Biomedical subjects

John R Roth

Publications and source records attributed to John R Roth.

27 records · Page 2Linked to original sources

PubMed2004

A pH-sensitive function and phenotype: evidence that EutH facilitates diffusion of uncharged ethanolamine in Salmonella enterica.

The eutH gene is part of an operon that allows Salmonella enterica to use ethanolamine as a sole source of nitrogen, carbon, and energy. Although the sequence of EutH suggests a role in transport, eutH mutants use ethanolamine normally under standard conditions (pH 7.0). These mutants fail to use ethanolamine at a low pH. Evidence is presented that protonated ethanolamine (Eth0) does not enter cells, while uncharged ethanolamine (Eth0) diffuses freely across the membrane. The external concentration of Eth0 varies with the pH (pK=9.5). At pH 7.0, the standard ethanolamine concentration (41 mM) provides enough Eth0 for an influx rate that can support growth with or without EutH. When a lowered pH and/or ethanolamine concentration reduced the Eth0 concentration below 25 microM, EutH was needed to facilitate diffusion. EutH+ cells grew normally at Eth0 concentrations above 3 microM, close to the Km (9 microM) of the first degradative enzyme, ethanolamine ammonia lyase. It is suggested that EutH facilitates diffusion of Eth0. As predicted for a transporter, EutH contributed to the toxicity of ethanolamine seen under some conditions; furthermore, fusion of EutH to fluorescent Yfp protein provided evidence that EutH is a membrane protein.

Bacterial Proteins↗

PubMed2004

Evidence that a B12-adenosyl transferase is encoded within the ethanolamine operon of Salmonella enterica.

Adenosylcobalamin (Ado-B12) is both the cofactor and inducer of ethanolamine ammonia lyase (EA-lyase), a catabolic enzyme for ethanolamine. De novo synthesis of Ado-B12 by Salmonella enterica occurs only under anaerobic conditions. Therefore, aerobic growth on ethanolamine requires import of Ado-B12 or a precursor (CN-B12 or OH-B12) that can be adenosylated internally. Several known enzymes adenosylate corrinoids. The CobA enzyme transfers adenosine from ATP to a biosynthetic intermediate in de novo B12 synthesis and to imported CN-B12, OH-B12, or Cbi (a B12 precursor). The PduO adenosyl transferase is encoded in an operon (pdu) for cobalamin-dependent propanediol degradation and is induced by propanediol. Evidence is presented here that a third transferase (EutT) is encoded within the operon for ethanolamine utilization (eut). Surprisingly, these three transferases share no apparent sequence similarity. CobA produces sufficient Ado-B12 to initiate eut operon induction and to serve as a cofactor for EA-lyase when B12 levels are high. Once the eut operon is induced, the EutT transferase supplies more Ado-B12 during the period of high demand. Another protein encoded in the operon (EutA) protects EA-lyase from inhibition by CN-B12 but does so without adenosylation of this corrinoid.

Alkyl and Aryl Transferases↗

PubMed2003

Adaptive mutation: general mutagenesis is not a programmed response to stress but results from rare coamplification of dinB with lac.

In a particular genetic system, selection stimulates reversion of a lac mutation and causes genome-wide mutagenesis (adaptive mutation). Selection allows rare plated cells with a duplication of the leaky lac allele to initiate clones within which further lac amplification improves growth rate. Growth and amplification add mutational targets to each clone and thereby increase the likelihood of reversion. We suggest that general mutagenesis occurs only in clones whose lac amplification includes the nearby dinB+ gene (for error-prone DNA polymerase IV). Thus mutagenesis is not a programmed response to stress but a side effect of amplification in a few clones; it is not central to the effect of selection on reversion.

Bacterial Proteins↗

PubMed2003

Regulating general mutation rates: examination of the hypermutable state model for Cairnsian adaptive mutation.

In the lac adaptive mutation system of Cairns, selected mutant colonies but not unselected mutant types appear to arise from a nongrowing population of Escherichia coli. The general mutagenesis suffered by the selected mutants has been interpreted as support for the idea that E. coli possesses an evolved (and therefore beneficial) mechanism that increases the mutation rate in response to stress (the hypermutable state model, HSM). This mechanism is proposed to allow faster genetic adaptation to stressful conditions and to explain why mutations appear directed to useful sites. Analysis of the HSM reveals that it requires implausibly intense mutagenesis (10(5) times the unselected rate) and even then cannot account for the behavior of the Cairns system. The assumptions of the HSM predict that selected revertants will carry an average of eight deleterious null mutations and thus seem unlikely to be successful in long-term evolution. The experimentally observed 35-fold increase in the level of general mutagenesis cannot account for even one Lac(+) revertant from a mutagenized subpopulation of 10(5) cells (the number proposed to enter the hypermutable state). We conclude that temporary general mutagenesis during stress is unlikely to provide a long-term selective advantage in this or any similar genetic system.

Adaptation, Biological↗

PubMed2003

Formation of an F' plasmid by recombination between imperfectly repeated chromosomal Rep sequences: a closer look at an old friend (F'(128) pro lac).

