In vitro site-directed mutagenesis: Generation and properties of an infectious extracistronic mutant of bacteriophage Qβ

An infectious extracistronic mutant of phage Qβ has been prepared by site-directed mutagenesis. Qβ RNA minus strands containing the mutagenic base analog N⁴-hydroxy-CMP instead of UMP at position 39 from the 5′ end were synthesized in vitro and used as template for Qβ replicase to synthesize one generation of plus strands. E. coli spheroplasts were infected with the newly synthesized plus strands and phage recovered from single plaques. RNA sequence analysis revealed that four out of the eighteen phage clones analyzed contained RNA with an A → G transition at position 40 from the 3′-end (which corresponds to position 39 of the minus strand). Thus, the viability of phage Qβ does not depend on a unique nucleotide sequence in the 3′-extracistronic RNA segment.Upon in vivo propagation of mutant 40, spontaneous true revertants arose with high frequency and overgrew the parental clone within about 10 passages, indicating a selective disadvantage of the extracistronic mutant. Replication of mixtures of wild type and mutant RNA in vitro resulted in a decrease of the proportion of mutated RNA in the progeny plus strands. The fact that Qβ RNA containing an A → G transition in nucleotide −40 of Qβ RNA is less efficiently replicated in vitro may explain the selective disadvantage of the mutant phage in vivo.The preparation of an infectious mutated RNA by site-directed mutagenesis shows that the method is suitable to produce specific nucleotide exchanges without impairing the biological competence of the RNA.     

Intercellular Transfer by Phage Receptor Site Lipopolysaccharide

TREATMENT of Escherichia coli cells with lysozyme and EDTA partially removes the outer layer of the cell wall containing lipopolysaccharide (LPS), leaving osmotically unstable spheroplasts¹. These can be infected with phage nucleic acid² and can produce viable phage particles. Removal of LPS-containing phage receptor sites^3–5, however, leaves spheroplasts resistant to infection by intact phages¹. We now show that LPS, obtained from phage-sensitive cells by aqueous phenol extraction, can provide functional phage receptor sites to spheroplasts prepared from cells lacking receptor sites.     

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.     

How does lysozyme penetrate through the bacterial outer membrane?

Lysozyme fails to penetrate through the outer membrane of stationary phase cells of Escherichia coli when it is simply added to suspensions of plasmolyzed cells. Lysozyme penetrates the outer membrane only when these cells are exposed to a mild osmotic shock in the presence of EDTA and lysozyme.In the presence of Mg²⁺, the outer membrane is stabilized sufficiently so that there is no lysozyme penetration during osmotic shock. If Mg²⁺ is added after an osmotic shock has been used to cause lysozyme to penetrate a destabilized outer membrane, the outer membrane is stabilized once again. In this case however, cells are converted to spheroplasts by the lysozyme which has gained access to the murein layer prior to the addition of Mg²⁺. Mg²⁺ stabilizes the outer membranes of these spheroplasts sufficiently so that they remain immune to lysis even in the absence of osmotic stabilizers such as sucrose.These results are discussed in terms of current information on the structure of the murein layer and the outer membrane.     

Purification of cytoplasmic membranes and outer membranes from Proteus mirabilis

Summary A crude cell envelope suspension has been prepared from Proteus mirabilis after osmotic shock of penicillin-induced spheroplasts. Employing discontinuous sucrose gradients this cell envelope suspension can be fractionated into four fractions. Besides a pellet of remaining spheroplasts and an intermediate fraction with mixed composition a highly purified cytoplasmic membrane fraction and an outer membrane fraction have been obtained. The cytoplasmic membrane fraction is not contaminated with mucopeptide or outer membrane material. It has a buoyant density of 1.13 g/ml and a protein content of 38%. The specific activities of formate dehydrogenase and nitrate reductase and the content of cytochrome b₁ have increased sixfold in comparison with the crude cell envelope suspension. The outer membrane fraction contains only few contaminations with cytoplasmic membrane components and with mucopeptide.The gradient fractions have been characterized by electron microscopy and by polyacrylamide gel electrophoresis.     

The interaction betweenE. coli spheroplasts and plant protoplasts: A proposed procedure to deliver foreign genes into plant cells

Summary E. coli spheroplasts can be used to deliver DNA vectors into plant protoplasts. The use of fluorescent dyes showed that 25–100% of the protoplast population was associated with 1–9 spheroplasts following incubation with several fusogens. Electron microscopy demonstrated spheroplasts attached to protoplasts via a plasma membrane protrusion after high pH/Ca²⁺ treatment, but PEG-high pH/Ca²⁺ promoted endocytosis of spheroplasts into a plasma membrane bounded vesicle. Ultrastructural profiles showed that fusion between spheroplasts and protoplasts did not occur. Immunofluorescence studies detectedE. coli antigens associated with tobacco protoplasts, and after fusogen treatment the antigens were dispersed within the peripheral cytoplasm. The elimination of residual contaminatingE. coli cells from protoplasts was achieved by lysozyme and antibiotic treatment, thus allowing DNA vector assessment in axenic culture.     

