Control of lytic development in the Streptomyces temperate phage φC31

The repressor gene. c, is required for maintenance of lysogeny in the Streptomyces phage φC31. The c gene expresses three in-frame N-terminally different protein isoforms at least one of which is thought to bind to a 17bp highly conserved inverted repeat (CIR) sequence found at 18 (or more) loci throughout the φC31 genome. Here we present evidence that one of these loci, CIR6, and its interaction with the products of the repressor gene are critical in the control of the lytic pathway in φC31. To the right of CIR6, according to the standard map of φC31, an ‘immediate-early’ promoter. ap1, was discovered after insertion of a fragment containing CIR6 upstream of a promoterless kanamycin-resistance gene. aphll, to form pCIA2. pCIA2 conferred kanamycin resistance upon Streptomyces coelicolor A3(2) but not upon a φC31 lysogen of S. coelicolor. Operator-constitutive (O^c) mutants of pCIA2 were isolated and the mutations lay in CIR6, i.e. CIR6:G14T and CIR6:C2A. Primer extension analysis of RNA prepared from an induced, temperature-sensitive lysogen of S. coelicolor localized a mRNA 5′ endpoint 21 bp to the right of CIR6. The importance of the ap1/CIR6 region in the regulation of lytic growth was demonstrated by the analysis of a virulent mutant, φC31 vir1, capable of forming plaques on an S. coelicolorφC31 lysogen, φC31 vir1 contained a DNA inversion with the breakpoints lying within the integrase gene (which lies approximately 7kbp to the right of CIR6) and in the essential early region between CIR6 and the -10 sequence for ap1. The separation of ap1 from its operator was thought to be the basis for the virulent phenotype in φC31 vir1. Band-shift assays and DNase I footprinting experiments using purified 42kDa repressor isoform confirmed that CIRs 5 and 6 were indeed the targets for binding of this protein. The 42 kDa repressor bound to CIR6 with higher affinity than to CIR5 in spite of their identical core sequences. Repressor bound at CIR6 facilitated binding at CIR5. The high-affinity binding to CIR6 was abolished with the O^c mutant, CIR6:G14T. Hydroxyl radical footprinting and dimethyl sulphate methylation protection of the 42 kDa repressor–CIR6 interaction suggested that the protein bound in the major groove and to one face of the DNA.     

Tail-associated structural protein gp61 of Staphylococcus aureus phage φMR11 has bifunctional lytic activity

A tailed bacteriophage, φMR11 (siphovirus), was selected as a candidate therapeutic phage against Staphylococcus aureus infections. Gene 61, one of the 67 ORFs identified, is located in the morphogenic module. The gene product (gp61) has lytic domains homologous to CHAP (corresponding to an amidase function) at its N-terminus and lysozyme subfamily 2 (LYZ2) at its C-terminus. Each domain of gp61 was purified as a recombinant protein. Both the amidase [amino acids (aa) 1–150] and the lysozyme (aa 401–624) domains but not the linker domain (aa 151–400) caused efficient lysis of S . aureus . Immunoelectron microscopy localized gp61 to the tail tip of the φMR11 phage. These data strongly suggest that gp61 is a tail-associated lytic factor involved in local cell-wall degradation, allowing the subsequent injection of φMR11 DNA into the host cytoplasm. Staphylococcus aureus lysogenized with φMR11 was also lysed by both proteins. Staphylococcus aureus strains on which φMR11 phage can only produce spots but not plaques were also lysed by each protein, indicating that gp61 may be involved in 'lysis from without'. This is the first report of the presence of a tail-associated virion protein that acts as a lysin, in an S. aureus phage.     

Survival of the temperate actinophage φC31 and Streptomyces lividans in soil and the effects of competition and selection on lysogens

Abstract Lysogenic infections were demonstrated in microcosms of sterile soil inoculated with Streptomyces lividans and the φC31 derivative, KC301, in free state or via lysogenized hosts. Intermittent soil mixing caused liberation of KC301 due to lysis of germinating lysogenized and uninfected spores. The presence of lysogenized host ensured that KC301 was maintained at a constant density. The lysogen S. lividans TK24 (KC301) achieved a population density lower than that of its non-lysogenized counterpart. Thiostrepton in the soil did not select for the thiostrepton resistance gene KC301. The long-term survival in soil of a temperate actinophage was demonstrated.     

Structural characteristics of the Streptomyces bacteriophage φC31

Abstract Some structural characteristics of the Streptomyces phage φC31 were analyzed. A simpler and at least 50 times more efficient method than those previously described for isolating the phage is reported. The phage is naked, showing a polyhedral head 53 nm wide, a long non-contractile tail 100 × 5 nm, a basal plate 15 nm in diameter with at least one pin, and a prominent knot between the head and the tail. Up to 17 polypeptides had been found in the virion. Four of them, of 51, 38.5, 29.5 and 28 kDa, make up around 84% of the total protein of the particle.     

