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PubMed1999

The complete genome sequence of the Streptomyces temperate phage straight phiC31: evolutionary relationships to other viruses.

The completed genome sequence of the temperate Streptomyces phage straight phiC31 is reported. straight phiC31 contains genes that are related by sequence similarities to several other dsDNA phages infecting many diverse bacterial hosts, including Escherichia, Arthrobacter, Mycobacterium, Rhodobacter, Staphylococcus, Bacillus, Streptococcus, Lactobacillus and Lactococcus. These observations provide further evidence that dsDNA phages from diverse bacterial hosts are related and have had access to a common genetic pool. Analysis of the late genes was particularly informative. The sequences of the head assembly proteins (portal, head protease and major capsid) were conserved between straight phiC31, coliphage HK97, staphylococcal phage straight phiPVL, two Rhodobacter capsulatus prophages and two Mycobacterium tuberculosis prophages. These phages and prophages (where non-defective) from evolutionarily diverse hosts are, therefore, likely to share a common head assembly mechanism i.e. that of HK97. The organisation of the tail genes in straight phiC31 is highly reminiscent of tail regions from other phage genomes. The unusual organisation of the putative lysis genes in straight phiC31 is discussed, and speculations are made as to the roles of some inessential early gene products. Similarities between certain phage gene products and eukaryotic dsDNA virus proteins were noted, in particular, the primase/helicases and the terminases (large subunits). Furthermore, the complete sequence clarifies the overall transcription map of the phage during lytic growth and the positions of elements involved in the maintenance of lysogeny.

Amino Acid Sequence↗
PubMed1978

Base specific fractionation of double stranded DNA: affinity chromatography on a novel type of adsorbant.

A material suitable for base-pair-specific "affinity chromatography" of double stranded DNA is described. The synthesis of this material involves two successive polymerization reactions yielding solid particles of cross-linked bisacrylamide to which base-pair-specific dyes are covalently attached by spacers of polyacrylamide chains of different length. Materials with immobilized A.T-specific malachite green or G.C-specific phenyl neutral red were used for base-pair-specific fractionation of sheared DNA from bacterial sources, as well as of higher molecular weight Calf thymus DNA or defined fragments of coliphage lambda DNA. The excellent resolving power of this method of "affinity chromatography" of nucleic acids is comparable only to DNA fractionation in buoyant density gradients in the presence of base-pair-specific ligands. A great advantage of this material is its simple handling and its chemical stability, which allows repeated re-use of the same column.

Animals↗
PubMed1978

Extracellular nucleases of pseudomonas BAL 31. III. Use of the double-strand deoxyriboexonuclease activity as the basis of a convenient method for the mapping of fragments of DNA produced by cleavage with restriction enzymes.

We have previously characterized an extracellular nuclease from Pseudomonas BAL 31 which, in addition to other activities, displays a double-strand exonuclease activity which progressively shortens both strands of linear duplex DNA molecules from both termini. This degradation is accomplished without the introduction of detectable scissions away from the ends of the duplexes. When this nuclease is used to produce a series of progressively shortened samples from a linear duplex DNA, subsequent digestion of these samples with a site-specific restriction endonuclease and analysis of the resulting fragments by gel electrophoresis permits the rapid establishment of the order of the restriction enzyme fragments through the entire genome. This is accomplished by noting from the electropherograms the order in which the various restriction enzyme fragments become noticeably shortened or disappear. Using this method, the five cleavage sites for the endonuclease Hpa I and the single cleavage sites for the nucleases Hpa II and Pst I have been mapped in PM2 bacteriophage DNA. In a more stringent test of the method, 18 of the 24 fragments produced by cleavage of coliphage lambdab2b5c DNA with the Pst I nuclease have been mapped, and five of the six remaining fragments have been assigned to small regions of the genome.

DNA Restriction Enzymes↗
PubMed2002

Sequence analysis of bacteriophage T4 DNA packaging/terminase genes 16 and 17 reveals a common ATPase center in the large subunit of viral terminases.

