Robert E. Webster

Translational control of bacteriophage f1 gene II and gene X proteins by gene V protein

The gene II region of bacteriophage f1 DNA codes for two proteins, the 46 kd gene II protein and the 13 kd gene X protein, which results from an in-phase start at codon 300 of gene II. Using antigene II protein IgG, we show that the intracellular concentration of both proteins is controlled by the phage gene V protein. In wild-type f1-infected cells, the amount of gene II protein reaches a plateau of about 1500 molecules per cell at 20 min after infection, as measured by blot immunoassay. Similarly, the amount of gene X protein reaches a peak of about 500 molecules per cell around 10 min after infection. In contrast, when the gene V protein is inactive, both gene II and gene X proteins continue to accumulate at a high rate for at least 40 min after infection. This difference is caused by decreased synthesis of gene II and gene X proteins in the presence of gene V protein, which represses the translation of these two proteins.

Visualization of the same PtK2 cytoskeletons by both immunofluorescence and low power electron microscopy

Abstract A procedure is described which allows the examination of the cytoskeleton of a single PtK2 cell first by immunofluorescence and then by electron microscopy after staining with uranyl acetate. The immunofluorescent patterns of these detergent resistant cytoskeletons elicited with various monospecific antibodies closely resemble the patterns found in whole cells. Comparison of the immunofluorescence and electron micrographs directly supports the previous assignments of actin, myosin, filamin, α-actinin and tropomyosin as proteins associated with microfilament bundles in non-muscle cells. Actin is also found associated with a fine lattice-like structure present both in the ruffles and lying above the microfilament bundles in the cell body. The tonofilament bundles present in PtK2 cytoskeletons are not decorated by antibodies directed against the proteins associated with microfilament bundles. Antibodies directed against tonofilaments decorate specifically this system and not the microfilament bundles.

Abortive infection of Escherichia coli with the bacteriophage f1: Cytoplasmic membrane proteins and the f1 DNA-gene 5 protein complex

A partially purified cytoplasmic membrane fraction has been isolated from non-permissive Escherichia coli infected with various amber mutants of the bacteriophage f1. The bacteria were labeled with [ 14 C]lysine 20–40 min after infection and the proteins extracted and analyzed by SDS polyacrylamide gel electrophoresis and radio-autography. The f1 coat protein was present in the membrane of bacteria infected with all f1 amber mutants except gene 8. A complex composed of f1 single-stranded DNA and the f1 gene 5 protein was isolated with the inner membrane fraction. Approximately 300 molecules of this complex were found in bacteria infected with f1 wild type and all f1 amber mutants except those in gene 2 and gene 5. In bacteria infected with f1 wild type, the f1 single-stranded DNA was chased from the complex into phage and the displaced gene 5 protein appeared able to associate with another f1 single-stranded DNA molecule. In contrast, the f1 single stranded DNA-gene 5 protein complex was stable in the amber mutant infected bacteria. Two other proteins associated with the membrane of the amber mutant infected bacteria were tentatively identified as the products of gene 2 and gene 4.

Bacteriophage Coat Protein as Repressor

It seems that viral coat protein acts as a repressor of protein synthesis at the level of transcription rather than translation.

Orientation of the DNA in the filamentous bacteriophage f1

Abstract The filamentous bacteriophage f1 consists of a molecule of circular single-stranded DNA coated along its length by about 2700 molecules of the B protein. Five molecules of the A protein and five molecules of the D protein are located near or at one end of the virion, while ten molecules of the C protein are located near or at the opposite end. The two ends of the phage can be separated by reacting phage fragments, which have been generated by passage of intact phage through a French press, with antibody directed against the A protein (Grant et al. , 1981 a ). By hybridizing the DNA isolated from either end of 32 P-labeled phage to specific restriction fragments of fl replicative form I DNA, we have determined that the single-stranded DNA of the filamentous bacteriophage f1 is oriented within the virion. For wild-type phage, the DNA that codes for the gene III protein is located at the A and D protein end and that which corresponds to the intergenic region is located close to the C protein end of the particle. The intergenic region codes for no protein but contains the origins for both viral and complementary strand DNA synthesis. Analysis of the DNA orientation in phage in which the plasmid pBR322 has been inserted into different positions within the intergenic region of fl shows that the C protein end of all sizes of filamentous phage particles appears to contain a common sequence of phage DNA. This sequence is located near the junction of gene IV and the intergenic region, and probably is important for normal packaging of phage DNA into infectious particles. There appears to be no specific requirement for the origins of viral and complementary strand DNA synthesis to be at the end of a phage particle.