Characterization of Streptococcus sanguis Isolated from Patients with Behçet's Disease

The DNA homology and cell wall sugar constituents of eight Streptococcus sanguis(-like) strains, three isolated from the patients with Behçet's disease (BD114-23, BD113-20, BD118-1), two from patients with Kawasaki disease (MCLS-1, MCLS-2), and three type and reference strains of ATCC (ATCC10556^T: S. sanguis, ATCC10557: S. oralis, and ATCC10558^T: S. gordonii) were analyzed. Strains BD114-23 and BD118-1 showed high DNA homology to ATCC10556^T, and their cell wall constituents were identical. Conversely, BD113-20, MCLS-1, MCLS-2, and ATCC10557 showed little DNA homology to ATCC10556^T and ATCC10558^T, but showed approximately 50 to 60% homology to each other. The cell wall constituents of BD113-20, MCLS-1, MCLS-2, and ATCC10557, however, were somewhat different, indicating that some of the clinical isolates have different characters from those of the three ATCC strains.     

DNA polymerase template switching at specific sites on the φ29 genome causes the in vivo accumulation of subgenomic φ29 DNA molecules

The accumulation of subgenomic phage φ29 DNA molecules with specific sizes was observed after prolonged infection times with delayed lysis phage mutants. Whereas the majority of the molecules had a size of 4 kb, additional DNA species were observed with sizes of 8.2, 6.5, 2.3, 2 and 1 kb. Most of the molecules were shown to originate from the right end of the linear Bacillus subtilis phage φ29 genome. The nature of the 4, 2.3, 2 and 1 kb molecules was studied. The 2 kb molecules were shown to be single-stranded self-complementary strands forming hairpin structures. The other molecules consisted of palindromic linear double-stranded DNA molecules. Most probably, the subgenomic DNA molecules were formed when the moving phage replication fork from the right origin encountered a block that induces the DNA polymerase to switch template. Once formed, the subgenomic molecules are then amplified in vivo . Determination of the centres of symmetry of the 4 and 1 kb molecules revealed that both contained the almost 16 bp perfect dyad symmetry element (DSE): 5'-TGTTtCAC-GTGgAACA-3' being a likely candidate for a protein binding site. Database analysis showed that this sequence occurs four times in the φ29 genome. In addition, the almost identical sequence 5'-TgGTTTCAC-GTGGAAtCA-3' was found once. These five DSEs are all located in the right half of the φ29 genome, and the same sequences are also present in the linear DNA of related B. subtilis phages. Most interestingly, this sequence is also found in the spoOJ gene of the B. subtilis chromosome. Recently, it has been shown that the SpoOJ protein is associated in vivo with the same DSE. As the same subgenomic φ29 DNA molecules accumulate after infection of B. subtilis spoOJ deletion strains, it is likely that, in addition to and/or independently of SpoOJ, other protein(s) bind to DSE.     

A super lytic actinophage system as a pre-treatment in the isolation of non-streptomycete actinomycetes from soil

P.F. LONG AND G.E. AMPHLETT. 1996. An increase in the abundance and diversity of non-streptomycete actinomycetes isolated from soils was achieved after the number of streptomycetes was reduced by pre-incubating the soil with streptomycete-specific actinophage. This method is described as a super lytic actinophage system.     

Genetic analysis of the φC31 -specific phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2)

The phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2) was shown to be specific to φC31 homo-immune phages, and to be absent from the closely related strain Streptomyces Iividans. A 16 kb fragment of S. coelicolor A3(2) DNA was isolated which complemented the Pgl^? phenotype of J1501, a pgl mutant derivative of the Pgl^tsS. coelicolor strain M130. The cloned DNA complemented only half of the available pgl mutants, which therefore represented at least two groups, designated Pgl class A and class B strains. It follows that more than one kind of high-frequency genetic event can lead to the Pgl^? phenotype. Crosses between class A and class B strains yielded high frequencies of Pgl⁺ recombinants. Crosses between strains of the same class gave no Pgl⁺ recombinants. The cloned DNA was altered by deletion or apparent point mutation upon passage through the two class B strains tested, such that it was no longer capable of complementing class A strains. This accumulation of mutations might suggest that the expression of the cloned DNA is toxic to at least some class B strains. The nature of the genetic instability associated with the Pgl system was not detectable by Southern blot analysis.     

