The lysis function of RNA bacteriophage Qbeta is mediated by the maturation (A2) protein

Complete or partial cDNA sequences of the RNA bacteriophage Qbeta were cloned in plasmids under the control of the lambdaP(L) promoter to allow regulated expression in Escherichia coli harbouring the gene for the temperature-sensitive lambdaCI857 repressor. Induction of the complete Qbeta sequence leads to a 100-fold increase in phage production, accompanied by cell lysis. Induction of the 5'-terminal sequence containing the intact maturation protein (A2) cistron also causes cell lysis. Alterations of the A2 cistron, leading to proteins either devoid of approximately 20% of the C-terminal region or of six internal amino acids, abolish the lysis function. Expression of other cistrons in addition to the A2 cistron does not enhance host lysis. Thus, in Qbeta, the A2 protein, in addition to its functions as maturation protein, appears to trigger cell lysis. This contrasts with the situation in the distantly related group I RNA phages such as f2 and MS2 where a small lysis polypeptide is coded for by a region overlapping the end of the coat gene and the beginning of the replicase gene.     

Structural relatedness of lysis proteins from colicinogenic plasmids and icosahedral coliphages

The host-lysis-inducing functions of phi X174 protein E and MS2 protein L were recently shown to reside on the N-terminal and C-terminal halves of the two respective lysis proteins. In the present study it is shown that the small lysis proteins encoded in various colicinogenic plasmids share local sequence similarities and certain structural characteristics with the essential peptides of their coliphage-coded counterparts. Despite their dissimilar sizes and origins, it is suggested that the colicinogenic lysis proteins are functionally analogous and evolutionarily related to those of icosahedral single- stranded DNA and RNA phages.      

Properties of Caulobacter Ribonucleic Acid Bacteriophage φCb5

The ribonucleic acid (RNA) bacteriophage phiCb5, which specifically infects only one form of the dimorphic stalked bacterium Caulobacter crescentus, has been obtained in high yield. Since the phage is extremely salt-sensitive, a purification procedure was devised which avoided contact with solutions of high ionic strength. Phage phiCb5 was studied with respect to the physical and chemical properties of both the phage and its RNA. In an electron microscope, the phage particles appear as small polyhedra, 23 nm in diameter. The phage is similar to the Escherichia coli RNA phages in that it (i) sediments at an S(20, w) of 70.6S, (ii) is composed of a single molecule of single-stranded RNA and a protein coat, (iii) contains two structural proteins, and (iv) apparently contains the genetic capacity to code for a coat protein subunit, a maturation-like protein, and an RNA polymerase. Phage phiCb5 differs from the E. coli RNA phages in (i) host specificity, (ii) salt sensitivity, and (iii) the presence of histidine, but not methionine, in the coat protein.     

RNA recognition site of PP7 coat protein

The coat proteins of different single-strand RNA phages use a common protein tertiary structural framework to recognize different RNA hairpins and thus offer a natural model for understanding the molecular basis of RNA-binding specificity. Here we describe the RNA structural requirements for binding to the coat protein of bacteriophage PP7, an RNA phage of Pseudomonas. Its recognition specificity differs substantially from those of the coat proteins of its previously characterized relatives such as the coliphages MS2 and Qbeta. Using designed variants of the wild-type RNA, and selection of binding-competent sequences from random RNA sequence libraries (i.e. SELEX) we find that tight binding to PP7 coat protein is favored by the existence of an 8 bp hairpin with a bulged purine on its 5' side separated by 4 bp from a 6 nt loop having the sequence Pu-U-A-G/U-G-Pu. However, another structural class possessing only some of these features is capable of binding almost as tightly.     

Lysis gene t of T-even bacteriophages: evidence that colicins and bacteriophage genes have common ancestors.

