Ongoing phenotypic and genomic changes in experimental coevolution of RNA bacteriophage Qβ and Escherichia coli

According to the Red Queen hypothesis or arms race dynamics, coevolution drives continuous adaptation and counter-adaptation. Experimental models under simplified environments consisting of bacteria and bacteriophages have been used to analyze the ongoing process of coevolution, but the analysis of both parasites and their hosts in ongoing adaptation and counter-adaptation remained to be performed at the levels of population dynamics and molecular evolution to understand how the phenotypes and genotypes of coevolving parasite-host pairs change through the arms race. Copropagation experiments with Escherichia coli and the lytic RNA bacteriophage Qβ in a spatially unstructured environment revealed coexistence for 54 days (equivalent to 163-165 replication generations of Qβ) and fitness analysis indicated that they were in an arms race. E. coli first adapted by developing partial resistance to infection and later increasing specific growth rate. The phage counter-adapted by improving release efficiency with a change in host specificity and decrease in virulence. Whole-genome analysis indicated that the phage accumulated 7.5 mutations, mainly in the A2 gene, 3.4-fold faster than in Qβ propagated alone. E. coli showed fixation of two mutations (in traQ and csdA) faster than in sole E. coli experimental evolution. These observations suggest that the virus and its host can coexist in an evolutionary arms race, despite a difference in genome mutability (i.e., mutations per genome per replication) of approximately one to three orders of magnitude.     

Site-directed mutagenesis of a family 42 β-galactosidase from an antarctic bacterium

Site directed mutagenesis was used to modify the active site of a cold active beta-galactosidase taken from an Antarctic psychrotolerant Planococcus Bacterial isolate. The goal was to modify the active site such that there would be an increase in activity on certain substrates which showed little to no activity with the wild type enzyme. A total of 5 mutant enzymes were constructed with amino acid changes based on an analysis done via homology modeling. All 5 modified enzymes were assayed using 14 different nitrophenol substrates. In most cases there was a loss of activity on substrates that showed activity with the wild type enzymes. None of the expected activity was observed with any of the mutants, possibly in part due to a decrease in hydrogen bonding between the active site and the substrates. With the substrates p-nitrophenyl-β-d-galacturonide and p-nitrophenyl-α-d-glucopyranoside we saw increased activity. With one of the mutants we measured a 320% increase in activity on p-nitrophenyl-β-d-galacturonide. Two other mutants showed activity on p-nitrophenyl-α-d-glucopyranoside, which showed no activity at all with the wild type enzyme.     

The binding site for coat protein on bacteriophage Qβ RNA

The site of interaction of phage Qβ coat protein with Qβ RNA was determined by ribonuclease T₁ degradation of complexes of coat protein and [^{3 2}P]-RNA obtained by codialysis of the components from urea into buffer solutions. The degraded complexes were recovered by filtration through nitrocellulose filters, and bound [^{3 2}P] RNA fragments were extracted and separated by polyacrylamide gel electrophoresis. Fingerprinting and further sequence analysis established that the three main fragments obtained (chain lengths 88, 71 and 27 nucleotides) all consist of sequences extending from the intercistronic region to the beginning of the replicase cistron. These results suggest that in the replication of Qβ, as in the case of R17, coat protein acts as a translational repressor by binding to the ribosomal initiation site of the replicase cistron.     

Quantitative analysis of the bacteriophage Qβ infection cycle

In this study, the infection cycle of bacteriophage Qβ was investigated. Adsorption of bacteriophage Qβ to Escherichia coli is explained in terms of a collision reaction, the rate constant of which was estimated to be 4 × 10^− 10 ml/cells/min. In infected cells, approximately 130 molecules of β-subunit and 2 × 10⁵ molecules of coat protein were translated in 15 min. Replication of Qβ RNA proceeded in 2 steps—an exponential phase until 20 min and a non-exponential phase after 30 min. Prior to the burst of infected cells, phage RNAs and coat proteins accumulated in the cells at an average of up to 2300 molecules and 5 × 10⁵ molecules, respectively. An average of 90 infectious phage particles per infected cell was released during a single infection cycle up to 105 min.     