Calibration of the change in thermal stability of DNA duplexes and degree of base pair mismatch

Summary One method of determining the degree of base pair divergence between two sources of DNA (different strains, species, etc.) is to determine the decrease in thermal stability of hybrid duplex DNA due to mismatching of base pairs. Attempts to calibrate the change in median melting temperature (T_m) to base pair mismatch have led to conflicting results. We have studied the T_m between DNAs of known sequence over a range of from 0.55% to 7.2% base pair mismatch. The relationship of T_m and percent base pair mismatch is remarkably linear over this range with a correlation coefficient >0.98. A T_m of 1°C corresponds to 1.7% base pair mismatch. This conversion is higher than that usually assumed and, therefore, rates of DNA evolution estimated by DNA-DNA hybridization studies are likely faster than previously thought.     

Isolation and characterization of a novel Enterococcus faecalis bacteriophage φEF24C as a therapeutic candidate

Vancomycin-resistant Enterococcus faecalis (VRE) has become a significant threat in nosocomial settings. Bacteriophage (phage) therapy is frequently proposed as a potential alternative therapy for infections caused by this bacterium. To search for candidate therapeutic phages against Enterococcus faecalis infections, 30 Enterococcus faecalis phages were isolated from the environment. One of these, virulent phage φEF24C, which has a broad host range, was selected for analysis. The plaque-forming ability of φEF24C was virtually unaffected by differences in the clinical host strains. Furthermore, the phage had a shorter latent period and a larger burst size than ordinary tailed phages, indicating that φEF24C has effective lytic activity against many Enterococcus faecalis strains, including VRE. Morphological and genomic analyses revealed that φEF24C is a large myovirus (classified as family Myoviridae morphotype A1) with a linear double-stranded DNA genome of c . 143 kbp. Analyses of the N-terminal amino acid sequences of the virion proteins, together with the morphology and the genome size, speculated that φEF24C is closely related to other myoviruses of Gram-positive bacteria that have been used experimentally or practically for therapy or prophylaxis. Considering these results, φEF24C may be a potential candidate therapeutic phage against Enterococcus faecalis infections.     

Induction and preliminary characterization of intracellular β-glucosidases from a cellulolytic Streptomyces strain

Abstract The cellulolytic actinomycete Streptomyces sp. QM-B814 posasses an intracellular β-glucosidase system which is induced by cellobiose and carboxymethylcellulose. Maximal β-glucosidase activity was attained 8–10 h after inducer addition to exponential phase growing cultures. The induction is depressed in the presence of glucose. The system is composed of two electrophoretically different β-glucosidase forms showing relative molecular masses of about 60 and 35 kDa, and p I values in the range 4.2–4.5. Both β-glucosidases are synthesized de novo. The enzymes share substrate preference and are both inhibited by δ-gluconolactone and p -chloromercuribenzoate. The induction pattern and glucose inhibition are similar for both enzymes.     

Spectroscopic characterization of the electronic changes in the active site of Streptomyces antibioticus tyrosinase upon binding of transition state analogue inhibitors

The dinuclear copper enzyme tyrosinase (Ty) from genetically engineered Streptomyces antibioticus has been investigated in its paramagnetic half-met form [Cu(I)-Cu(II)]. The cw EPR, pulsed EPR, and hyperfine sublevel correlation spectroscopy (HYSCORE) experiments on the half-met-Ty and on its complexes with three different types of competitive inhibitor are reported. The first type includes p-nitrophenol, a very poor substrate for the monooxygenase activity of Ty. The second type comprises hydroxyquinones, such as kojic acid and l-mimosine, and the third type of inhibitor is represented by toluic acid. The electronic and structural differences of the half-met-Ty form induced at the cupric site by the different inhibitors have been determined. Probes of structural effects are the hyperfine coupling constants of the non coordinating Ndelta histidyl nitrogens. By using the available crystal structures of hemocyanin as a template in combination with the spectroscopic results, a structural model for the active site of half-met-Ty is obtained and a model for the binding modes of both mono- and diphenols could be proposed.     

DNA-DNA dot hybridization technique used as DNA determination method in the alkaline elution analysis of DNA damage

A DNA-DNA (‘Southern’) dot hybridization technique was adapted for use as a quantitative DNA detection method during alkaline elution analysis of irradiated rat cell material. In comparison to standard microfluorometric methods, similar γ-ray-dose-response relationships were obtained with less than 1% of the cell material when the dot hybridization assay was used. When a highly repetitive, long interspersed DNA element of the rat genome is used as a hybridization probe, as few as 10⁴ cells of rat tissue or rat cell culture cells per sample with approx. 50 ng of DNA were sufficient to detect single-strand breaks and protein cross-links in the DNA of rat hepatocytes and cells of the nasal epithelium after in vitro γ-irradiation. Since highly repetative DNA elements are available from nearly all higher eukaryotes, this alternative approach of detecting DNA in alkaline elution analysis is generally proposed for tissues which yield only low amounts of cell material and/or which are difficult to label by radioactive DNA precursors.