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 the result of periplasmic oxidation of glucose to gluconic acid via the quinoprotein glucose dehydrogenase (GDH), a component of the direct oxidation pathway. Escherichia coli K-12 derivatives synthesize apo-GDH but not the cofactor pyrroloquinoline-quinone (PQQ) essential for formation 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 cepacia open reading frame (designated gabY) in E. coli JM109 (a K-12 derivative) was sufficient to induce the Mps+ phenotype and production of gluconic acid. We present the nucleotide sequence of this open reading frame which coded for a protein (GabY) with a deduced M(r) of 14,235. Coupled transcription-translation of a plasmid (pSLY4 or pGAB1) carrying gabY resulted in production of a protein with an M(r) of 14,750. Disruption of the open reading frame of gabY via site-directed mutagenesis changed the phenotype to Mps- and eliminated gluconic acid production. The deduced amino acid sequence of gabY has no apparent homology with those of previously cloned direct oxidation pathway genes but does share regions highly homologous with the histidine permease system membrane-bound protein HisQ as well as other proteins in this family. In the presence of 1 microM exogenous PQQ, both JM109(pSLY4) and JM109(pGAB1) produced 10 times as much gluconic acid as was seen with either the plasmid or exogenous PQQ alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Babu-Khan
S
S
Department of Biology, California State University at Los Angeles 90032.
Yeo
T C
TC
Martin
W L
WL
Duron
M R
MR
Rogers
R D
RD
Goldstein
A H
AH
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D026923:Amino Acid Transport Systems, Basic; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D016956:Burkholderia cepacia / Q000201:enzymology / Q000235:genetics*; D002130:Calcium Phosphates / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D005942:Gluconates / Q000378:metabolism; D008565:Membrane Proteins; D026901:Membrane Transport Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D010641:Phenotype; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012995:Solubility
D018528:ATP-Binding Cassette Transporters; D026923:Amino Acid Transport Systems, Basic; D001426:Bacterial Proteins; D002130:Calcium Phosphates; C093078:GabY protein, Burkholderia cepacia; D005942:Gluconates; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C043828:histidine permease, Bacteria; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous; C030691:gluconic acid