Marjorie Russel

An improved filamentous helper phage for generating single-stranded plasmid DNA ☆

Gene cloning in plasmid vectors that contain a filamentous phage intergenic region presents several advantages. However, technical difficulties have been a problem, primarily low yields of packaged single stranded (ss) plasmid DNA from the rapid, small scale procedures usually employed, and ambiguities in sequencing reactions attributed to the contamination by helper phage ss DNA. We report here the construction and some properties of a new f1 helper phage. Using this phage, R408, plasmid ss DNA is packaged and exported preferentially over phage ss DNA, and the absolute yield of plasmid ss DNA is usually increased.

Filnmentous phage assembly

Filamentous phages present a genetically well‐defined system for studying the ordered membrane assembly of five different phage‐encoded proteins around the circular single‐stranded DNA phage genome. Assembly occurs at high efficiency in vivo, catalysed by two phage‐encoded membrane proteins and al least one host protein, thioredoxin. This review presents a description of the virion and its cytoplasmic precursor and summarizes the results of genetic and biochemical experiments that are beginning to elucidate the rote of the three morphogenetic proteins. The recent discovery of bacterial transport proteins with homology to a phage morphogenetic protein located in the outer membrane suggests the existence of a common mechanism for moving complex macromolecules across bacterial membranes.

The Salmonella typhimurium InvH protein is an outer membrane lipoprotein required for the proper localization of InvG.

The secretion of pathogenicity factors by Salmonella typhimurium is mediated by a type III secretion system that includes an outer membrane protein of the secretin family. Related secretins are also required for f1 phage assembly and type II secretion. When the C‐terminal 43 amino acids of the S. typhimurium secretin InvG are added to f1 pIV, the chimeric f1 pIV‐′InvG43 protein becomes dependent on the co‐expression of another gene, invH, for function in phage assembly. [3H]‐palmitic acid labelling, globomycin sensitivity and density gradient flotation were used to demonstrate that InvH is an outer membrane lipoprotein that is processed by signal peptidase II. A complex between chimeric f1 pIV‐′InvG43 and InvH was demonstrated in vivo. InvH was shown to be required for the proper localization of InvG in the outer membrane and for the secretion of the virulence factor SipC. These results suggest that InvH and InvG are part of the functional outer membrane translocation complex in type III secretion systems.

Filamentous phage assembly: variation on a protein export theme

Biogenesis of both filamentous phage and type-IV pili involves the assembly of many copies of a small, integral inner membrane protein (the phage major coat protein or pilin) into a helical, tubular array that passes through the outer membrane. The occurrence of related proteins required for assembly and export in both systems suggests that there may be similarities at the mechanistic level as well. This report summarizes the properties of filamentous phage and the proteins required for their assembly, with particular emphasis on features they may share with bacterial protein export and pilus biogenesis systems, and it presents evidence that supports the hypothesis that one of the phage proteins functions as an outer membrane export channel.

Moving through the membrane with filamentous phages.

Filamentous phages are small, highly evolved parasites that can reproduce and disseminate without killing their host. During assembly, virion proteins are transferred from the host membrane to the single-stranded DNA phase genome and simultaneously secreted from the cell. Filamentous phage assembly shares certain features with bacterial processes responsible for the assembly of cell-surface structures and for extracellular protein secretion.

Structure of the Filamentous Phage pIV Multimer by Cryo-electron Microscopy

The homo-multimeric pIV protein constitutes a channel required for the assembly and export of filamentous phage across the outer membrane of Escherichia coli. We present a 22 A-resolution three-dimensional reconstruction of detergent-solubilized pIV by cryo-electron microscopy associated with image analysis. The structure reveals a barrel-like complex, 13.5 nm in diameter and 24 nm in length, with D14 point-group symmetry, consisting of a dimer of unit multimers. Side views of each unit multimer exhibit three cylindrical domains named the N-ring, the M-ring and the C-ring. Gold labeling of pIV engineered to contain a single cysteine residue near the N or C terminus unambiguously identified the N-terminal region as the N-ring, and the C-terminal region was inferred to make up the C-ring. A large pore, ranging in inner diameter from 6.0 nm to 8.8 nm, runs through the middle of the multimer, but a central domain, the pore gate, blocks it. Moreover, the pore diameter at the N-ring is smaller than the phage particle. We therefore propose that the pIV multimer undergoes a large conformational change during phage transport, with reorganization of the central domain to open the pore, and widening at the N-ring in order to accommodate the 6.5 nm diameter phage particle.