Secondary structure of Qβ RNA

The complete set of possible secondary structures of a variant Qβ RNA sequenced by Schaffner has been found using a computer program which allows G-U pairing as well as the usual Watson-Crick A-U and G-C pairing. Of special interest are those secondary structures with the highest double-strandedness. Omitting G-U pairing, we find the structure with the maximum double-strandedness has a pairing of 62% and exhibits a similarity to the clover leaf structure of tRNA. Including G-U pairing, the complementary strands of RNA are asymmetrical. We find maximum pairings of 71% for both the plus and minus strands. These structures also exhibit a cloverleaf structure. A similar analysis has been carried out for the secondary structure of a larger Qβ variant sequenced by Mills, Kramer and Spiegelman, but in this case there are a large number of secondary structures with the same maximum number of pairs and it is therefore not possible to select a unique structure with the maximum double-strandedness.     

Oligonucleotide Sequence of Replicase Initiation Site in Qβ RNA

THE single stranded RNA genome of bacteriophage Qβ has been variously estimated to consist of from 3,500¹ to 4,500² nucleotides. It contains three known cistrons³, which correspond to three of the four Qβ-specific proteins synthesized in vivo and in vitro^4–6. These are: (1) the gene for the maturation or A protein (molecular weight 41,000 (refs. 4, 5)), (2) that for the major coat protein of the virus (molecular weight 14,000 (ref. 9)) and (3) the gene for the phage-specific subunit of the Qβ replicase (molecular weight 64,000 (ref. 10) or 69,000 (ref. 24)), listed in the probable order^7,8 that they occur on the Qβ RNA. The fourth Qβ-specific protein, A₁ or IIb (molecular weight 36,000 (refs. 4–6, 10)), has recently been shown by Weiner and Weber to have an N-terminal sequence which is identical (for eight amino-acids) to that of the coat protein⁷. Because increased amounts of A₁ appear in virus particles grown in cells containing a UGA suppressor, Weiner and Weber postulate⁷ that this protein is the product of natural read-through at the UGA termination signal of the Qβ coat cistron. Such read-through (involving about 600 nucleotides) could occur entirely within a large “intercistronic” region between the coat and replicase genes, or could involve translation, either in or out of phase, of the replicase cistron. In hopes of distinguishing between these alternatives, I have isolated and examined the nucleotide sequence of the region surrounding the initiator codon of the Qβ replicase gene.     

Site-directed mutagenesis in DNA: Generation of point mutations in cloned β globin complementary DNA at the positions corresponding to amino acids 121 to 123

We have utilized the principle of site-directed mutagenesis, previously applied to the RNA of bacteriophage Qβ, to generate nucleotide transitions in a predetermined region of DNA. Plasmid PβG, which contains an almost complete DNA copy of rabbit β globin messenger RNA, was nicked at the EcoRI site which is located within the globin gene, in a region corresponding to amino acids 121 and 122. Substrate-limited nick translation using DNA polymerase I and N⁴-hydroxydCTP, dCTP and dATP led to the replacement of TMP residues by the nucleotide analog in the immediate vicinity of the nicks. The substituted DNA was amplified in vivo, treated with EcoRI and retransfected. 1.9% of the amplified DNA was found to be EcoRI-resistant. Nucleotide sequence analysis of the critical region of six EcoRI-resistant isolates revealed that two plasmids had one, three had two and one had three A · T → G · C transitions, all located within the substituted region. No point mutations (< 3 × 10^?3%) were found in control preparations; however, a small number of deletion mutants, lacking the EcoRI site, were isolated.     

Site-directed mutagenesis of putative active-site residues of Bacillus stearothermophilus α-amylase

Putative catalytic residues of the thermostable Bacillus stearothermophilus -amylase derived by sequence analysis and computer modeling were tested by site-directed mutagenesis. The conservative mutations produced were Asp-234-Glu, Glu-264-Asp, and Asp-331-Asn. The corresponding amino acids have been proposed to act in acid-base catalysis in the Aspergillus oryzae and porcine pancreatic -amylase. Isoelectric focusing and immunodiffusion studies showed that, although inactive, the mutant proteins have conformations similar to the wild type enzyme. The cause of inactivation is presumably a steric clash or alteration of a catalytic amino acid in the case of Asp-234-Glu and a mutation of a catalytic residue in the mutants Glu-264-Asp and Asp-331-Asn.Abbreviations BStA Bacillus stearothermophilus -amylase - PPA porcine pancreatic -amylase - TAA Aspergillus oryzae -amylase     

