CHARACTERIZATION OF A BINDING-PROTEIN-DEPENDENT, ACTIVE-TRANSPORT SYSTEM FOR SHORT-CHAIN AMIDES AND UREA IN THE METHYLOTROPHIC BACTERIUM METHYLOPHILUS-METHYLOTROPHUS

Three genes (fmdCAB) encoding an outer-membrane porin for short-chain amides and urea, formamidase, and a putative regulatory protein in Met hylophilus methylotrophus have previously been cloned and characterise d. Three genes have now been identified downstream of fmdB, viz fmdD e ncoding a hydrophilic protein containing an N-terminal signal sequence , and fmdEF encoding hydrophobic transmembrane proteins. The derived a mino acid sequence of mature FmdD (predicted molecular mass 41870 Dal was similar to the cytoplasmic, amide-binding protein (AmiC) from Pseu domonas aeruginosa and to several periplasmic, solute-binding proteins from other bacteria. Mature FmdD was purified and shown to be a monom er (40-45 kDa) with the predicted N-terminal amino acid sequence (ADYP TA-). Equilibrium dialysis showed that the purified protein bound shor t-chain amides and urea with high affinity (K-d 7.2 mu M for [C-14]ure a). SDS/PAGE and western blotting using antiserum to mature FmdD showe d it was induced by short-chain amides and urea, and repressed by exce ss ammonia. The derived amino acid sequences of FmdE (32822 Dal and Fm dF (incomplete; >25435 Dal were similar to the transmembrane proteins BraD/LivH and BraE/LivM, respectively, in various leucine/isoleucine/v aline transport systems. Uptake of [C-14]urea by washed cells was inhi bited by the uncoupling agent carbonyl cyanide p-trifluoromethoxypheny lhydrazone and unlabelled formamide. It is concluded that FmdDEF compr ise part of a high-affinity, binding-protein-dependent active-transpor t system for short-chain amides and urea in M. methylotrophus.