Phage DNA packaging is believed to be driven by a rotary device coupled to an ATPase 'motor'. Recent evidence suggests that the phage DNA packaging motor is one of the strongest force-generating molecular motors reported to date. However, the ATPase center that is responsible for generating this force is unknown. In order to identify the DNA translocating ATPase, the sequences of the packaging/terminase genes of coliphages T4 and RB49 and vibriophages KVP40 and KVP20 have been analyzed. Alignment of the terminase polypeptide sequences revealed a number of functional signatures in the terminase genes 16 and 17. Most importantly, the data provide compelling evidence for an ATPase catalytic center in the N-terminal half of the large terminase subunit gp17. An analogous ATPase domain consisting of conserved functional signatures is also identified in the large terminase subunit of other bacteriophages and herpesviruses. Interestingly, the putative terminase ATPase domain exhibits some of the common features found in the ATPase domain of DEAD box helicases. Residues that would be critical for ATPase catalysis and its coupling to DNA packaging are identified. Com binatorial mutagenesis shows that the predicted threonine residues in the putative ATPase coupling motif are indeed critical for function.

Adenosine Triphosphatases↗
PubMed2002

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.

Base Sequence↗
PubMed2003

Bidirectional replication from an internal ori site of the linear N15 plasmid prophage.

The prophage of coliphage N15 is not integrated into the chromosome but exists as a linear plasmid molecule with covalently closed hairpin ends (telomeres). Upon infection the injected phage DNA circularizes via its cohesive ends. Then, a phage-encoded enzyme, protelomerase, cuts the circle and forms the hairpin telomeres. N15 protelomerase acts as a telomere-resolving enzyme during prophage DNA replication. We characterized the N15 replicon and found that replication of circular N15 miniplasmids requires only the repA gene, which encodes a multidomain protein homologous to replication proteins of bacterial plasmids replicated by a theta-mechanism. Replication of a linear N15 miniplasmid also requires the protelomerase gene and telomere regions. N15 prophage replication is initiated at an internal ori site located within repA and proceeds bidirectionally. Electron microscopy data suggest that after duplication of the left telomere, protelomerase cuts this site generating Y-shaped molecules. Full replication of the molecule and subsequent resolution of the right telomere then results in two linear plasmid molecules. N15 prophage replication thus appears to follow a mechanism that is distinct from that employed by eukaryotic replicons with this type of telomere and suggests the possibility of evolutionarily independent appearances of prokaryotic and eukaryotic replicons with covalently closed telomeres.

Bacteriophages↗
PubMed2000

Fluorescence microscopic visualization of a DNA-cationic fullerene complex.

Cationic fullerene derivative 1, which is soluble in aqueous medium, bound to double stranded DNA. Observation of Coliphage T4 DNA in the absence and presence of 1 by fluorescence microscopy suggested that 1 bound to DNA along its groove. Upon irradiation of visible light on the DNA complex of 1, DNA was cleaved dramatically. The process of DNA photo-cleavage could be monitored continuously by fluorescence microscopy.

Bacteriophage T4↗
PubMed1994

Differentiation of Bacillus anthracis from other Bacillus cereus group bacteria with the PCR.

Variation among isolates of Bacillus anthracis was examined by using restriction fragmentation patterns and the PCR performed with arbitrary and sequence-specific oligonucleotide primers. The patterns were compared with the patterns generated from strains of closely related species belonging to the "Bacillus cereus group" of bacteria, including B. cereus, Bacillus thuringiensis, and Bacillus mycoides. All B. anthracis profiles were identical for each of 18 restriction enzymes, each of 10 arbitrary PCR primers, and a repetitive extragenic palindrome-specific PCR primer. The PCR profiles generated with a coliphage M13-based primer exhibited slight pattern variation in a 400- to 500-bp band region. The B. anthracis profiles were unique compared with the profiles of the other species examined. In these other species, strain-to-strain variations were observed. Our results showed that isolates of B. anthracis are almost completely homogeneous, indicating a clonal lineage, and are distinct from other members of the B. cereus group and that B. anthracis, as a species in its own right, may have evolved only relatively recently.

