Protein Kinase Induction in Escherichia coli by Bacteriophage T7

Abstract

After bacteriophage T7 infection, a protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) activity can be demonstrated in E. coli in vivo by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Cell-free extracts catalyzed the transfer of the terminal phosphoryl group of [-y_32P]ATP to endogenous protein acceptor or to added histone. The bond between phosphate and protein shows the characteristies of serine phosphate: it is stable in 1 N HCl (1000) and cleaved by 1 N KOH (370) and by alkaline phosphatase treatment. Moreover, after partial acid hydrolysis, radiophosphate migrates with marker O-phosphoserine on polyethyleneimine-cellulose thin-layer chromatograms. Enzyme activity in uninfected cells is negligible. Ultraviolet irradiation of the phage genome prevents the appearance of the protein kinase; irradiation of the host genome does not. The enzyme activity occurs 4 min after infection and its gene maps in the early region (promoter proximal to gene 1). Ribosomal proteins are phosphorylated in vivo and are substrates in vitro. Enzyme activity in vitro is not changed by addition of cyclic AMP or cyclic GMP.Bacteriophage systems have been used extensively to study the regulation of gene expression (1,2). One particular aspect of these systems is the rapidity of changes in the pattern of gene expression; phage T7 induces a whole series of control mechanisms within 4-5 min (2, 3). This rapidity of change in gene expression prompted our search for group transfers after phage infection (4-6). A phage-induced group transfer to components of the existing host machinery of gene expression would allow both a fast and an economical control.In our previous work on phage T7, we reported that labeled phosphate is incorporated into ribosomal protein after T7 infection (6,23 were then subject to SDS slab-gel electrophoresis (9); the gels were dried and placed on x-ray film overnight. Assay for Protein Kinase In Vitro. Brij-lysozyme cell extracts (10) were used as enzyme sources. The incubations were carried out at 300 for 5 min in a total volume of 0.1 ml. The incubation mixtures contained 30A]. of cell extract, 3 mg/ml of type II histone, and either (1) 15 mM sodium phosphate (pH 7.0), 0.01 MMgCl2, 0.14 mM ATP (35.7 Ci ['y32-P]ATP/mol) or (2) 25 mM Tris HCl (pH 7.0), 5 mM MgCI2, 0.1 mM EDTA, 44,AM, ATP (114 Ci of [32P]ATP/mol). The incubations were started by the addition of enzyme (cell extract) and stopped by addition of 1.5 ml of 10% trichloroacetic acid.The precipitates were boiled in trichloroacetic acid for 15 min, chilled in ice for 10 min, and collected on glass-fiber filter discs. The discs were washed 5 times with 10 ml of 10% trichloroacetic acid and once with 10 ml of methanol. After the filters were dried, radioactivity on the filters was measured by liquid scintillation spectrometry. In selected experiments, the 10% trichloroacetic acid suspensions of precipitate were heated to 900 for 20 min, cooled to ambient temperature, and centrifuged at 2500 X g. These precipit...

A Novel Strategy for Exploitation of Host RNase E Activity by a Marine Cyanophage

“…In contrast to T4, the subsequent T7 expression program is coordinated by the activity of a phage RNAP (Maniloff and Ackermann 1998). Nevertheless, direct manipulation of host proteins plays a crucial role for the gene expression program of T7.The gene product of the early T7 0.7 gene codes for a protein kinase (Rahmsdorf et al 1974) and is responsible for phosphorylation and, thus, functional manipulation of .90 host proteins (Robertson et al 1994). For example, phosphorylation of host RNAP b9-subunit (Zillig et al 1975) results in reduced processivity, which is believed to assist in the coordinated transition of expression from T7 cluster 1 to cluster 2 genes (Severinova and Severinov 2006).…”

Characterization of the phosphoproteins of Escherichia coli cells by electrophoretic analysis

“…Thus, the phosphorylating activity originally described in Escherichia coli extracts [6] was later found to be a polyphosphate kinase rather than a protein kinase [7,8]. Also, the protein kinase activity detected in bacteria after infection with bacteriophage T7 [9] was shown to be specifically a viral gene product naturally absent in host cells [lo]. In other experiments [l 1 -131 no definite conclusion could be drawn mainly because of the incomplete characterization of the phosphorylated moiety of proteins.…”

Coliphage BA14: a New Relative of Phage T7

“…The supernatant fluid was used as enzyme source. The following procedures were then employed, with minor modifications: the method of Gefter et al (1966) for assaying S-adenosyl-methioninecleaving enzyme (SAMase) activity; the method of Chamberlin et al (1970) for assaying RNA polymerase activity; the method of Rahmsdorf et al (1974) for assaying protein kinase activity.…”