Cloning and expression in Escherichia coli K-12 of the structural gene for outer membrane PhoE protein from Enterobacter cloacae

Abstract

SUMMARYIn Escherichia coli K-12, the phoE gene encodes an outer membrane pore protein, which is induced by phosphate starvation. The co~esponding gene of ~~tero~acter cfoucae was transferred to E. coli K-12 by using RP4 : : mini Mu plasmid pULB 113 and selecting for R-prime plasmids that carry the genesproA andpro& which are closely linked to phoE in E. coli K-12. The phoE gene was subcloned into the multicopy vector pACYC 184, and the location of the gene was determined by analysis of in vitro constructed deletion plasmids and mutant plasmids generated by $5 insertions. The E. CioacaephoE gene is normally expressed in E. coli K-12, and the regulation of the expression is similar to that of the E. coiipho~ gene. Function~ly, the products of the phoE genes of E. coli K-12 and E. cloacae behave very similarly since they form pores in the outer membrane with a recognition site for negatively charged compounds and they serve as (part of) the receptor for phage TC45.The outer membrane of Enterobacteriaceae functions as a barrier for harmful compounds. The

Phosphate-starvation-induced outer membrane proteins of members of the families Enterobacteriaceae and Pseudomonodaceae: demonstration of immunological cross-reactivity with an antiserum specific for porin protein P of Pseudomonas aeruginosa

“…Physiological (11,22) and mutant (11,23) Phosphate-regulated outer membrane proteins which have been identified in other bacteria include the PhoE proteins of E. coli (19), Salmonella typhimurium (2), and Enterobacter cloacae (28) and a 36,000-molecular-weight (36K) protein of Klebsiella aerogenes (25). All except the 36K protein, which has not yet been examined, have been demonstrated to form weakly anion-selective channels (2,3,20,28) consistent with their proven (15) or presumed roles in phosphate acquisition. We report here that a number of bacterial species demonstrate phosphate-starvation-induced outer membrane proteins which cross-react immunologically.…”

Characterization of the Citrobacter freundii phoE gene and development of C. freundii-specific oligonucleotides

“…The E. coli strains used were MXR containing plasmid pULBll3 [12], strain CE1226, containing as relevant markers A (proB-proA-phoE-gpt), phoS, recA, hsdR, hsdM [13] and strain CEl195 [14]. Citrobacter strains used were C. amalonaticus strains ATCC 25405 and 25406, C. dic, ersus strains ATCC 27156, 29936 and 27028, a C. freundii isolate described earlier [15] and now designated CE1345 and ATCC strains 8454, 8090, 29935, 6750 and 10625.…”

A comparative study on the phoE genes of three enterobacterial species

“…The E. coli K-12 strains used were the hsdS phoS phoE strain CE1226 [22], the pore-protein-deficient phoR strain CE1248 [23], and JM103 [24]. Cells of the latter strain, infected with phage M13 derivatives mplO and mpll [25], were grown in yeast broth [26].…”

“…Sensitivity to the PhoE-specific phages TC45 [28] and TC45 hrN3 [15] was determined by cross-streaking. Plasmids pEC17 [22] and pJP29 [29] were used as source of the E. cloacae and E. coli phoE genes, respectively. A derivative of the RP4: :miniMu plasmid pULBll3, carrying the phoE gene of K. pneumoniae, was obtained from C. Verhoef.…”

“…An in vivo cloning procedure with the RP4::miniMu plasmid pULBll3 has been used previously to transfer the proA-proB-phoE regions of the E. cloacae [22] and K. pneumoniae (C. Verhoef, personal communication) genome to E. coli K-12. Both phoE genes were found to be normally regulated in E. coli K-12, since they were expressed only under phosphate limitation in a Pho' background and constitutively in the phoS strain CE1226 [22] (and C. Verhoef, personal communication). In both cases the amounts of the foreign PhoE proteins present in the outer membrane were similar to that of the E. coii protein.…”