Bacillus anthracis↗
PubMed1987

The head-tail linker protein of bacteriophage T5: genetic and immunological studies.

We investigated the properties of amber mutants of coliphage T5 defective in a late stage of phage assembly. The non-functional heads synthesized by mutants am25 or am158 were unable to combine with functional tails to produce viable phage particles. These mutants were shown to lack a single protein, designated the head-tail linker protein (HTLp), which was identified by polyacrylamide gel electrophoresis and Western blot analysis and had an Mr of 18,000. An extract containing the HTLp, when added to the HTLp-deficient heads, restored their ability to combine with functional tails.

Electrophoresis, Polyacrylamide Gel↗
PubMed2002

In vivo generation of hybrids between different species of RNA phages.

Hybrids between different species or genera of the single-stranded RNA coliphages have not been found in nature. Here, it has been shown that viable hybrids between different phage species can easily be generated in the laboratory by in vivo recombination. cDNA of species I phage MS2 located on a plasmid and lacking part of its 5' untranslated leader (5' UTR) was complemented with another plasmid carrying the 5' half of the genome of fr, a species I phage, or of KU1, a species II representative with low sequence similarity. When the two plasmids were present in the same cell there was spontaneous production of hybrid phages. Interestingly, these hybrids did not arise by a double or single crossover that would replace the missing MS2 sequences with those of fr or KU1. Rather, hybrids arose by attaching the complete 5' UTR of fr or KU1 to the 5' terminus of the defective MS2 phage. Several elements of the 5' UTR then occurred twice, one from KU1 (or fr) and the other from MS2. These redundant elements are in most cases deleted upon evolution of the hybrids. As a result, the 5' UTR of KU1 (or fr) then replaced that of MS2. It was earlier shown that this 5' UTR could assume two alternating structures that facilitated transient translation of the proximal maturation gene. Apparently, this timer function of the 5' UTR was exchangeable and could function independently of the rest of the genome. When hybrids were competed against wild-type, they were quickly outgrown, probably explaining their absence from natural isolates.

5' Untranslated Regions↗
PubMed1977

The mechanism of action of nitro-heterocyclic antimicrobial drugs. Primary target of 1-methyl-2-nitro-5-vinylimidazole is DNA.

The antimicrobial drug 1-methyl-2-nitro-5-vinylimidazole (MEV) preferentially blocked DNA synthesis, was mutagenic and induced coliphage lambda in Escherichia coli. The antibacterial effects of MEV are the consequences of repairable damage to DNA, as shown by hypersensitivity of recA and uvr strains to MEV and related drugs, stimulation by MEV of DNA turnover which was dependent on the product of the uvrA gene, and the presence of cross-links in DNA from MEV-treated bacteria.

Anti-Bacterial Agents↗
PubMed1980

Protein synthesis in cell-free extracts of Coxiella burnetti.

Some aspects of protein biosynthesis were investigated in extracts of the obligate intracellular bacterium Coxiella burnetti. Sucrose gradient analysis revealed small quantities of 30S and 50S ribosomal subunits, few 70S ribosomes and no polysomes. Functional endogenous mRNA was not detected. In translation of exogenously added poly(U), extracts required Mg2+ (17 mM) and NH4+ (60 mM) for optimal polyphenylalanine synthesis; the optimum MG2+ requirement differed from that of Escherichia coli. The translation of coliphage Q beta RNA by C. burnetti extracts required Mg2+ (13 mM), NH4+ (60 mM) and an energy source for polypeptide synthesis, and was sensitive to chloramphenicol but not to cycloheximide. Under optimal conditions, the translation of Q beta RNA proceeded at a rate and to an extent equal to that obtained in a conventional E. coli system. Electrophoretic analysis of translation products made during incubation of C. burnetti extracts with polycistronic Q beta RNA revealed a major product with a molecular weight of about 14 000; this product co-electrophoresed with the coat protein extracted from Q beta phage propagated in E. coli. The results suggested that the extracellular form of the rickettsia-like organism, C. burnetti, possessed the full array of components necessary for the initiation, elongation and termination of polypeptides.