Spackle and Immunity Functions of Bacteriophage T4

Cells of Escherichia coli B infected with the immunity-negative (imm2) mutant of bacteriophage T4 are able to develop a substantial level of immunity to superinfecting phage ghosts if the ghost challenge is made late in infection. This background immunity is not seen in infections with phage carrying the spackle (s) mutation in addition to the imm2 lesion. The level of immunity in s⁻ infections is intermediate between that of imm⁻ and wild-type infections under standard assay conditions. With respect to genetic exclusion of superinfecting phage, cells infected with imm⁻ phage are completely deficient, whereas infections with the s⁻ phage are only partially deficient compared to wild-type infections. Whereas s⁻-infected cells are unable to resist lysis from without by a high multiplicity of infection (MOI) of superinfecting phage, cells infected with imm⁻ phage show less than wild-type levels of resistance and the majority of cells remaining intact are unable to incorporate leucine or form infective centers. Under conditions of superinfection by low MOI of homologous phage, imm⁻-infected cells are lysis inhibited, whereas s⁻-infected cells do not show this property. Superinfecting phage inject their DNA into imm⁻-infected cells with the same efficiency as seen in wild-type infections, but this efficiency is reduced when the cells are first infected with s⁻ phage. The s function of T4 appears not only to affect the host cell wall as previously postulated by Emrich, but may also affect the junctures of cell wall and membrane with consequences similar to those of the imm function.     

In Vitro Evolution and Affinity-Maturation with Coliphage Qβ Display

The Escherichia coli bacteriophage, Qβ (Coliphage Qβ), offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV). DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb) SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.     

Some properties of cefoxitin-induced spheroplasts of Proteus species, including sensitivity to colistin

Spheroplasts of various Proteus species could be induced by cefoxitin in nutrient broth alone or in broth containing 0–33 mol/l sucrose and 4 times 10^-3 mol/l magnesium sulphate. Spheroplasts induced in the latter medium were lysed when diluted into water, but osmotically stable if transferred to broth or other appropriate menstrua. High concentrations of colistin were needed to induce lysis of cefoxitin-induced spheroplasts and it was concluded that, contrary to earlier findings with benzylpenicillin-induced spheroplasts, peptidoglycan had little, if any, role to play in acting as a barrier to the entry of polymyxins into Proteus cells.     

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.     

Lactococcal Bacteriophages Require a Host Cell Wall Carbohydrate and a Plasma Membrane Protein for Adsorption and Ejection of DNA

The mechanism of the initial steps of bacteriophage infection in Lactococcus lactis subsp. lactis C2 was investigated by using phages c2, ml3, kh, l, h, 5, and 13. All seven phages adsorbed to the same sites on the host cell wall that are composed, in part, of rhamnose. This was suggested by rhamnose inhibition of phage adsorption to cells, competition between phage c2 and the other phages for adsorption to cells, and rhamnose inhibition of lysis of phage-inoculated cultures. The adsorption to the cell wall was found to be reversible upon dilution of the cell wall-adsorbed phage. In a reaction step that apparently follows adsorption to the cell wall, all seven phages adsorbed to a host membrane protein named PIP. This was indicated by the inability of all seven phages to infect a strain selected for resistance to phage c2 and known to have a defective PIP protein. All seven phages were inactivated in vitro by membranes from wild-type cells but not by membranes from the PIP-defective, phage c2-resistant strain. The mechanism of membrane inactivation was an irreversible adsorption of the phage to PIP, as indicated by adsorption of [³⁵S] methionine-labeled phage c2 to purified membranes from phage-sensitive cells but not to membranes from the resistant strain, elimination of adsorption by pretreatment of the membranes with proteinase K, and lack of dissociation of ³⁵S from the membranes upon dilution. Following membrane adsorption, ejection of phage DNA occurred rapidly at 30°C but not at 4°C. These results suggest that many lactococcal phages adsorb initially to the cell wall and subsequently to host cell membrane protein PIP, which leads to ejection of the phage genome.     

Transfection: enhancement by assembly protein of bacteriophage R17.