The lysis gene t of the T-even-like bacteriophage K3 has been cloned and sequenced. The gene codes for a protein with a predicted molecular weight of 25,200. Expression of the complete lysis protein was impossible, but peptides complementing T4 amber mutants in t are described. No known lysis protein of other phages is homologous to protein T. Also, the Escherichia coli phospholipase A is different from protein T. CelB, the lysis protein of the colicin E2 operon, shows a similarity to protein T. Sequences of colicins A, E1, and E2 are related to gene 38 sequences, the gene preceding t and coding for the phage adhesin. A common origin for colicin genes and phage genes is discussed, and a protein region in colicins that is responsible for receptor recognition is predicted.     

Recognition of diverse RNAs by a single protein structural framework

The coat proteins of different single-strand RNA phages utilize a common structural framework to recognize different RNA targets, making them suitable models for studies of RNA-protein recognition generally, especially for the class of proteins that bind RNA on a beta-sheet surface. Here we show that structurally distinct molecules are capable of satisfying the requirements for binding to Qbeta coat protein. Although the predicted secondary structures of the RNAs differ markedly, we contend that they are approximately equivalent structurally in their complexes with coat protein. Based on our prior observations that the RNA-binding specificities of Qbeta and MS2 coat proteins can be interconverted with as few as one amino acid substitution each, and taking into account details of the structures of complexes of MS2 coat protein with wild-type and aptamer RNAs, we propose a model for the Qbeta coat protein-RNA complex.     

Two-stage model for integration of the lysis protein E of ΦX174 into the cell envelope of Escherichia coli

Abstract: As a tool for determining the topology of the small, 91-amino acid ΦX174 lysis protein E within the envelope complex of Escherichia coli , a lysis active fusion of protein E with streptavidin (E-FXa-StrpA) was used. The E-FXa-StrpA fusion protein was visualised using immune electron microscopy with gold-conjugated anti-streptavidin antibodies within the envelope complex in different orientations. At the distinct areas of lysis characteristic for protein E, the C-terminal end of the fusion protein was detected at the surface of the outer membrane, whereas at other areas the C-terminal portion of the protein was located at the cytoplasmic side of the inner membrane. These results suggest that a conformational change of protein E is necessary to induce the lysis process, an assumption supported by proteinase K protection studies. The immune electron microscopic data and the proteinase K accessibility studies of the E-FXa-StrA fusion protein were used for the working model of the E-mediated lysis divided into three phases: phase 1 is characterised by integration of protein E into the inner membrane without a cytoplasmic status in a conformation with its C-terminal part facing the cytoplasmic side; phase 2 is characterised by a conformational change of the protein transferring the C-terminus across the inner membrane; phase 3 is characterised by a fusion of the inner and outer membranes and is associated with a transfer of the C-terminal domain of protein E towards the surface of the outer membrane of E. coli.     

The host-dependent restriction of growth of an RNA coliphage FI.

Phage FIC is a spontaneous host-dependent mutant of phage FI which is classified into the fourth group of RNA Escherichia coli phages (RNA coliphages). The mutant phage (FIC) grows normally in E. coli strain Q13 (permissive host), but poorly in strain A/lambda (non-permissive host) (9). Attempts to elucidate the regulatory mechanism of growth of the mutant phage in the non-permissive host revealed the following: (a) growth of the mutant phage was specifically restricted in E. coli strains that have certain suppressor genes for amber mutation; (b) the mutant phage RNA (FIC-RNA) could not produce progeny in the spheroplasts of the non-permissive host; (c) adsorption of the mutant phage to, and penetration of the mutant phage RNA into, the non-permissive host were normal; and (d) biosynthesis of the phage-specific late protein and RNA did not occur in the non-permissive host. Based on these results we conclude that phage FIC is a spontaneous azure-type mutant of the fourth group of RNA coliphage FI.     

