Kensuke Horiuchi

Mutants of the bacteriophage f2: V. On the production of noninfectious phage particles

Abstract One class of conditional lethal mutants of the RNA bacteriophage f2 produces, when growing under nonpermissive conditions, a normal-sized yield of defective, nonviable phage particles. This class includes both host-dependent and temperature-sensitive mutants. The nonviable phage particles form a heterogeneous band in CsCl density gradients, and lack the normal amount of RNA, varying from almost a complete amount to about 50% less. They are immunologically identical to wild-type phage. However, unlike viable phage, they do not adsorb to bacteria nor to cellulose nitrate filter pads. The mutation does not appear to be in a phage gene controlling either the synthesis of the viral RNA polymerase or the viral coat protein. Temperature shift experiments indicate that the mutation is in a late functioning gene and is involved with the assembly of intact viral RNA and of coat protein into virions. Interruption of protein synthesis late in infection by wild-type phage produces nonviable phage particles similar to those produced by the mutant.

Mutants of the bacteriophage f2: VI. Homology of temperature-sensitive and host-dependent mutants

Temperature-sensitive mutants of phage f2 have been isolated and classified into three groups by physiological criteria. One group is blocked at an early stage of growth and synthesizes neither phage RNA nor phage antigen at high temperature. A second group of mutants is blocked in a late function, forms antigenic but defective phage particles. The mutation is related to the maturation of phage particles. Mutants in the other group also affect a late function, do not form antigen, and are assumed to be coat protein mutants. These mutants can be related to a series of amber mutants of f2.

Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon

Abstract SulA is induced in Escherichia coli by the SOS response and inhibits cell division through interaction with FtsZ. To determine which region of SulA is essential for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletions of SulA and a series of alanine substitution mutants. Arginine at position 62, leucine at 67, tryptophan at 77 and lysine at 87, in the central region of SulA, were all essential for the inhibitory activity. Residues 3–27 and the C-terminal 21 residues were dispensable for the activity. The mutant protein lacking N-terminal residues 3–47 was inactive, as was that lacking the C-terminal 34 residues. C-terminal deletions of 8 and 21 residues increased the growth-inhibiting activity in lon+ cells, but not in lon− cells. The wild-type and mutant SulA proteins were isolated in a form fused to E. coli maltose-binding protein, and tested in vitro for sensitivity to Lon protease. Lon degraded wild-type SulA and a deletion mutant lacking the N-terminal 93 amino acids, but did not degrade the derivative lacking 21 residues at the C-terminus. Futhermore, the wild-type SulA and the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did not. MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by Lon. When LacZ protein was fused at its C-terminus to 8 or 20 amino acid residues from the C-terminal region of SulA the protein was stable in lon+ cells. These results indicate that the C-terminal 20 residues of SulA permit recognition by, and complex formation with, Lon, and are necessary, but not sufficient, for degradation by Lon.

Osmoregulation of the fatty acid receptor gene fadL in Escherichia coli

The fadL gene of Escherichia coli codes for an outer membrane protein that is involved in the uptake of long-chain fatty acids. Uptake is regulated by environmental osmolarity, and decreases when the cells are grown under conditions of high osmolarity. A temperature-sensitive mutant that requires fatty acid for growth at 42° C was unable to grow at the high temperature even in the presence of fatty acid if the medium contained 10% sucrose. Promoter activity of the fadL gene in vivo was repressed by high osmolarity in a FadR repressor null mutant. Furthermore, in vitro transcription of the fadL gene was strongly repressed by the addition of OmpR and EnvZ proteins. The results of gel retardation and DNase I protection experiments indicated that OmpR, after incubation with the protein kinase EnvZ, specifically binds to at least four sites around the fadL promoter, two upstream and two downstream from the transcriptional start site. These results suggest that transcription of the fadL gene is osmotically regulated by the OmpREnvZ two-component system.

Cleavage map of bacteriophage f1: Location of the Escherichia coli B-specific modification sites☆

Abstract Replicative form DNA of bacteriophage f1 was cleaved into specific fragments by two endonucleases isolated from Hemophilus aegyptius and an endonuclease isolated from H. influenzae . The fragments were ordered so as to construct a circular map of the phage f1 genome by: (1) digesting the isolated restriction fragments with a second restriction enzyme; and (2) testing in a transfection system for the ability of the fragments to rescue amber mutations contained on single-stranded viral DNA that was hybridized to a particular fragment. The genome of bacteriophage f1 contains two SB sites, genetic sites which confer upon a DNA molecule susceptibility to the restriction-modification system of Escherichia coli B. Each of these sites was located on a specific restriction fragment by transfection experiments. In vitro modification of replicative form DNA of f1 and its SB mutants by endonuclease R · Eco B and the subsequent cleavage of the DNA by the Hemophilus endonucleases showed that the B-specific methylation occurs within at least 200 nucleotide pairs of the SB site.

