EVIDENCE FOR A 14-GENE, PHNC TO PHNP LOCUS FOR PHOSPHONATE METABOLISMIN ESCHERICHIA-COLI

The Escherichia coli phn (psiD) locus consists of a large gene cluster encoding proteins necessary for the use of phosphonates (Pn) as a sol e phosphorus source. On the basis of nucleotide (nt) sequence analysis , the phn locus contains a 12. 6-kb operon of seventeen genes named, i n alphabetical order, phnA to phnQ Chen et al., J. Biol. Chem. 265 (1 990) 4461-4471!. New Pn+ plasmids were made which are suitable for mut ational analysis of this gene cluster. These plasmids contain the R6K origin for DNA replication, can be conjugatively transferred, contain the tetAR genes, and therefore provide a way for allele replacement. T he construction of these plasmids showed that phnA and phnB have no ro le in Pn metabolism. Also, these plasmids were employed to introduce n onpolar phnD::lacZ and phnD::uidA fusions into the chromosome, which a llowed us to show that phnD probably has a role in transport. In addit ion, it was shown that phnP is the most distal gene required for Pn us e. This was done by testing the effect of phn::uidA insertions in or n ear the 3' end of phnP on Pn use. Altogether, these results show that all genes required for Pn use are in the 10. 9-kb, fourteen-gene, phnC DEFGHIJKLMNOP locus.