Rhizobium (Sinorhizobium) meliloti phn genes: characterization and identification of their protein products.
In Escherichia coli, the phn operon encodes proteins responsible for the uptake and breakdown of phosphonates. The C-P (carbon-phosphorus) lyase enzyme encoded by this operon which catalyzes the cleavage of C-P bonds in phosphonates has been recalcitrant to biochemical characterization. To advance the understanding of this enzyme, we have cloned DNA from Rhizobium (Sinorhizobium) meliloti that contains homologues of the E. coli phnG, -H, -I, -J, and -K genes. We demonstrated by insertional mutagenesis that the operon from which this DNA is derived encodes the R. meliloti C-P lyase. Furthermore, the phenotype of this phn mutant shows that the C-P lyase has a broad substrate specificity and that the organism has another enzyme that degrades aminoethylphosphonate. A comparison of the R. meliloti and E. coli phn genes and their predicted products gave new information about C-P lyase. The putative R. meliloti PhnG, PhnH, and PhnK proteins were overexpressed and used to make polyclonal antibodies. Proteins of the correct molecular weight that react with these antibodies are expressed by R. meliloti grown with phosphonates as sole phosphorus sources. This is the first in vivo demonstration of the existence of these hitherto hypothetical Phn proteins.
Parker
G F
GF
School of Animal and Microbial Sciences, University of Reading, Reading RG6 6AJ, United Kingdom.
Higgins
T P
TP
Hawkes
T
T
Robson
R L
RL
D000595:Amino Acid Sequence; D000906:Antibodies; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008190:Lyases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D005810:Multigene Family; D009876:Operon; D063065:Organophosphonates / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016962:Sinorhizobium meliloti / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity
D000906:Antibodies; D001426:Bacterial Proteins; D017931:DNA Primers; D063065:Organophosphonates; D008190:Lyases; C117101:PhnI protein, Rhizobium meliloti; C051328:carbon-phosphorus lyase