Replication of a plasmid containing two origins of bacteriophage f1

Abstract A chimeric plasmid was constructed that contains a tandem duplication of the bacteriophage f1 origin of DNA replication. This plasmid replicates stably in the absence of phage. When cells carrying this plasmid are infected with f1, two new plasmid-derived DNA species are generated: a smaller, chimeric plasmid containing only one f1 origin of replication, and a miniphage, the genome of which consists of the f1 fragment that was located between the two f1 origins of the original plasmid. These data support the hypothesis (Horiuchi, 1980) that the nucleotide sequence recognized for initiation of plus strand synthesis in f1 DNA replication is also the signal for its termination.

Construction of a microphage variant of filamentous bacteriophage

The intergenic region in the genome of the Ff class of filamentous phage (comprising strains fl, fd and M13) genome constitutes 8% of the viral genome, and has essential functions in DNA replication and phage morphogenesis. The functional domains of this region may be inserted into separate sites of a plasmid to function independently. Here, we demonstrate the construction of a plasmid containing, sequentially, the origin of (+)-strand synthesis, the packaging signal and a terminator of (+)-strand synthesis. When host cells harboring this plasmid (pLS7) are infected with helper phage they produce a microphage particle containing all the structural elements of the mature, native phage. The microphage is 65 A in diameter and about 500 A long. It contains a 221-base single-stranded circle of DNA coated by about 95 copies of the major coat protein (gene 8 protein).

Physical map of defective interfering particles of bacteriophage f1.

Multiple passages of bacteriophage f1 at high multiplicities of infection lead to the accumulation of mutants that overgrow the wild-type phage in a mixed infection but are unable to complete an infectious cycle by themselves. These mutants (defective interfering particles) lack more than 70% of the genome and contain no intact rans-acting cistrons. Genetic and physical mapping of several defectives shows that they contain the origin of phage DNA replication and the large intercistronic space that surrounds this region. A marked selective advantage is given to some of these mutants by a partial duplication in this region of the genome.

Interaction between the replication origin and the initiator protein of the filamentous phage f1: Binding occurs in two steps☆

The replication initiator protein of bacteriophage f1 (gene II protein) binds to the phage origin and forms two complexes that are separable by polyacrylamide gel electrophoresis. Complex I is formed at low gene II protein concentrations, and shows protection from DNase I of about 25 base-pairs (from position +2 to +28 relative to the nicking site) at the center of the minimal origin sequence. Complex II is produced at higher concentrations of the protein, and has about 40 base-pairs (from -7 to +33) protected. On the basis of gel mobility, complex II appears to contain twice the amount of gene II protein as does complex I. The 40 base-pair sequence protected in complex II corresponds to the minimal origin sequence as determined by in-vivo analyses. The central 15 base-pair sequence (from +6 to +20) of the minimal origin consists of two repeats in inverted orientation. This sequence, when cloned into a plasmid, can form complex I, but not complex II. We call this 15 base-pair element the core binding sequence for gene II protein. Methylation interference with the formation of complex I by the wild-type origin indicates that gene II protein contacts six guanine residues located in a symmetric configuration within the core binding sequence. Formation of complex II requires, in addition to the core binding sequence, the adjacent ten base-pair sequence on the right containing a third homologous repeat. A methylation interference experiment performed on complex II indicates that gene II protein interacts homologously with the three repeats. In complex II, gene II protein protects from DNase I digestion not only ten base-pairs on the right but also ten base-pairs on the left of the sequence that is protected in complex I. Footprint analyses of various deletion mutants indicate that the left-most ten base-pairs are protected regardless of their sequence. The site of nicking by gene II protein is located within this region. A model is presented for the binding reaction involving both protein-DNA and protein-protein interactions.

Restructuring the bacteriophage f1 genome: Expression of gene VIII in the intergenic space

Abstract Using in vitro recombinant DNA techniques, we have constructed a derivative of bacteriophage f1 which contains an amber mutation in the major coat protein gene (gene VIII) plus an additional wild-type copy of this gene in the intergenic space near the origin of phage DNA replication. The gene is functional in this new location and is expressed at the same level relative to available transcriptional template DNA (RF) as the corresponding gene in wild-type f1. The implication of this finding in terms of current models of gene expression in the filamentous coliphages is discussed. Intertypic mixing of coat protein monomers derived from the two genes occurs when this phage is grown in an amber suppressor host. The effect of this DNA insertion on replication of the f1 genome is discussed. The concept of restructuring the f1 genome as a means of studying its regulation and expression is also discussed.