Glycerol and glucose fermentation redox routes by Escherichia coli and their regulation by oxidizing and reducing reagents were investigated at different pHs. Cell growth was followed by decrease of pH and redox potential (E ( h )). During glycerol utilization at pH 7.5 ∆pH, the difference between initial and end pH, was lower compared with glucose fermentation. After 8 h growth, during glycerol utilization E ( h ) dropped down to negative values (-150 mV) but during glucose fermentation it was positive (+50 mV). In case of glycerol H(2) was evolved at the middle log phase while during glucose fermentation H(2) was produced during early log phase. Furthermore, upon glycerol utilization, oxidizer potassium ferricyanide (1 mM) inhibited both cell growth and H(2) formation. Reducing reagents DL-dithiothreitol (3 mM) and dithionite (1 mM) inhibited growth but stimulated H(2) production. The findings point out the importance of reductive conditions for glycerol fermentation and H(2) production by E. coli.

Escherichia coli is able to ferment glycerol and produce H2 by different Hyds (hydrogenases). Wild-type whole cells were shown to extrude H+ through the F1Fo-ATPase and by other means with a lower rate compared with that under glucose fermentation. At pH 7.5, H+ efflux was stimulated in fhlA mutant (with defective transcriptional activator of Hyd-3 or Hyd-4) and was lowered in hyaB or hybC mutants (with defective Hyd-1 or Hyd-2) and hyaB hybC double mutant; DCCD (dicyclohexylcarbodi-imide)-sensitive H+ efflux was observed. At pH 5.5, H+ efflux in wild-type was lower compared with that at pH 7.5; it was increased in fhlA mutant and absent in hyaB hybC mutant. Membrane vesicle ATPase activity was lower in wild-type glycerol-fermented cells at pH 7.5 compared with that in glucose-fermented cells; 100 mM K+ did not stimulate ATPase activity. The latter at pH 7.5, compared with that in wild-type, was lower in hyaB and less in hybC mutants, stimulated in the hyaB hybC mutant and suppressed in the fhlA mutant; DCCD inhibited ATPase activity. At pH 5.5, the ATPase activities of hyaB and hybC mutants had similar values and were higher compared with that in wild-type; ATPase activity was suppressed in hyaB hybC and fhlA mutants. The results indicate that during glycerol fermentation, H+ was expelled also via F1Fo. At pH 7.5 Hyd-1 and Hyd-2 but not FhlA or Hyd-4 might be related to F1Fo or have their own H+-translocating ability. At pH 5.5, both Hyd-1 and Hyd-2 more than F1Fo might be involved in H+ efflux.

Determination of the number of active motors pulling a single MT or bead during motility assays has proven difficult. Traditional protein concentration assays, such as Bradford, cannot distinguish between active and inactive motors. We attach a superparamagnetic bead to the (þ) end of a microtubule. When placed in a magnet with uniform magnetic field gradient, the bead pulls on the MT with a controllable 0-10 pN force. If the force is perpendicular to the gliding direction of the MT, a short section of the MT ''pops off'' the surface every 2 to 5 s, as shown in the diagram. This detachment is characterized by rapid motion of the superparamagnetic bead in the direction of higher magnetic field gradient followed by normal microtubule gliding velocity when the MT is pulled taut. The length of the short section between ''popoffs'' is the distance between active kinesins along a microtubule.

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