copies information from one chromosome to another generating recombinant chromosomes in normal cells in vivo. This alternative mechanism can occur independently of known Holliday junction cleaving proteins, requires DNA polymerase III, and produces recombined DNA molecules that carry newly replicated DNA. The replicational mechanism underlies about half the recombination of linear DNA in E. coli; the other half occurs by breakage and reunion, which we show requires resolvases, and is replication-independent. The data also indicate that accumulation of recombination intermediates promotes replication dramatically.

Bacterial Proteins↗
PubMed1999

Mechanisms of genome-wide hypermutation in stationary phase.

Stationary-phase mutation (a subset of which was previously called adaptive mutation) occurs in apparently nondividing, stationary-phase cells exposed to a nonlethal genetic selection. In one experimental system, stationary-phase reversion of an Escherichia coli F'-borne lac frameshift mutation occurs by a novel molecular mechanism that requires homologous recombination functions of the RecBCD system. Chromosomal mutations at multiple loci are detected more frequently in Lac+ stationary-phase revertants than in cells that were also exposed to selection but did not become Lac+. Thus, mutating cells represent a subpopulation that experiences hypermutation throughout the genome. This paper summarizes current knowledge regarding stationary-phase mutation in the lac system. Hypotheses for the mechanism of chromosomal hypermutation are discussed, and data are presented that exclude one hypothetical mechanism in which chromosomal mutations result from Hfr formation.

DNA Repair↗
PubMed1999

Mismatch repair is diminished during stationary-phase mutation.

This paper is an invited Response to a recent Commentary [P.L. Foster, Rev. Mut. Res. 436 (1999) 179-184] entitled "Are adaptive mutations due to a decline in mismatch repair? The evidence is lacking". The Commentary argues that no evidence exists supporting the idea that mismatch repair is limiting specifically during stationary-phase mutation. A primary concern of the author is to question the method that we used previously to measure growth-dependent mutation. In this method, mutation rates are calculated using counts of mutant colonies taken at times when those colonies arise, rather than at a predetermined, fixed time. Here we show further data that illustrate why this must be done to ensure accurate mutation measurements. Such accuracy was necessary for our published determination that mismatch repair proteins are not limiting during growth-dependent mutation, but become so during stationary-phase mutation. We review the evidence supporting the idea that stationary-phase reversion of a lac frameshift mutation occurs in an environment of decreased mismatch repair capacity. Those data are substantial. The data presented in the Commentary, in apparent contradiction to this idea, do not justify the conclusion presented there.

Adenosine Triphosphatases↗
PubMed1998

Transient and heritable mutators in adaptive evolution in the lab and in nature.

Major advances in understanding the molecular mechanism of recombination-dependent stationary-phase mutation in Escherichia coli occurred this past year. These advances are reviewed here, and we also present new evidence that the mutagenic state responsible is transient. We find that most stationary-phase mutants do not possess a heritable stationary-phase mutator phenotype, although a small proportion of heritable mutators was found previously. We outline similarities between this well-studied system and several recent examples of adaptive evolution associated with heritable mutator phenotype in a similarly small proportion of survivors of selection in nature and in the lab. We suggest the following: (1) Transient mutator states may also be a predominant source of adaptive mutations in these latter systems, the heritable mutators being a minority (Rosenberg 1997); (2) heritable mutators may sometimes be a product of, rather than the cause of, hypermutation that gives rise to adaptive mutations.

Adaptation, Biological↗
PubMed1998

Mismatch repair in Escherichia coli cells lacking single-strand exonucleases ExoI, ExoVII, and RecJ.

In vitro, the methyl-directed mismatch repair system of Escherichia coli requires the single-strand exonuclease activity of either ExoI, ExoVII, or RecJ and possibly a fourth, unknown single-strand exonuclease. We have created the first precise null mutations in genes encoding ExoI and ExoVII and find that cells lacking these nucleases and RecJ perform mismatch repair in vivo normally such that triple-null mutants display normal mutation rates. ExoI, ExoVII, and RecJ are either redundant with another function(s) or are unnecessary for mismatch repair in vivo.

Bacterial Proteins↗
PubMed1997

Mismatch repair protein MutL becomes limiting during stationary-phase mutation.

Postsynthesis mismatch repair is an important contributor to mutation avoidance and genomic stability in bacteria, yeast, and humans. Regulation of its activity would allow organisms to regulate their ability to evolve. That mismatch repair might be down-regulated in stationary-phase Escherichia coli was suggested by the sequence spectrum of some stationary-phase ("adaptive") mutations and by the observations that MutS and MutH levels decline during stationary phase. We report that overproduction of MutL inhibits mutation in stationary phase but not during growth. MutS overproduction has no such effect, and MutL overproduction does not prevent stationary-phase decline of either MutS or MutH. These results imply that MutS and MutH decline to levels appropriate for the decreased DNA synthesis in stationary phase, whereas functional MutL is limiting for mismatch repair specifically during stationary phase. Modulation of mutation rate and genetic stability in response to environmental or developmental cues, such as stationary phase and stress, could be important in evolution, development, microbial pathogenicity, and the origins of cancer.

Adenosine Triphosphatases↗
PubMed1997

Genome-wide hypermutation in a subpopulation of stationary-phase cells underlies recombination-dependent adaptive mutation.

Stationary-phase mutation in microbes can produce selected ('adaptive') mutants preferentially. In one system, this occurs via a distinct, recombination-dependent mechanism. Two points of controversy have surrounded these adaptive reversions of an Escherichia coli lac mutation. First, are the mutations directed preferentially to the selected gene in a Lamarckian manner? Second, is the adaptive mutation mechanism specific to the F plasmid replicon carrying lac? We report that lac adaptive mutations are associated with hypermutation in unselected genes, in all replicons in the cell. The associated mutations have a similar sequence spectrum to the adaptive reversions. Thus, the adaptive mutagenesis mechanism is not directed to the lac genes, in a Lamarckian manner, nor to the F' replicon carrying lac. Hypermutation was not found in non-revertants exposed to selection. Therefore, the genome-wide hypermutation underlying adaptive mutation occurs in a differentiated subpopulation. The existence of mutable subpopulations in non-growing cells is important in bacterial evolution and could be relevant to the somatic mutations that give rise to cancers in multicellular organisms.

Adaptation, Biological↗