dye (arc) mutants: insights into an unexplained phenotype and its suppression by the synthesis of poly (3-hydroxybutyrate) in Escherichia coli recombinants

Abstract

arcA codes for a central regulator in Escherichia coli that responds to redox conditions of growth. Mutations in this gene, originally named dye, confer sensitivity to toluidine blue and other redox dyes. However, the molecular basis for the dye-sensitive phenotype has not been elucidated. In this work, we show that toluidine blue redirects electrons to O2 and causes an increase in the generation of reactive O2 species (ROS). We also demonstrate that synthesis of poly (3-hydroxybutyrate) suppresses the Dye phenotype in E. coli recombinants, as the capacity to synthesize the polymer reduces sensitivity to toluidine blue, O2 consumption and ROS production levels.

CytochromedBut Not CytochromeoRescues the Toluidine Blue Growth Sensitivity ofarcMutants ofEscherichia coli

“…Moreover, the fact that the dye phenotype is observed only in the presence of oxygen raises the possibility that TBO perturbs the electron transport chain by diverting electrons to redox cycling, thereby restricting energy production. This is supported by the previous observation demonstrating that cyanide-treated E. coli membranes that are unable to respire recover oxygen consumption in the presence of TBO (50) and also by the fact that the presence of glucose reverts the TBO-dependent growth defect of the arc mutants. Interestingly, a protective role for cytochrome d in generating an intracellular anaerobic environment has previously been observed with Azotobacter vinelandii, which uses the terminal oxidase to protect nitrogenase, an oxygen-sensitive enzyme, enabling nitrogen fixation even during aerobic growth (35).…”

“…Later on, a similar phenotype was described for mutants of arcB (32) and cydAB (15). Redox dyes, such as TBO and methylene blue, in the presence of light are able to produce ROS that were suggested to be the cause for the dye sensitivity of the arcA mutants (50). However, the results presented in this study indicate that the dye phenotype of arc mutants but also the TBO-dependent production of ROS is light independent.…”

“…In a recent study, it was proposed that TBO results in a significant increase of ROS in an arcA mutant and that this increase in ROS is the cause of the dye phenotype (50). Moreover, the heterologous expression of poly 3-hydroxybutyrate was shown to be able to suppress the dye sensitivity in arcA mutants by diminishing O 2 consumption and the production of ROS (50).…”

The Legacy of HfrH: Mutations in the Two-Component System CreBC Are Responsible for the Unusual Phenotype of an Escherichia coli arcA Mutant

“…arcA2 and ⌬arcA mutants were also observed to differ in other traits: arcA deletion mutants are very sensitive to redox dyes, such as toluidine blue (4), while strains bearing the arcA2 mutation produce medium-sized colonies on toluidine blue agar. Besides, arcA2 strains show a higher respiratory capacity (18,21) and show a higher NADH/NAD ϩ ratio when glycerol is used as a carbon substrate (19); as a consequence, enhanced yields of poly(3-hydroxybutyrate) and ethanol were observed. The different behaviors of these mutants were intriguing, and a deeper analysis was necessary in order to explain the molecular basis for the differences in phenotypes.…”

Production of Short Chain Length Polyhydroxyalkanoates by Bacillus megaterium PHB29 from Starch Feed Stock

“…They act as carbonenergy reserves and also as electron sinks, which enhance the fitness of the bacteria and help in maintaining the redox balance (López et al, 1995;Madison and Huisman 1999;Ruiz et al, 2006). These materials are biocompatible, non-toxic and considered as alternatives for conventional petroleum derived plastics (Chen, 2010).…”