Genetic analysis of bacteriophage N4 adsorption

Abstract

We isolated six mutants of Escherichia coli K-12 that were defective in bacteriophage N4 adsorption. We mapped the mutations to four loci designated nfrA through nfrD (N four resistance). nfrA and nfrB were tightly linked to each other and were mapped to min 12 of the E. coli linkage map. nfrC was mapped to min 85, and nfrD was mapped between min 44 and 58. We isolated a clone carrying both nfrA and nfrB and identified its gene products through maxicell analysis of plasmid subclones. The nfrA gene product was an outer membrane protein of 96,000 apparent molecular weight, whereas nfrB encoded an inner-membrane protein of 69,500 apparent molecular weight. The nfrBI mutation did not affect the export of the nfrA gene product to the outer membrane and did not affect the alkaline phosphatase activity of an nfrA-phoA fusion. We propose that nfrA encodes the structural receptor for N4 and that the nfrB gene product may be required for irreversible adsorption and injection of the phage genome and virion-encapsulated RNA polymerase through the inner membrane.Bacteriophage N4 is a lytic phage specific for Escherichia coli K-12 (see reference 15 for a review). It is unique among DNA-containing bacteriophages because transcription of the early region of the genome is independent of the activity of the host RNA polymerase. Indeed, each virion contains, in addition to a 72-kilobase-pair, double-stranded DNA genome, a single-subunit (Mr, 320,000), DNA-dependent RNA polymerase that is responsible for the transcription of the phage early genes. Therefore, the first event in N4 infection requires the injection not only of its genome but also of this very large polypeptide. The study of disruptions of this event may provide information about E. coli membrane structure and penetration of large macromolecular species such as the phage genome and the RNA polymerase through the outer and inner membranes.In this paper we describe the isolation and characterization of E. coli mutants that were defective in N4 adsorption. Unexpectedly, the mutations were mapped to at least four different loci, indicating the involvement of several gene products and a relatively complex process. We describe the characterization of two of these gene products, NfrA and NfrB, in detail and propose roles for each in the process of N4 adsorption and penetration. MATERIALS AND METHODSBacterial strains. heim Biochemicals, Indianapolis, Ind., and New England BioLabs, Inc., Beverly, Mass.Isolation of N4-resistant mutants. E. coli K-12 strain SC1100 was grown to the early stationary phase in LB at 37°C. Cultures (0.1 ml) were plated onto LB medium previously spread with about 3 x 109 PFU of N4. Colonies appearing after overnight incubation at 37°C were purified by restreaking for single colonies onto LB plates spread with N4. Purified colonies were tested for N4 resistance by cross-streaking at 37°C.Genetic techniques. Standard techniques for bacterial conjugation, P1 transduction, and X induction were used (23, 31).We used ATnphoA (9, 20) to construct a gene fusio...

A cytoplasmic protein, NfrC, is required for bacteriophage N4 adsorption

“…We screened a XD69 (30) library of E. coli DNA (generously provided by S. Gottesman) for potential clones of nfrC by using the approach described previously (20). We plated the library for single plaques on a lawn of strain KW8801 (carrying the nfrC2 mutation) which had been seeded with N4.…”

“…Selection for spontaneously occurring mutations which confer N4 resistance has consistently resulted in mutations mapping to four different loci (20), suggesting the involvement of several E. coli gene products and a relatively complex process. Approximately 60% of the mutants map to nfrA and nfrB, two tightly linked loci at 12 min on the E. coli linkage map (2) whose coding regions overlap (21).…”

Two overlapping genes encoding membrane proteins required for bacteriophage N4 adsorption

“…No other open reading frames are present in this region. The calculated molecular sizes for the predicted peptides are 85 kDa for NfrB and 122 kDa for NfrA, which is larger than the apparent molecular sizes of 69.5 and 96 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of maxicell products (12 (5,8).…”

“…The early events occurring during N4 infection require the injection of both its double-stranded DNA genome and a virion-encapsulated 320,000-Da RNA polymerase required for transcription of the early genes. A genetic analysis of N4 adsorption has identified four genes, nfrA to -D, required for this event (10,12). We have previously shown that the two tightly linked genes, ifl-A and nflB, encode an outer membrane protein with an apparent molecular size of 96 kDa and an inner membrane protein with an apparent molecular size of 69.5 kDa, respectively.…”

DNA Sequencing and Transcriptional Analysis of the Kasugamycin Biosynthetic Gene Cluster from Streptomyces kasugaensis M338-M1

“…The deduced KasQ protein (413 amino acids, M r of 45,121, pI of 9.45) had significant similarity (41ϳ45% identity) to UDP-N-acetylglucosamine 2-epimerases from Streptomyces verticillus (Protein ID: AAF68965.1), Escherichia coli (Protein ID: AAC36847.1) [16], Yersinia pestis (Protein ID: CAC93332.1) [17], Ralstonia solanacearum (Protein ID: AAA91626.1) [18] and Salmonella enterica (Protein ID: CAD09396.1) [19]. Bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible epimerization at C-2 of UDP-Nacetylglucosamine (UDP-GlcNAc).…”