Characterization of the Salmonella typhimurium phoE gene and development of Salmonella-specific DNA probes

• Published in: Gene Vol. 122; no. 1; pp. 45 - 52
• Main Authors: Spierings, Gonnie, Elders, Rietje, van Lith, Bart, Hofstra, Harm, Tommassen, Jan
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 01.12.1992; Amsterdam Elsevier; New York, NY
• Subjects: Amino Acid Sequence; Autoradiography; Bacterial Outer Membrane Proteins - genetics; Base Sequence; Biological and medical sciences; Cloning, Molecular; DNA Probes; DNA, Bacterial; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Fundamental and applied biological sciences. Psychology; Genes. Genome; Ion Channels - metabolism; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; pho regulon; PhoE protein; Plasmids; Polymerase Chain Reaction; pore protein; porin; Porins; Recombinant DNA; Restriction Mapping; RP4::miniMu; Salmonella - genetics; Salmonella typhimurium - genetics; Sequence Homology, Nucleic Acid; Species Specificity; proA; PhoE protein; proB; Km; pore protein; E; nt; Cm; Recombinant DNA; K; bp; Ap; ds; Tc; tsp; RP4::miniMu; R; kb; pho regulon; oligo; polymerase chain reaction; aa; ss; RBS; SSC; d; S., Salmonella; porin; PAGE; PhoE; Rif; mAb; p; PA; LB; Sm; : PCR; Membrane protein; Porin; Nucleotide sequence; Escherichia coli; Bacteria; Homology; Molecular probe; Species specificity; Molecular cloning; Salmonella typhimurium; Enterobacteriaceae
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(92)90030-S

In Escherichia coli K-12, the phoE gene, encoding a phosphate-limitation-inducible outer membrane pore protein (PhoE), is closely linked to the genes proA and proB. When the corresponding fragment of the Salmonella typhimurium chromosome was transferred to E. coli K-12 using an RP4::miniMu plasmid, pULB113, no production of S. typhimurium PhoE could be detected. Nevertheless, DNA hybridization studies revealed that the corresponding plasmid did contain S. typhimurium phoE. Production of S. typhimurium PhoE in E. coli was detected only after subcloning the gene in a multicopy vector. Nucleotide (nt) sequence analysis showed extensive homology of S. typhimurium phoE to the E. coli gene and suggested possible explanations for the low expression of S. typhimurium phoE in E. coli. In addition, the sequence information was used to develop Salmonella-specific DNA probes. Two oligodeoxyribonucleotides were synthesized based on nt sequences encoding the fifth and eighth cell-surface-exposed regions of PhoE. When used in polymerase chain reactions, these probes turned out to be specific i.e., no crossreactions occurred with the non- Salmonella strains, whereas 132 out of 133 tested Salmonella strains were recognized.