Ammonia↗
PubMed1986

The expression of the Escherichia coli lacZ gene in Streptomyces.

The Escherichia coli lacZ gene was stably introduced into phi C31-based phage cloning vectors in Streptomyces lividans. However, lacZ could not be stably introduced into S. lividans on the plasmid vectors pIJ702 or pIJ41. Studies of the expression of lacZ in S. lividans and S. coelicolor were facilitated by the use of mutants and/or growth conditions in which endogenous beta-galactosidase activity was low or absent. Plaques and lysogens of phi C31::lacZ constructs involving transcriptional fusions to the pBR322 tet gene contained beta-galactosidase activity. Activities were markedly higher in S. lividans than in S. coelicolor. Insertion of the major transcriptional terminator of coliphage fd between the tet promoter and lacZ reduced lacZ expression more in lysogens than in lytically infected cultures, suggesting the existence of a phage-specified anti-termination function. Several phi C31 derivatives suitable for the construction of different kinds of transcriptional and translational fusions in Streptomyces were derived. The expression of lacZ in one transcriptional fusion vector (KC659) was increased both in plaques and in lysogens by the appropriately positioned insertion of a previously characterized promoter from the aph gene of S. fradiae. When phi C31 DNA fragments were inserted into KC659, at least one recombinant phage gave high beta-galactosidase activity in lytic infections, but not in lysogens. The potential usefulness of lacZ fusions in Streptomyces is discussed.

Bacterial Proteins↗
PubMed1990

Yersinia enterocolitica immunodominant 60 kDa antigen, common to a broad range of bacteria, is a heat-shock protein.

Monoclonal antibodies (mAbs) against the Yersinia enterocolitica immunodominant 60 kDa antigen, termed cross-reacting protein antigen (CRPA), were obtained by fusion of spleen cells from mice immunized with CRPA with murine myeloma cells. The reactivities of the mAbs were examined by Western blotting against extracts of Y. enterocolitica and 23 other species of Gram-positive and Gram-negative bacteria. Cross-reactions were recognized with a wide range of bacteria, but not with Gram-positive cocci. The reactivities were different for each mAb, suggesting that both species-specific and multiple cross-reactive epitopes were present on the CRPA molecule. CRPA was produced under heat-shock conditions in Y. enterocolitica and was shown to correspond immunologically to the GroEL protein in Escherichia coli, a protein involved in the morphogenesis of coliphage. In addition to CRPA, at least nine other major heat-shock proteins were detected by two-dimensional gel electrophoresis of extracts of heat-shocked Y. enterocolitica.

Antigens, Bacterial↗
PubMed1998

The transcriptional regulator gene phrR in Sinorhizobium meliloti WSM419 is regulated by low pH and other stresses.

The phrR gene in Sinorhizobium meliloti (previously known as Rhizobium meliloti) WSM419, directly downstream from actA, is induced by low pH or certain stresses (e.g. high concentrations of Zn2+, Cu2+, H2O2 or ethanol), but not in stationary phase or by other stresses (e.g. phosphate limitation, elevated temperature, high concentrations of sucrose or iron). A DNA fragment containing the wild-type phrR gene could not be cloned and inverse PCR was therefore used to amplify a 3.5 kb BamHI fragment containing phrR from the mutant S. meliloti TG2-6 (actA::Tn5). DNA fragments from a BamHI/SalI digest of the amplified product were cloned into pUK21 and sequenced. The phrR open reading frame contiguous to actA appears to code for a 15.2 kDa protein showing significant identity with the proteins encoded by y4wC and y4aM in Rhizobium sp. NGR234. All three proteins resemble transcriptional regulators in containing a DNA-binding helix-turn-helix motif similar to that reported for URF4 in Rhodospirillum rubrum and repressors in coliphage.

Amino Acid Sequence↗
PubMed2000

Microvirus of chlamydia psittaci strain guinea pig inclusion conjunctivitis: isolation and molecular characterization.