Morphogenesis of f1 filamentous bacteriophage. Increased expression of gene I inhibits bacterial growth.

We have cloned the gene I sequence of the filamentous bacteriophage f1 downstream from the lambda leftward promoter on a plasmid that also contains the temperature-sensitive lambda repressor, cI857. Temperature induction of gene I protein (pI) resulted in rapid cessation of growth. This inhibition appears to involve a rapid decrease in synthesis of host protein and RNA. The ability of pI to cause this inhibition is not dependent on thioredoxin, a host factor that is necessary for phage morphogenesis and has been shown by genetic data to interact with pI. The inhibition does not appear to be mediated by the amino half of the protein, as induction of an identical plasmid construction of an amber mutant positioned two-thirds along gene I, does not affect cell growth. Analysis of the transcription products from the cloned gene I confirmed previous suggestions that a transcription terminator exists in the amino-terminal portion of the gene. In addition, there is no detectable promoter activity in the 152 bases immediately upstream from the gene. These data and the inability to overproduce pI argue for down-regulation of pI production. Radioactive labeling of proteins in maxi-cells and normal Escherichia coli cells identifies pI as a protein of about 39,000 Mr that partitions with the cell envelope. Pulse-chase experiments suggest that pI is not processed to any appreciable extent.

f1 Coat protein synthesis and altered phospholipid metabolism in f1 infected Escherichia coli

Abstract The phospholipid composition of cytoplasmic membranes prepared from bacteria grown in the presence of [ 32 P]phosphate and infected with f1 wild type and f1 amber mutant bacteriophages was determined. Ninety minutes after infection with f1 amber mutants in genes 1, 3, 4, 5, 6, and 7 the percentage of cardiolipin was increased from the level in uninfected bacteria of 5% to about 20–35%, and the percentage of phosphatidylethanolamine was decreased from 70% to about 50–60%. The phospholipid composition of cytoplasmic membranes from bacteria infected with a phage containing an amber mutation in the coat cistron (gene 8) did not differ from that of uninfected bacteria. Although late in infection there were no detectable alterations in phospholipid metabolism in wild type infected bacteria, transient alterations in phospholipid metabolism occurred in these bacteria 10 to 20 min after infection. During this time period, the f1 coat protein was found to be rapidly synthesized but was not being packaged into mature phage and released from bacteria. Both the long-term alterations of phospholipid metabolism found in the amber mutant infected bacteria and the transient alterations found in wild-type infected bacteria resulted from an increase in the rate of synthesis of phosphatidylglycerol and cardiolipin and a decrease in the rate of synthesis of phosphatidylethanolamine. These results are discussed in terms of the relationship between the accumulation of f1 coat protein in infected bacteria and the observed alterations in phospholipid metabolism.

A new species of small covalently closed f1 DNA in Escherichia coli infected with an amber mutant of bacteriophage f1

Infection of nonpermissive cells with an amber mutant in gene 5 of fl leads to the accumulation of covalently closed circles of double-stranded DNA about one-fourth the size of f1 RF DNA. These circles appear to contain a unique fraction of the fl chromosome.

Minor protein content of the gene V protein/phage single-stranded DNA complex of the filamentous bacteriophage f1

The gene V protein/phage single-stranded (SS) DNA complex is an intermediate in the assembly of the filamentous bacteriophage f1. The minor protein content of this complex isolated from wild-type and amber mutant phage-infected Escherichia coli bacteria has been analyzed. Other than the gene V protein, none of the proteins found in purified samples of the complex correspond to any known phage gene products. In particular, the minor coat proteins found in the mature phage particle do not appear to be components of the cytoplasmic gene V protein/f1 SS DNA complex. However, approximately 1-3 molecules of E. coli single-stranded DNA binding protein (SSB) copurify with the complex and may be stably associated with this structure in vivo.

Nucleotide sequence of the F plasmid gene, traC, and identification of its product

The traC gene of the F plasmid tra operon is required for the assembly of mature F-pilin subunits into extended F pili. The nucleotide sequence of traC was determined with a determined with a deduced coding region of 875 amino acids (aa) and 99066 Da. The traC1044 mutant allele, which allows filamentous phage infection in the absence of piliation, contains a C-to-T transition leading to an Arg----Cys substitution. Confirmation of the translational start came from the direct N-terminal aa sequencing of a TraC-alkaline phosphatase fusion protein.