CLONING OF A MINERAL PHOSPHATE-SOLUBILIZING GENE FROM PSEUDOMONAS-CEPACIA

We have recently shown that the ability of some gram-negative bacteria to dissolve poorly soluble calcium phosphates (Mps(+) phenotype) is t he result of periplasmic oxidation of glucose to gluconic acid via the quinoprotein glucose dehydrogenase (GDH), a component of the direct o xidation pathway. Escherichia coli K-12 derivatives synthesize apo-GDH but not the cofactor pyrroloquinoline-quinone (PQQ) essential for for mation of the holoenzyme, Therefore, in the absence of exogenous PQQ, these strains do not produce gluconic acid and are Mps(-). Evidence is presented to show that expression of a single 396-base Pseudomonas ce pacia open reading frame (designated gabY) in E. coli JM109 (a K-12 de rivative) was sufficient to induce the Mps(+) phenotype and production of gluconic acid, We present the nucleotide sequence of this open rea ding frame which coded for a protein (GabY) with a deduced M(r) of 14, 235. Coupled transcription-translation of a plasmid (pSLY4 or pGAB1) c arrying gabY resulted in production of a protein with an M(r) of 14,75 0. Disruption of the open reading frame of gabY via site-directed muta genesis changed the phenotype to Mps(-) and eliminated gluconic acid p roduction. The deduced amino acid sequence of gabY has no apparent hom ology with those of previously cloned direct oxidation pathway genes b ut does share regions highly homologous with the histidine permease sy stem membrane-bound protein HisQ as well as other proteins in this fam ily. In the presence of 1 mu M exogenous PQQ, both JM109(pSLY4) and JM 109(pGAB1) produced 10 times as much gluconic acid as was seen with ei ther the plasmid or exogenous PQQ alone. The presence of pGAB1 was als o sufficient to cause production of gluconic acid in E. coli HB101 (a K-12-B hybrid), In AG121, an apoGDH(-), Tn5 mutant of HB101, the prese nce of pGAB1 did not cause the production of gluconic acid.