Assembly protein was isolated by DEAE cellulose chromatography from disrupted R17 bacteriophage and reconstituted with purified R17 phage RNA. Following reconstitution, ¹²⁵I labeled assembly protein co-sediments with 27S R17 phage RNA in a sucrose gradient. SDS-polyacrylamide gel analysis of the 27S ¹²⁵I labeled protein-RNA complex confirmed that assembly protein was the only phage protein associated with the RNA. The specific infectivity (PFU/μg RNA) of the R17 phage RNA-assembly protein complex was 35-fold greater than that of R17 phage RNA when assayed on $\text{Escherichia coli}$ spheroplasts. Infectivity of both preparations was destroyed by treatment with pancreatic ribonuclease A. Furthermore, the assembly protein-RNA complex was infectious for intact cells whereas phage RNA was not infectious. Infectivity of this 27S complex for intact cells was totally eliminated by pretreatment with ribonuclease.     

Action of penicillin onSerratia marcescens

The action of penicillin onSerratia marcescens was studied. In culture media containing sucrose (0.33m) and in the presence of magnesium ions, cell wall lesions occurred giving rise to osmotically fragile spheroplasts. However, in the absence of sucrose and magnesium ions it was still possible to induce some spheroplast formation. Quantitative aspects of the conversion of rods into spheroplasts were studied as well as physical properties of the spheroplasts.     

Degradation and turnover of bacterial cell wall mucopeptides in growing bacteria

The mucopeptide layer of the cell wall ofBacillus megaterium is broken down into separate components during growth of the cells. The released diaminopimelic acid is partly decarboxylated to lysine, which is incorporated in the proteins and partly used for cell wall resynthesis. The smaller portion of the degraded mucopeptide is released into the medium in the form of non-utilized fragments. The rate of the mucopeptide turnover is a function of the rate of growth of the culture. About 15–20% of the rigid layer of the cell wall is degraded during on cell division. The sensitivity ofBacillus megaterium to lysozyme and the rate of its conversion to protoplasts is also proportionate to the rate of growth of the culture. There is no measurable mucopeptide turnover in non-growing cells, either in the stationary phase of the culture or in starvation in nitrogen-free medium. The resistance of the cell wall to lysozyme also increases during the stationary phase. The rigid component of the cell wall is probably also broken down during growth ofBacillus cereus andEscherichia coli cultures.     

Damage and mutagenesis of E. coli and bacteriophage λ induced by oxathiolane and aziridinyl steroids

λ-Escherichia coli complexes exhibited remarkable sensitivity to the treatment with test steroidal derivatives in the presence of Cu(II). The decline in plaque-forming units after steroid treatment was more pronounced in complexes with some of the irradiation repair-defective mutants of E. coli K-12, i.e., recA, lexA and polA, as compared to uvrA and wild-type strains. The red gene of λ phage and recA gene of E. coli seem to have a complementary effect on the steroid-induced lesions. An enhanced level of mutagenesis was observed when steroid-treated E. coli cells were transformed with steroid-treated pBR322 plasmid DNA. A remarkable degree of c mutation was also observed when steroid I-treated phage particles were allowed to adsorb on steroid-treated wild-type bacteria. Moreover, the oxathione steroid treatment of λcI857-E. coli lysogen resulted in prophage induction in nutrient broth even at 32°C. Thus on the basis of these results, the role of SOS repair system in steroid-induced mutagenesis and repair of DNA lesions in E. coli and bacteriophage λ has been suggested.     

Localization of an Amikacin 3′-Phosphotransferase in Escherichia coli

A plasmid-encoded enzyme reported by us to phosphorylate amikacin in a laboratory strain of Escherichia coli has been localized in the bacterial cell. More than 88% of this amikacin phosphotransferase (APH) activity was retained in spheroplasts formed by ethylenediaminetetraacetate-lysozyme treatment of an APH-containing E. coli transconguant known to form spheroplasts readily. By comparison, the spheroplasts retained 94% of deoxyribonucleic acid polymerase I and 98% of glutamyl-transfer ribonucleic acid synthetase, two internal markers, whereas less than 10% of the activity of a periplasmic marker, acid phosphatase, was present in spheroplasts. Treatment of whole cells of the transconjugant with chemical probes incapable of crossing the plasma membrane obliterated acid phosphatase activity, whereas the internal markers deoxyribonucleic acid polymerase I, glutamyl-transfer ribonucleic acid synthetase, and β-galactosidase were virtually unaffected after treatment for 5 min; more than 60% of the APH activity remained. As a control, similar chemical treatment of sonic extracts, in which enzymes were not protected by bacterial compartmentalization, produced more extensive reduction in the activities of all test enzymes, including APH. Spheroplasts preincubated with adenosine triphosphatase were shown by thin-layer chromatography to phosphorylate amikacin. Spheroplasts of cells grown in the presence of H₃³²PO₄ were shown to utilize internally generated adenosine 5′-triphosphate in the phosphorylation of amikacin. The absence of ³²P-phosphorylated amikacin after incubation of [γ-³²P]adenosine 5′-triphosphate with spheroplasts confirmed that exogenous adenosine 5′-triphosphate was not used in the reaction. These results suggest an internal location for APH. This conclusion has implications for the role of such enzymes in aminoglycoside resistance of gram-negative bacteria.     