The C‐terminal domain of the secretin PulD contains the binding site for its cognate chaperone, PulS, and confers PulS dependence on pIVf1 function

Related outer membrane proteins, termed secretins, participate in the secretion of macromolecules across the outer membrane of many Gram‐negative bacteria. In the pullulanase‐secretion system, PulS, an outer membrane‐associated lipoprotein, is required both for the integrity and the proper outer membrane localization of the PulD secretin. Here we show that the PulS‐binding site is located within the C‐terminal 65 residues of PulD. Addition of this domain to the filamentous phage secretin, pIV, or to the unrelated maltose‐binding protein rendered both proteins dependent on PulS for stability. A chimeric protein composed of bacteriophage f1 pIV and the C‐terminal domain of PulD required properly localized PulS to support phage assembly. An in vivo complex formed between the pIV‐PulD65 chimera and PulS was detected by co‐immunoprecipitation and by affinity chromatography.

Secretion and membrane integration of a filamentous phage-encoded morphogenetic protein☆

The filamentous phage-encoded gene IV protein is required at high levels for virus assembly, although it is not a constituent of the virion. It is an integral membrane protein that does not contain an extended hydrophobic region of the kind often required for stable integration in the inner membrane. Rather, like a number of Escherichia coli outer membrane proteins, pIV is rich in charged amino acid residues and is predicted to consist of extensive beta-sheet structures. In phage-producing cells, pIV is primarily detected in the outer membrane, while in cells that produce it from the cloned gene, pIV is found in both the inner and outer membranes. The protein is synthesized as a precursor. Following cleavage of the signal sequence and translocation into the periplasm, the mature form is initially found as a soluble species. Soluble pIV then integrates into the membrane with a half-time of one to two minutes. Neither phage assembly nor other phage proteins are needed for this membrane integration, and phage assembly does not require the presence of the soluble form. The gene IV protein may be part of the structure through which the assembling phage is extruded.

Mutants at conserved positions in gene IV, a gene required for assembly and secretion of filamentous phages.

The filamentous phage protein pIV is required for assembly and secretion of the virus and possesses regions homologous to those found in a number of Gram‐negative bacterial proteins that are essential components of a widely distributed extracellular protein‐export system. These proteins form multimers that may constitute an outer membrane channel that allows phage/protein egress. Three sets of f1 gene IV mutants were isolated at positions that are absolutely (G355 and P375) or largely (F381) conserved amongst the 16 currently known family members. The G355 mutants were non‐functional, interfered with assembly of plV+ phage, and made Escherichia coli highly sensitive to deoxycholate. The P375 mutants were non‐functional and defective in multimerization. Many of the F381 mutants retained substantial function, and even those in which charged residues had been introduced supported some phage assembly. Some inferences about the roles of these conserved amino acids are made from the mutant phenotypes.

Processing of filamentous phage pre-coat protein. Effect of sequence variations near the signal peptidase cleavage site.

Abstract The amber lesion of am 8H1, the only conditional lethal mutant in a filamentous phage coat protein gene, lies two codons after the signal peptidase cleavage site (Boeke & Model, 1979). We sequenced the DNA of 15 independently isolated pseudorevertants of this mutant. We studied the production of unprocessed and processed coat protein in pseudorevertant-infected cells and in amber mutant-infected suppressor strains. These studies show that serine, glutamine, tyrosine or leucine residues can replace the glutamic acid residue found in the wild-type coat protein at position 2. Reversion to tyrosine or leucine was always accompanied by a second mutation, which leads to the replacement of asparagine by aspartic acid at position 12. Leucine and, to a lesser extent, tyrosine seem to inhibit processing since pre-coat protein accumulates in the infected cells. Filamentous phage particles were shown to migrate on agarose gels with a mobility that reflects the charge of the “external”, N-terminal domain of their major coat protein.