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

Abstract

At least four genes are required for irreversible adsorption of bacteriophage N4. nfrA and nfrB have been characterized previously and encode an outer membrane protein and inner membrane protein, respectively. The nfrC gene product is characterized in detail in this study. We have mapped the nfrD locus to min 52 on the Escherichia coli linkage map. Maxicell analysis of nfrC and a null allele (nfrC2) cloned into a high-copy-number plasmid shows its gene product to be 42 kDa in size. We determined the nfrC nucleotide sequence which predicts a gene product of 42 kDa. Western blots (immunoblots) of Escherichia coli proteins after cellular fractionation show NfrC to be a cytoplasmic protein which is required for irreversible bacteriophage N4 adsorption, an event occurring at the cell surface.Bacteriophage N4 is a lytic phage specific for Escherichia coli K-12 (19). It is unique among the DNA-containing bacteriophages because transcription of the early region of the genome is independent of the activity of the host RNA polymerase. Instead, transcription of the early phage genes is carried out by a virion-encapsulated DNA-dependent RNA polymerase (12). Therefore, the first event in N4 infection requires the injection of both its 72-kbp double-stranded DNA genome and the virion RNA polymerase, a large single-subunit enzyme (M,, 320,000). We have been conducting a genetic analysis of N4 adsorption to learn more about E. coli membrane structure and the penetration of large macromolecular species such as the phage genome and RNA polymerase through the outer and inner membranes.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). These genes have been previously characterized as an outer membrane protein (NfrA) which presumably is the structural receptor for N4 and an inner membrane protein (NfrB). The remaining mutations map to two unlinked loci, nfrC at 85 min and nfrD, which, until this study, had not been precisely mapped.In this article, we characterize the nfrC gene product in detail and present its cloning and the determination of its nucleotide sequence. MATERIALS AND METHODSBacterial strains. (29,38).Cloning nfrC. 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. Clear plaques representing phage potentially complementing the adsorption defect were picked and purified. Thosc that were turbid in the absence of N4 after replating were lysogenized into KW8801 and tested for their ability to complement the nfrC2 mutation and to integrate at ...

Characterization of the locus encoding the Streptococcus pneumoniae type 19F capsular polysaccharide biosynthetic pathway

“…RffE is a UDP-N-acetylglucosamine-2-epimerase, and functions in the synthesis of UDP-N-acetylmannosamine, which is a component of the enterobacterial common antigen (Meier-Dieter et al, 1990). Whilst the precise mechanism is not understood, E. coli rffE (nfrC ) mutants are known to be resistant to infection with bacteriophage N4 (Kiino et al, 1993). To examine whether cps19fK is capable of complementing rffE mutations, we transformed the rffE mutant KI8828 (Kiino et al, 1993) with pJCP470 (which contains only the complete cps19fK ORF), or with pGEM-7Zf(þ).…”

“…Whilst the precise mechanism is not understood, E. coli rffE (nfrC ) mutants are known to be resistant to infection with bacteriophage N4 (Kiino et al, 1993). To examine whether cps19fK is capable of complementing rffE mutations, we transformed the rffE mutant KI8828 (Kiino et al, 1993) with pJCP470 (which contains only the complete cps19fK ORF), or with pGEM-7Zf(þ). The various strains, including the rffE wild-type parent E. coli MC4100 (Silhavy et al, 1984), were then examined for susceptibility to infection with N4 phage (Fig.…”

A Complementary Bioinformatics Approach to Identify Potential Plant Cell Wall Glycosyltransferase-Encoding Genes

“…Furthermore, a DxD motive (as described below) is not found in ATUXS2. Whereas the E. coli UDP-N-acetyl-Dglucosamine 2-epimerase (Kiino et al 1993; P27828) as expected do not adopt a typical type II membrane protein structure when run through the TMHMM version 2.0 server (Filter I), it is predicted to belong to the UDP-glycosyltransferase/glycogen phosphorylase superfamily by the SUPERFAMILY prediction server and is predicted to adopt a GT-B fold by mGen-THREADER. However, a DxD motif as described below is not found.…”

Two overlapping genes encoding membrane proteins required for bacteriophage N4 adsorption

“…Both host and phage proteins are thought to constitute the channels. To our knowledge, however, only N4 has been shown to require a specific cytoplasmic protein for the process of adsorption (10). Since N4 must translocate both its DNA genome and the virion RNA polymerase, the singular requirement for numerous gene products may be a reflection of the complexity of the N4 life cycle.…”

“…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.…”