MUTATIONAL ANALYSIS OF AN ESCHERICHIA-COLI 14-GENE OPERON FOR PHOSPHONATE DEGRADATION, USING TNPHOA' ELEMENTS

All genes for phosphonate (Pn) utilization in Escherichia coli are in a large cluster of 14 genes named, in alphabetical order, phnC to phnP . Plasmids carrying these genes were mutagenized by using TnphoA'-1, a nd 43 mutants containing simple insertions were studied in detail. The ir insertion sites were defined by restriction mapping and by DNA sequ encing. One or more mutations in each phn gene was identified. In 23 m utants, expression of the TnphoA'-1 lacZ gene was phosphate starvation inducible. These mutants had TnphoA'-1 oriented in line behind the ph nC promoter, i.e., in the + orientation. In 20 mutants, the TnphoA'-1 lacZ gene was expressed at a low basal level. These mutants had insert ions in the opposite orientation. All 43 phn::TnphoA'-1 insertions wer e recombined onto the chromosome to test for mutational effects, and t heir structures on the chromosome were verified by DNA hybridization. Those in the + orientation were switched to TnphoA'-9, which has an ou tward promoter for expression of downstream genes. These insertions we re tested for polar effects by measuring beta-glucuronidase synthesis from a uidA gene transcriptionally fused to the 3' end of the phnP gen e. The results indicate the following: (i) the phnC-to-phnP gene clust er is an operon of 14 genes, and the phnC promoter is the sole psi pro moter; (ii) three gene products (PhnC, PhnD, and PhnE) probably consti tute a binding protein-dependent Pn transporter; (iii) seven gene prod ucts (PhnG, PhnH, PhnI, PhnJ, PhnK, PhnL, and PhnM) are required for c atalysis and are likely to constitute a membrane-associated carbon-pho sphorus (C-P) lyase; (iv) two gene products (PhnN and PhnP) are not ab solutely required and may therefore be accessory proteins for the C-P lyase; and (v) two gene products (PhnF and PhnO) are not required for Pn use and may have a regulatory role because they have sequence simil arities to regulatory proteins. The mechanism for breaking the C-P bon d by a lyase is discussed in light of these results.