Oxidase and periplasmic cytochrome assembly in Escherichia coli K-12: CydDC and CcmAB are not required for haem-membrane association

The mechanism(s) that bacteria use to transport haem into and across the cy toplasmic membrane to complete the assembly of periplasmic cytochromes is u nknown. The authors have tested directly the role(s) of two ATP-binding cas sette (ABC) transporters - the cydDC and ccmAB gene products - in Escherich ia coil by measuring haem uptake in everted (inside-out) membrane vesicles, If haem is exported to the periplasm in vivo, the same process should resu lt in active accumulation in such everted vesicles. [C-14]Haemin (chloride) with bovine serum albumin (BSA) as a carrier protein was accumulated in in tact everted membrane vesicles by an energy-independent mechanism. The kine tics of this process were biphasic: rapid uptake/binding was followed by a slower uptake of haem, which was inhibited by a large excess of unlabelled haemin-BSA, but not by BSA, However, accumulated haemin was not chased out of the vesicles by unlabelled haemin-BSA, suggesting specific binding of ha emin with the membrane or transport into the lumen of the vesicle, Neither ATP nor a protonmotive force (Delta p) generated by lactate oxidation was r equired for haemin binding or subsequent transport, and carbonyl cyanide m- chlorophenylhydrazone (CCCP), sodium vanadate and monensin had no effect on haemin transport. The rate of haemin uptake following the initial rapid bi nding was proportional to the external haemin concentration, suggesting tha t the uptake process was driven by the haemin concentration gradient across the cell membrane. The kinetics of [C-14]haemin uptake were similar in wil d-type and cydD1 or Delta ccmA mutants, suggesting that the activity of nei ther the CydDC nor CcmAB transporters is essential for haem export to the p eriplasm, Cytochrome d levels were unaffected by mutations in trxB (encodin g thioredoxin reductase), trxA (thioredoxin), or grx (glutaredoxin), sugges ting that the CydDC transporter does not export these components of reducin g pathways for cytochrome assembly.