The authors report the isolation and molecular characterization of a bacteriophage, φCPG1, which infects CHLAMYDIA: psittaci strain Guinea pig Inclusion Conjunctivitis. Purified virion preparations contained isometric particles of 25 nm diameter, superficially similar to spike-less members of the φX174 family of bacteriophages. The single-stranded circular DNA genome of φCPG1 included five large ORFs, which were similar to ORFs in the genome of a previously described CHLAMYDIA: bacteriophage (Chp1) that infects avian C. psittaci. Three of the ORFs encoded polypeptides that were similar to those in a phage infecting the mollicute Spiroplasma melliferum, a pathogen of honeybees. Lesser sequence similarities were seen between two ORF products and the major capsid protein of the φX174 coliphage family and proteins mediating rolling circle replication initiation in phages, phagemids and plasmids. Phage φCPG1 is the second member of the genus CHLAMYDIAMICROVIRUS:, the first to infect a member of a CHLAMYDIA: species infecting mammals. Similarity searches of the nucleotide sequence further revealed a highly conserved (75% identity) 375 base sequence integrated into the genome of the human pathogen Chlamydia pneumoniae. This genomic segment encodes a truncated 113 residue polypeptide, the sequence of which is 72% identical to the amino-terminal end of the putative replication initiation protein of φCPG1. This finding suggests that C. pneumoniae has been infected by a phage related to φCPG1 and that infection resulted in integration of some of the phage genome into the C. pneumoniae genome.

Amino Acid Sequence↗
PubMed2002

The cell surface protein Ag43 facilitates phage infection of Escherichia coli in the presence of bile salts and carbohydrates.

It was found that infection of Escherichia coli by bacteriophage lambda is inhibited in the presence of certain bile salts and carbohydrates when cells are in the "OFF" state for production of the phase-variable cell surface protein antigen 43 (Ag43). The inhibition of phage growth was found to be due to a significant impairment in the process of phage adsorption. Expression of the gene encoding Ag43 (agn43) from a plasmid or inactivation of the oxyR gene (encoding an activator of genes important for defence against oxidative stress) suppressed this inhibition. A mutation, rpoA341, in the gene encoding the alpha subunit of RNA polymerase also facilitated phage adsorption in the presence of bile salts and carbohydrates. The rpoA341 mutation promoted efficient production of Ag43 in a genetic background that would otherwise be in the "OFF" phase for expression of the agn43 gene. Analysis of a reporter gene fusion demonstrated that the promoter for the agn43 gene was more active in the rpoA341 mutant than in the otherwise isogenic rpoA(+) strain. The combined inhibitory action of bile salts and carbohydrates on phage adsorption and the abolition of this inhibition by production of Ag43 was not restricted to lambda, as a similar phenomenon was observed for the coliphages P1 and T4.

Adhesins, Bacterial↗
PubMed2006

In vivo hydrolysis of S-adenosylmethionine induces the met regulon of Escherichia coli.

Regulation of methionine biosynthesis in Escherichia coli involves a complex of the MetJ aporepressor protein and S-adenosylmethionine (SAM) repressing expression of most genes in the met regulon. To test the role of SAM in the regulation of met genes directly, SAM pools were depleted by the in vivo expression of the cloned plasmid vector-based coliphage T3 SAM hydrolase (SAMase) gene. Cultures with in vivo SAMase activity were assayed for expression of the metA, B, C, E, F, H, J, K and R genes in cells grown in methionine-rich complete media as well as in defined media with and without l-methionine. In vivo SAMase activity dramatically induced expression between 11- and nearly 1000-fold depending on the gene assayed for all but metJ and metH, and these genes were induced over twofold. metJ : : Tn5 (aporepressor defective) and metK : : Tn5 (SAM synthetase impaired; produces <5 % of wild-type SAM) strains containing in vivo SAMase activity produced even higher met gene activity than that seen in comparably prepared cells with wild-type genes for all but metJ in a MetJ-deficient background. The SAMase-mediated hyperinduction of metH in wild-type cells and of the met genes assayed in metJ : : Tn5 and metK : : Tn5 cells provokes questions about how other elements such as the MetR activator protein or factors beyond the met regulon itself might be involved in the regulation of genes responsible for methionine biosynthesis.

Artificial Gene Fusion↗