Complete genomic sequence of bacteriophage phiEcoM-GJ1, a novel phage that has myovirus morphology and a podovirus-like RNA polymerase

The complete genome of phiEcoM-GJ1, a lytic phage that attacks porcine enterotoxigenic Escherichia coli of serotype O149:H10:F4, was sequenced and analyzed. The morphology of the phage and the identity of the structural proteins were also determined. The genome consisted of 52,975 bp with a G+C content of 44% and was terminally redundant and circularly permuted. Seventy-five potential open reading frames (ORFs) were identified and annotated, but only 29 possessed homologs. The proteins of five ORFs showed homology with proteins of phages of the family Myoviridae, nine with proteins of phages of the family Podoviridae, and six with proteins of phages of the family Siphoviridae. ORF 1 encoded a T7-like single-subunit RNA polymerase and was preceded by a putative E. coli sigma(70)-like promoter. Nine putative phage promoters were detected throughout the genome. The genome included a tRNA gene of 95 bp that had a putative 18-bp intron. The phage morphology was typical of phages of the family Myoviridae, with an icosahedral head, a neck, and a long contractile tail with tail fibers. The analysis shows that phiEcoM-GJ1 is unique, having the morphology of the Myoviridae, a gene for RNA polymerase, which is characteristic of phages of the T7 group of the Podoviridae, and several genes that encode proteins with homology to proteins of phages of the family Siphoviridae.     

Roles and applications of small heat shock proteins in the production of recombinant proteins in Escherichia coli

Proteome profiling of the inclusion body (IB) fraction of recombinant proteins produced in Escherichia coli suggested that two small heat shock proteins, IbpA and IbpB, are the major proteins associated with IBs. In this study, we demonstrate that IbpA and IbpB facilitate the production of recombinant proteins in E. coli and play important roles in protecting recombinant proteins from degradation by cytoplasmic proteases. We examined the cytosolic production, and Tat- or Sec-dependent secretion of the enhanced green fluorescent protein (EGFP) in wild type, ibpAB(-) mutant, and ibpAB-amplified E. coli strains. Analysis of fluorescence histograms and confocal microscopic imaging revealed that over-expression of the ibpA and/or ibpB genes enhanced cytosolic EGFP production whereas knocking out the ibpAB genes enhanced secretory production. This strategy seems to be generally applicable as it was successfully employed for the enhanced cytosolic or secretory production of several other recombinant proteins in E. coli.     

φX174 lysis requires slyD, a host gene which is related to the FKBP family of peptidyl-prolyl cis-trans isomerases

Abstract: Recessive mutations in the slyD (sensitivity to ly sis) gene were isolated by selecting for survival after induction of the cloned lysis gene E of bacteriophage φX174 [1]. The slyD ⁻ mutation, transduced into the normal φX174 host, Escherichia coli C, confers an absolute block on the plaque-forming ability of the wild-type phage, indicating that slyD is required for E function. slyD encodes a protein with 196 residues. A segment corresponding to the first 142 residues of the predicted SlyD protein has significant similarity throughout its length to the FKBP family of peptidyl-prolyl cis-trans isomerases, or rotamases. The C-terminal 46 codons of slyD encode a remarkable histidine-rich peptide which is a metal-binding domain [2]. This sequence is dispensable for slyD function in E -mediated lysis. Although there is no obvious phenotype associated with the slyD ⁻ genotype other than the resistance to E -mediated lysis, overexpression of slyD causes cells to filament and to increase significantly in diameter. Mutations in φX174 can restore the plaque-forming ability of the phage on a slyD ⁻ host. These pos ( p lates on s lyD) mutants plate on E. coli C wild-type and slyD ⁻. A model for SlyD involvement in E function and the role of SlyD in the cell is discussed.     