Plasmid F'(128) was formed by an exchange between chromosomal Rep sequences that placed lac near dinB between many pairs of Rep sequences. Plasmid F'(128) is critical for selection-enhanced lac reversion (adaptive mutation), which requires prior lac amplification. The structure of F'(128) supports the idea that amplification is initiated by Rep-Rep recombination and that general mutagenesis requires coamplification of dinB (error-prone polymerase) with lac.

Adenosine Triphosphatases↗

PubMed2002

Amplification-mutagenesis: evidence that "directed" adaptive mutation and general hypermutability result from growth with a selected gene amplification.

When a particular lac mutant of Escherichia coli starves in the presence of lactose, nongrowing cells appear to direct mutations preferentially to sites that allow growth (adaptive mutation). This observation suggested that growth limitation stimulates mutability. Evidence is provided here that this behavior is actually caused by a standard Darwinian process in which natural selection acts in three sequential steps. First, growth limitation favors growth of a subpopulation with an amplification of the mutant lac gene; next, it favors cells with a lac(+) revertant allele within the amplified array. Finally, it favors loss of mutant copies until a stable haploid lac(+) revertant arises and overgrows the colony. By increasing the lac copy number, selection enhances the likelihood of reversion within each developing clone. This sequence of events appears to direct mutations to useful sites. General mutagenesis is a side-effect of growth with an amplification (SOS induction). The F' plasmid, which carries lac, contributes by stimulating gene duplication and amplification. Selective stress has no direct effect on mutation rate or target specificity, but acts to favor a succession of cell types with progressively improved growth on lactose. The sequence of events--amplification, mutation, segregation--may help to explain both the origins of some cancers and the evolution of new genes under selection.

Electrophoresis, Agar Gel↗

PubMed2002

The effect of genomic position on reversion of a lac frameshift mutation (lacIZ33) during non-lethal selection (adaptive mutation).

In a system described by Cairns and Foster, starvation of a particular leaky lac mutant (lacIZ33) in the presence of lactose appears to direct mutation in non-growing cells to sites that allow growth (adaptive mutation). This behaviour requires that the lac operon be located on an F' plasmid. This position effect was investigated by placing the mutant lac operon at many sites in the genome of Salmonella enterica (Typhimurium; LT2) and testing reversion behaviour. Genomic position did not affect reversion during non-selective growth. When lac was at any of 550 chromosomal sites, starvation caused little or no enhancement of reversion. In the 28 strains with the lac on Salmonella's conjugative plasmid (pSLT), selection enhanced reversion strongly, just as seen for strains with lac on an F' plasmid. In 46 strains, the lac operon was inserted within a small chromosomal duplication, and selection stimulated RecA-dependent partial reversion by simple amplification (about 8x) of the mutant lac region. The position of lac on a conjugative plasmid is important to reversion because it allows more frequent gene duplication and amplification. These events are central to growth and reversion under selection because they increase the number of replicating lac alleles within each developing revertant clone.

Adaptation, Biological↗

PubMed2002

Evidence that selected amplification of a bacterial lac frameshift allele stimulates Lac(+) reversion (adaptive mutation) with or without general hypermutability.

In the genetic system of Cairns and Foster, a nongrowing population of an E. coli lac frameshift mutant appears to specifically accumulate Lac(+) revertants when starved on medium including lactose (adaptive mutation). This behavior has been attributed to stress-induced general mutagenesis in a subpopulation of starved cells (the hypermutable state model). We have suggested that, on the contrary, stress has no direct effect on mutability but favors only growth of cells that amplify their leaky mutant lac region (the amplification mutagenesis model). Selection enhances reversion primarily by increasing the mutant lac copy number within each developing clone on the selection plate. The observed general mutagenesis is attributed to a side effect of growth with an amplification-induction of SOS by DNA fragments released from a tandem array of lac copies. Here we show that the S. enterica version of the Cairns system shows SOS-dependent general mutagenesis and behaves in every way like the original E. coli system. In both systems, lac revertants are mutagenized during selection. Eliminating the 35-fold increase in mutation rate reduces revertant number only 2- to 4-fold. This discrepancy is due to continued growth of amplification cells until some clones manage to revert without mutagenesis solely by increasing their lac copy number. Reversion in the absence of mutagenesis is still dependent on RecA function, as expected if it depends on lac amplification (a recombination-dependent process). These observations support the amplification mutagenesis model.

Bacterial Proteins↗

PubMed2002

Effect of chromosome location on bacterial mutation rates.

In previous comparisons of enterobacterial sequences, synonymous substitution rates were higher in genes closer to the replication terminus, suggesting that mutation rates increase with distance from the replication origin. In order to directly test for the effects of chromosomal location on the rates of point mutations, we assayed the reversion rates of two lacZ alleles inserted at four positions in the Salmonella enterica chromosome. Mutation rates at an intermediate locus were significantly higher than those at loci nearer to and farther from the replication origin. The higher reversion rates at this locus were neither the result of an overall increase in mutation rates produced by the insertion at this location nor a function of the mutations' immediate neighbors, but rather a regional effect. At all loci, regardless of chromosome location, T.A --> G.C transversions were more frequent than A.T --> G.C transitions during the exponential phase.

Analysis of Variance↗