The 3′-termini of bacteriophage Qβ plus and minus strands

The 3′-end groups of bacteriophage Qβ plus and minus strands of different origin have been determined by terminal labelling with KB⁸H₄ (or NaB³H₄) following oxidation with sodium periodate. Qβ minus strands, as well as Qβ plus strands (extracted from the viral particle or from infected bacteria), terminate predominantly in adenosine, and to a lesser degree in cytidine. Qβ RNA synthesized in vitro by the purified Qβ replicase system also has mostly adenosine at the 3′-terminus, whether (a) Qβ RNA, (b) Qβ RNA from which the 3′-terminal nucleotide had been removed chemically, or (c) denatured double-stranded Qβ RNA (in the absence of factors), was used as template. The same enzyme system, however, was unable to transfer an adenylate residue from [α-³²P]ATP to Qβ RNA lacking the 3′-terminal adenosine. It is concluded that free, 3′-adenosineless Qβ RNA is not an intermediate in Qβ synthesis and that only nascent Qβ RNA can serve as an acceptor for the 3′-terminal pA.     

Ribosome Binding Site of Qβ RNA Polymerase Cistron

IN conditions of polypeptide chain initiation, 70S ribosomes bind to intact bacteriophage RNA predominantly at the initiation region of the coat protein cistron^1,2. The binding sites of the A protein and RNA polymerase cistrons are fully available only after modification of the secondary or tertiary structure of the RNA^2,3.     

The 3′-linked terminus of Qβ RNA

The 3′-linked terminal nucleotide, pppGp, of Qβ RNA was found only in the nonanucleotides when a pancreatic ribonuclease digest of uniformly ³²P-labeled Qβ RNA was chromatographed on DEAE-cellulose in the presence of 7 M urea (pH 7.4). The terminal oligonucleotide was distinguished from the nonanucleotides derived from internal positions of the RNA by dephosphorylation with alkaline phosphomonoesterase, and chromatography on DEAE-cellulose. Alkaline and enzymic degradation of the separated terminal oligonucleotide showed that the structure of this oligonucleotide is ppp(Gp)₄ApCp. Thus a 5′-phosphorylated compound of 11 negative charges migrates with typical nonanucleotides carrying 10 negative charges. In a number of preparations of Qβ RNA, no 3′-linked terminal pppGp was detected. One such preparation was actually found to terminate in unphosphorylated A−.     

Influence of SOS repair on the specificity of radiation mutagenesis in bacteriophage ?X174

Summary The ochre mutant oc9 of bacteriophage X174 was irradiated with -rays and the revertants were assayed on unirradiated and UV-irradiated host bacteria carrying an amber suppressor. The yield of revertants (amber+wild type) was higher on UV-irradiated than on unirradiated bacteria, showing that -irradiated X174 was subjected to W-mutagenesis.For oc9 -irradiated in the presence of oxygen the fraction of amber mutants among the revertants was lower when mutants were scored on UV-irradiated bacteria than when assayed on unirradiated indicator cells. The same fraction of ambers was obtained when mutants were assayed on unirradiated and UV-irradiated samples of a recA indicator strain. UV-irradiated X174 showed a similar phenomenon. These results suggest that the specificity with regard to insertion of bases opposite radiation damage in X174 DNA is different for host cells in which SOS repair has been induced and cells in which SOS repair is not operative.     

Crystal structure of the read-through domain from bacteriophage Qβ A1 protein

Bacteriophage Qβ is a small RNA virus that infects Escherichia coli. The virus particle contains a few copies of the minor coat protein A1, a C‐terminally prolonged version of the coat protein, which is formed when ribosomes occasionally read‐through the leaky stop codon of the coat protein. The crystal structure of the read‐through domain from bacteriophage Qβ A1 protein was determined at a resolution of 1.8 Å. The domain consists of a heavily deformed five‐stranded β‐barrel on one side of the protein and a β‐hairpin and a three‐stranded β‐sheet on the other. Several short helices and well‐ordered loops are also present throughout the protein. The N‐terminal part of the read‐through domain contains a prominent polyproline type II helix. The overall fold of the domain is not similar to any published structure in the Protein Data Bank.     

Structural basis for RNA-genome recognition during bacteriophage Qβ replication

Upon infection of Escherichia coli by bacteriophage Qβ, the virus-encoded β-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qβ replicase holoenzyme complex, which is responsible for amplifying the Qβ (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the β-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB_1–2) in the recognition of Qβ (+)-RNA by the Qβ replicase complex. We show how three basic residues of the β subunit form a patch located adjacent to the OB₂ domain, and use NMR spectroscopy to demonstrate for the first time that OB₂ is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Qβ replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the β-subunit and protein S1 cooperatively bind the (+)-stranded Qβ genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.