IMPROVEMENT OF ESCHERICHIA-COLI MICROAEROBIC OXYGEN-METABOLISM BY VITREOSCILLA HEMOGLOBIN - NEW INSIGHTS FROM NAD(P)H FLUORESCENCE AND CULTURE REDOX POTENTIAL

On-line NAD(P)H fluorescence and culture redox potential (CRP) measure ments were utilized to investigate the role of Vitreoscilla hemoglobin (VHb) in perturbing oxygen metabolism of microaerobic Escherichia col i. Batch cultures of a VHb-synthesizing E. coil strain and the isogeni c control under fully aerated conditions were subject to several high/ low oxygen transitions, and the NAD(P)H fluorescence and CRP were moni tored during these passages. The presence of VHb decreased the rate of net NAD(P)H generation by 2.4-fold under diminishing oxygen tension. in the absence of aeration, the strain producing VHb maintained a stea dy NAD(P)H level 1.8-fold less than that of the control, indicating th at the presence of VHb keeps E. coli in a more oxidized state under ox ygen-limited conditions. Estimated from CRP, the oxygen uptake rates n ear anoxia were 25% higher for cells with VHb than those without. Thes e results suggest that VHb-expressing cells have a higher microaerobic electron transport chain turnover rate. To examine how NAD(P)H utiliz ation of VHb-expressing cells responds to rapidly changing oxygen tens ion, which is common in large-scale fermentations, we pulsed air inter mittently into a cell suspension and recorded the fluorescence respons e to the imposed dissolved oxygen (DO) fluctuation. Relative to the co ntrol, cells containing VHb had a sluggish fluorescence response to su dden changes of oxygen tension, suggesting that VHb buffers intracellu lar redox perturbations caused by extracellular DO fluctuations. (C) 1 995 John Wiley and Sons, Inc.