Manganese Is Required for the Rapid Recovery of DNA Synthesis following Oxidative Challenge in Escherichia coli

ABSTRACT Divalent metals such as iron and manganese play an important role in the cellular response to oxidative challenges and are required as cofactors by many enzymes. However, how these metals affect replication after oxidative challenge is not known. Here, we show that replication in Escherichia coli is inhibited following a challenge with hydrogen peroxide and requires manganese for the rapid recovery of DNA synthesis. We show that the manganese-dependent recovery of DNA synthesis occurs independent of lesion repair, modestly improves cell survival, and is associated with elevated rates of mutagenesis. The Mn-dependent mutagenesis involves both replicative and translesion polymerases and requires prior disruption by H2O2 to occur. Taking these findings together, we propose that replication in E. coli is likely to utilize an iron-dependent enzyme(s) that becomes oxidized and inactivated during oxidative challenges. The data suggest that manganese remetallates these or alternative enzymes to allow genomic DNA replication to resume, although with reduced fidelity. IMPORTANCE Iron and manganese play important roles in how cell’s cope with oxygen stress. However, how these metals affect the ability of cells to replicate after oxidative challenges is not known. Here, we show that replication in Escherichia coli is inhibited following a challenge with hydrogen peroxide and requires manganese for the rapid recovery of DNA synthesis. The manganese-dependent recovery of DNA synthesis occurs independently of lesion repair and modestly improves survival, but it also increases the mutation rate in cells. The results imply that replication in E. coli is likely to utilize an iron-dependent enzyme(s) that becomes oxidized and inactivated during oxidative challenges. We propose that manganese remetallates these or alternative enzymes to allow genomic DNA replication to resume, although with reduced fidelity.

Adaptations to a Loss-of-Function Mutation in the Betaproteobacterium Aromatoleum aromaticum: Recruitment of Alternative Enzymes for Anaerobic Phenylalanine Degradation

ABSTRACT Anaerobic phenylalanine (Phe) degradation in the betaproteobacterium Aromatoleum aromaticum involves transamination and decarboxylation to phenylacetaldehyde, followed by oxidation to phenylacetate. The latter reaction is catalyzed simultaneously by two enzymes, a highly specific phenylacetaldehyde dehydrogenase (PDH) and a rather unspecific tungsten-dependent aldehyde oxidoreductase (AOR). Attempting to establish increased synthesis of AOR, we constructed a mutant lacking the gene for PDH. This mutant still grew on phenylalanine, exhibiting increased AOR activities on medium containing tungstate. In the absence of tungstate, the mutant showed initially severe growth deficiency, but it resumed growth on Phe after longer incubation times. Moreover, the growth rates of the mutant increased during several reinoculation cycles on either tungstate-proficient or -deficient media, reaching the same values as recorded in wild-type strains. We confirmed AOR as the major alternative enzyme serving Phe degradation under tungstate-supplied conditions and identified and characterized the alternative NAD-dependent aldehyde dehydrogenase AldB taking over the function under tungstate-deficient conditions. Sequence analysis of the respective genes from adapted cultures under either growth condition revealed a mutation in the upstream region of the aor operon and a mutation within the coding region of aldB, which are likely involved in the observed adaptation of the deletion mutant to regain fast growth on Phe. IMPORTANCE The betaproteobacterium Aromatoleum aromaticum degrades many aromatic compounds under denitrifying conditions. One of the steps of phenylalanine degradation is catalyzed by two simultaneously induced enzymes, a NAD(P)-dependent phenylacetaldehyde dehydrogenase and a W-containing aldehyde oxidoreductase. We report here that the latter fully complements a constructed deletion mutant lacking the gene for phenylacetaldehyde dehydrogenase and is overproduced after several reinoculations. Moreover, an alternative NAD-dependent dehydrogenase is recruited to resume growth in tungstate-free medium, which does not allow the production of aldehyde oxidoreductase. This alternative enzyme is overproduced and seems to have acquired a point mutation in the active center. Our research illustrates the flexibility of environmentally important bacteria in adapting their metabolic pathways to new challenges within only a few generations.

Evaluation of Selected Enzymes for Thiamine Determination

Seven commercially available enzymes were studied for suitability as substitutes in the AOAC thiamine determination, because the enzyme Takadiastase used in the method has not been available since 1976, and alternative enzymes were likewise unavailable or unsuitable for releasing thiamine from its phosphate esters. Four factors (substrate ester, enzyme level, time/temperature, and pH) at 2 levels were studied in a 24 factorial arrangement of treatments. Data were expressed in terms of mean percentage conversion (MPC) and were statistically evaluated by analysis of variance. Significant main effects and any interactions among treatments were calculated. Takadiastase and a-amylase (Miles) with MPCs of 101 and 102, respectively, appeared effective in dephosphorylation within method parameters. Potato phosphatase appeared marginally suitable. Wheat germ phosphatase, α-amylase (Sigma), Mylase 100, and Clarase 40,000 were judged unacceptable as enzyme substitutes.

Computer modeling identifies glucokinase as glucose sensor of pancreatic beta-cells

Previous work from this laboratory indicates that glucokinase serves as the glucose sensor of pancreatic islets. Here we show by nonlinear computer optimization that the kinetic properties of glucokinase (together with hexokinase, known to be present in islets) account for the observed glycolytic rates in islets as a function of glucose level. Alternative enzymes that have been suggested to perform the same function as glucokinase, N-acetyl-D-glucosamine kinase and glucose-6-phosphatase, are shown to have incompatible properties, including a poor fit, different curve shapes, and unreasonable parameter values resulting from optimization. Their activities in islets are shown to be too low to account for observed glucose usage rates. This work endorses our previous proposal that glucokinase acts as the glucose sensor in pancreatic islet cells.