ISOLATION, SEQUENCING AND OVERPRODUCTION OF THE SINGLE-STRANDED-DNA BINDING-PROTEIN FROM PSEUDOMONAS-AERUGINOSA PAO

The gene (ssb) encoding the single-stranded DNA binding (SSB) protein from Pseudomonas aeruginosa PAO was detected on a 2.1 kbp PstI-fragmen t of chromosomal DNA. The protein (PaeSSB) encoded by this gene consis ts of 165 aa and has a M(r) of 18 549. The genomic sequence was confir med by amino acid sequencing of the amino terminus of SSB protein isol ated from P. aeruginosa PAO. PaeSSB shows 68% homology to the respecti ve protein of E. coli. The nucleotide sequence upstream of the P. aeru ginosa ssb gene shows little homology to the regulatory region upstrea m of the ssb gene of E. coli. The ssb gene was located at a distance o f 690-870 kbp from the origin of replication on a physical map of P. a eruginosa PAO. In vivo PaeSSB could replace the SSB protein of E. coli (EcoSSB) if its production was controlled by the lac promoter on a hi gh-copy vector. PaeSSB was overproduced in E. coli. Both the overprodu ced protein and PaeSSB isolated from Pseudomonas aeruginosa PAO are po sttranslationally modified by cleavage of the first methionine. Analyt ical ultracentrifugation shows thar PaeSSB is a stable homotetramer. T he copy number of PaeSSB in P. aeruginosa is 1200+/-250 tetramers per cell. Preliminary characterization of the DNA binding properties shows PaeSSB to have a lower affinity for single-stranded DNA than EcoSSB.