Cloning and expression of the filamentous bacteriophage Pf1 major coat protein gene in Escherichia coli. Membrane protein processing and virus assembly.
A restriction fragment carrying the major coat protein gene (gene VIII) was excised from the replicative form (RF) DNA of the class II filamentous bacteriophage Pf1, which infects Pseudomonas aeruginosa. This fragment was cloned into the expression plasmid pKK223-3, where it came under the control of the tac promoter. In transformed Escherichia coli JM101 cells, in the presence of the inducer isopropyl-beta-D-thiogalactoside, the bacteriophage Pf1 gene was strongly expressed. The bacteriophage Pf1 coat protein displays the same pattern of negatively charged N-terminal region, hydrophobic middle region and positively charged C-terminal region as that of its counterpart in the class I bacteriophage fd, which infects E. coli, but otherwise the two proteins have no sequence homology. However, the Pf1 procoat protein was found to undergo processing and insertion into the E. coli cell inner membrane, like its fd counterpart, demonstrating that this part of the assembly process is the same for these different bacteriophages. The complete transcriptional unit, incorporating the tac promoter and rrnB transcription terminators flanking the Pf1 coat protein gene, was excised from the expression plasmid and cloned into the intergenic space of bacteriophage R252, an fd bacteriophage that carries an amber mutation in its own major coat protein gene. The Pf1 coat protein gene was again well expressed in infected E. coli cells but the chimeric bacteriophage had growth properties identical to those of the parent bacteriophage R252 on suppressor and non-suppressor strains of E. coli. The class I bacteriophage Pf1 coat protein evidently cannot be recognized by the class I bacteriophage assembly complex at or in the E. coli cell inner membrane, either at the point of initiation of assembly or during the elongation process.
Rowitch
D H
DH
Department of Biochemistry, University of Cambridge, England.
Perham
R N
RN
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D010957:Plasmids; D014176:Protein Biosynthesis; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics*
D014764:Viral Proteins