Crystal Structure of the Bacteriophage Qβ Coat Protein in Complex with the RNA Operator of the Replicase Gene

The coat proteins of single-stranded RNA bacteriophages specifically recognize and bind to a hairpin structure in their genome at the beginning of the replicase gene. The interaction serves to repress the synthesis of the replicase enzyme late in infection and contributes to the specific encapsidation of phage RNA. While this mechanism is conserved throughout the Leviviridae family, the coat protein and operator sequences from different phages show remarkable variation, serving as prime examples for the co-evolution of protein and RNA structure. To better understand the protein–RNA interactions in this virus family, we have determined the three-dimensional structure of the coat protein from bacteriophage Qβ bound to its cognate translational operator. The RNA binding mode of Qβ coat protein shares several features with that of the widely studied phage MS2, but only one nucleotide base in the hairpin loop makes sequence-specific contacts with the protein. Unlike in other RNA phages, the Qβ coat protein does not utilize an adenine-recognition pocket for binding a bulged adenine base in the hairpin stem but instead uses a stacking interaction with a tyrosine side chain to accommodate the base. The extended loop between β strands E and F of Qβ coat protein makes contacts with the lower part of the RNA stem, explaining the greater length dependence of the RNA helix for optimal binding to the protein. Consequently, the complex structure allows the proposal of a mechanism by which the Qβ coat protein recognizes and discriminates in favor of its cognate RNA.     

An auto-inducible Escherichia coli lysis system controlled by magnesium

The Escherichia coli (E. coli) prokaryotic expression system is widely used in the field of biology. The currently adopted processes for inducing cell wall rupture, in order to release the target protein, are complex and cumbersome. We developed an auto-inducible E. coli lysis system that is regulated by exogenous magnesium ion (Mg²⁺) concentration. This system is composed of a strictly Mg²⁺-regulated promoter Pmgt from the mgtB gene of Salmonella typhimurium, and the lysis genes from λ bacteriophage. Both the wild type and Sam7-mutant lysis genes were inducibly expressed in E. coli under Mg²⁺-depleted conditions. The former caused a rapid lysis, while the latter induced very mild lysis of the host strains. However, rapid lysis was observed when the latter was resuspended in Tris–EDTA buffer. Finally, the inducible lysis cassette containing wild type lysis gene was introduced into an expression plasmid expressing GFP gene and efficient lysis of the host E. coli strain and subsequent release of the target protein was achieved in Mg²⁺-depleted conditions. Collectively, the current study indicates that this novel inducible lysis system could have attractive applications in the field of protein expression and provides new insights for the development of bacterium-based vaccines.     

Complete genome sequence of the broad host range single-stranded RNA phage PRR1 places it in the Levivirus genus with characteristics shared with Alloleviviruses

Single-stranded RNA (ssRNA) bacteriophages of the family Leviviridae infect gram-negative bacteria. They are restricted to a single host genus. Phage PRR1 is an exception, having a broad host range due to the promiscuity of the receptor encoded by the IncP plasmid. Here we report the complete genome sequence of PRR1. Three proteins homologous with those of other ssRNA phages, i.e., maturation, coat, and replicase proteins, were identified. A fourth protein has a lysis function. Comparison of PRR1 with other members of the Leviviridae family places PRR1 in the genus Levivirus with some characteristics more similar to those of members of the genus Allolevivirus.     

The accuracy of Q beta RNA translation. 1. Errors during the synthesis of Q beta proteins by intact Escherichia coli cells

The fidelity of Q beta RNA translation by intact Escherichia coli cells has been studied. After infection, host protein synthesis was eliminated by adding rifampicin and the radioactive, phage-specified, proteins separated by one or two-dimensional gel electrophoresis. Labelled histidine and tryptophan were incorporated into the phage coat protein, whose message does not specify these amino acids, at a frequency of 0.09-0.13 per molecule. Errors leading to a change in the pI of the coat protein occurred at a rate of 0.05 per molecule, while the coat protein UGA stop codon was misread 6.5% of the time. These error rates are similar to data in some recent publications but much higher than the canonical 3-4 X 10(-4). They further provide a reference point in vivo to which the translation of the same message by E. coli extracts can be compared.