Production of protoplasts from Fusarium scirpi by lytic enzymes from Streptomyces tsusimaensis

Summary The ability of serveral strains of Streptomyces to degrade cell walls from Fusarium scirpi was tested by plating them on agar containing a cell wall preparation derived from the fungus. In this assay, S. tsusimaensis was most effective in producing a clear zone of lysis during growth on the opaque medium. This Streptomyce strain was subsequently grown in liquid culture containing cell walls as the sole carbon source and the exoenzymes were isolated from the culture broth. The enzyme preparation produces a clear zone of lysis when filled into wells in the cell wall agar and was used to prepare protoplasts from F. scirpi. The protoplast yield was 1x10⁹ protoplasts/ml of enzyme solution from 35 mg dry weight of Fusarium mycelium. Protoplasts could be regenerated at a frequency of up to 80%.     

UV-sensitivity and UV-mutability of infectious lambdaDNA: reactivation, protection and mutability in various assay systems

Summary The UV-sensitivity of phage and its infectious DNA have been compared in experiments involving infection of normal cells by phage and transfection of lysozyme-EDTA spheroplasts or Ca⁺⁺-treated cells by phage DNA. It is shown that UV-irradiated DNA undergoes extensive HCR. Since intact phage and free phage DNA have the same survival after UV-irradiation in Hcr^- spheroplasts and cells, resp., and since survival is also identical in Ca⁺⁺-treated Hcr⁺ cells it is concluded that DNA in solution or packaged in the phage head provides the same target for the induction of lethal UV lesions. This conclusion is supported by the observation that cysteamine provides a similar radioprotection to the intact phage and its free DNA. Spheroplasts of Hcr⁺ cells, however, have an HCR capacity reduced by about 20% when compared with normal or Ca⁺⁺-treated cells. Moreover, UV-reactivation of irradiated DNA, which is absent in spheroplasts, occurs efficiently in Ca⁺⁺-treated cells. Possible reasons for the physiological difference between spheroplasts and normal cells are discussed. c-mutations, which are readily induced by UV in phage assayed with E. coli mul ^-, could not be induced in DNA when assayed with spheroplasts or Ca⁺⁺-treated cells of this strain. No mutants were also found with DNA extracted from UV-irradiated phage. The significance of the mode of entry of UV-irradiated DNA into a cell for the production of mutations is discussed.     

Interaction of the isolated DNA of lambda phage with spheroplasts of E. coli treated with sturine]

The method of centrifugation in sucrose density gradient (30-55%) of the spheroplast membrane preparations treated and untreated with sturine and infected with phage lambda DNA demonstrated that sturine, treatment increased the phage lambda DNA absorption three-fold. About 50% of the lambda DNA molecules adsorbed by spheroplasts are bound with the cytoplasmic membrane of spheroplasts treated with sturine; 50% of the lambda DNA molecules are bound with the cell wall membrane on the sturine-untreated spheroplasts. The data obtained allow to conclude that the stimulating effect of sturine in E. coli spheroplasts transfection by lambda DNA is connected with redistribution of phage DNA absorbed on spheroplasts from the cell wall to the cytoplasmic membrane facilitating the penetration of DNA and its fastening on the membrane.     

Biochemical and Biophysical Characterization of an Unexpected Bacteriolytic Activity of VanX,a Member of the Vancomycin-resistance vanA Gene Cluster

VanX is a d-alanyl-d-alanine (d-Ala–d-Ala) dipeptidase encoded in the vancomycin-resistance vanA gene cluster. Here we report that strong bacteriolysis occurred when isolated VanX was expressed in Escherichia coli at temperatures lower than 30 °C, which was unexpected because the vanA operon confers vancomycin resistance by protecting the cell wall. Therefore, we monitored cell lysis by measuring sample turbidity with absorbance at 590 nm and VanX expression using SDS-PAGE. No cell lysis was observed when VanX was expressed, even in large quantities, in the cell inclusion bodies at 37 °C, suggesting that a natively folded VanX is required for lysis. In addition, VanX mutants with suppressed dipeptidase activity did not lyse E. coli cells, confirming that bacteriolysis originated from the dipeptidase activity of VanX. We also observed shape changes in E. coli cells undergoing VanX-mediated lysis with optical microscopy and classified these changes into three classes: bursting, deformation, and leaking fluid. Optical microscopic image analysis fully corroborated our interpretation of the turbidity changes in the samples. From a practical perspective, the finding that VanX expressed in isolation induces cell lysis suggests that inhibitors of VanA and VanH that act downstream from VanX could provide a new class of therapeutic chemicals against bacteria expressing the vancomycin-resistance gene cluster.