Properties of a filamentous phage which adsorbs to pili coded by plasmids of the IncI complex

A filamentous phage, PR64FS, which adsorbs to tips of I pili was isolated. PR64FS is shorter than the I pilus-adsorbing phage If1 and differs from it in plaque morphology. Phages PR64FS and Ifl are serologically related and, like the latter, PR64FS adsorbs to pili coded by all Inc groups of the I plasmid complex.     

The Phage Proteomic Tree: a genome-based taxonomy for phage

There are approximately 10(31) phage in the biosphere, making them the most abundant biological entities on the planet. Despite their great numbers and ubiquitous presence, very little is known about phage biodiversity, biogeography, or phylogeny. Information is limited, in part, because the current ICTV taxonomical system is based on culturing phage and measuring physical parameters of the free virion. No sequence-based taxonomic systems have previously been established for phage. We present here the "Phage Proteomic Tree," which is based on the overall similarity of 105 completely sequenced phage genomes. The Phage Proteomic Tree places phage relative to both their near neighbors and all other phage included in the analysis. This method groups phage into taxa that predicts several aspects of phage biology and highlights genetic markers that can be used for monitoring phage biodiversity. We propose that the Phage Proteomic Tree be used as the basis of a genome-based taxonomical system for phage.     

Phage viability in organic media: insights into phage stability

The stability of the filamentous phages derived from phagemid pG8H6 has been examined in a range of solvents and solvent mixtures. The results show an enhanced capacity to infect E. coli after exposure to various organic solvent-water mixtures. The dependence of stability upon solvent hydrophobicity was demonstrated. Furthermore, conditions have been identified which should allow the application of phage display libraries based upon pG8H6 in organic media.     

Phage formation in Staphylococcus muscae cultures; a factor necessary for phage formation

1. A factor necessary for the formation, of Staphylococcus muscae phage was found in acid digests of many highly purified proteins. 2. The factor is released from egg albumen and pepsin by peptic digestion. 3. No amino acids tried could replace the acid digests of proteins as a source of the factor. 4. The factor, when added to a multiplying bacteria-phage system, cannot be found in purified phage or in the lysate after complete lysis of the system has taken place.     

Characterization of a region of the IncHI2 plasmid R478 which protects Escherichia coli from toxic effects specified by components of the tellurite, phage, and colicin resistance cluster.

The IncHI2 plasmid R478 specifies resistance to potassium tellurite (Te(r)), to some bacteriophages (Phi), and to pore-forming colicins (PacB). The genes encoding the three phenotypes are linked, and an 8.4-kb fragment of R478 DNA encoding them cannot be subcloned unless cocloned with a second section of the plasmid. Subclone pKFW4A contains a 5.9-kb BamHI-EcoRI fragment which caused some toxicity when present in Escherichia coli cells. Bacterial cells containing freshly transformed pKFW4A, examined by light microscopy and electron microscopy, had a filamentous morphology consistent with a block in septation. Insertion of transposon Tn1000 into terZ, -A, -B, and -C genes of pKFW4A resulted in the loss of the filamentation phenotype. Deletion of several regions of the clone confirmed that these latter components are involved in the filamentation phenotype. The region specifying protection from toxicity caused by the larger 8.4-kb fragment (encompassing this cluster and the entire 5.9-kb section of pKFW4A) was sequenced and analyzed by T7 polymerase expression and Tn1000 mutagenesis. Three open reading frames, terW, terY, and terX, were identified in a 2.6-kb region. Two polypeptides with approximate molecular masses of 18 and 28 kDa were expressed in CSRDE3 cells and were consistent with TerW (17.1 kDa; 155 amino acids [aa]) and TerY (26.9 kDa; 248 aa), whereas a protein of 213 aa deduced from terX was not observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The terX gene product shows strong identity with the previously identified TerE, TerD, and TerZ polypeptides, and there is a conserved motif of 13 residues, GDN(R/L)TG(E/A)GDGDDE, within this group of polypeptides. Complementation analysis indicated that terW, located approximately 6.0 kb upstream of terZ, brings about protection of cells from toxic effects of components of the Te(r), Phi, and PacB cluster.     

Phage conversion of Panton-Valentine leukocidin in Staphylococcus aureus: molecular analysis of a PVL-converting phage, phiSLT

Staphylococcal Panton-Valentine leukocidin (PVL) is an important virulence factor, which causes leukocytolysis and tissue necrosis. Our previous report on the existence of the PVL genes (lukS-PV and lukF-PV) on the genome of prophage phiPVL in the Staphylococcus aureus strain V8 suggested the horizontal transmission of PVL genes by temperate bacteriophage among S. aureus (Kaneko, et al., 1998. Gene 215, 57-67). Here, we demonstrated the phage conversion of S. aureus leading to the production of PVL by discovery of a novel PVL-carrying phage, phiSLT (Staphylococcal Leukocytolytic Toxin) from a clinical isolate of S. aureus. phiSLT was able to lysogenize several clinical isolates of PVL-negative S. aureus strains as well as strain RN4220 at the conserved 29-bp sequence (attB) and all the lysogenized S. aureus strains had the ability to produce PVL. phiSLT had an elongated head of about 100x50 nm and a flexible tail of 400 nm long, that was quite different from phiPVL which had an isometric hexagonal head of about 60 nm diameter. The linear double-stranded phiSLT genome comprised 42,942 bp with 29-bp attachment core sequences and contained 62 open reading frames. Only 6.4 kbp region containing lysis cassette, PVL genes, attP, integrase, and orf204 of phiSLT was identical to that of phiPVL, while other regions were different from those of phiPVL. Thus, it can be concluded that PVL genes are carried by different temperate phages, which have the same attachment site.     

pIN32: a cointegrate plasmid with IncHI2 and IncFII components

An Enterobacter cloacae strain isolated from the faeces of a child with diarrhoea in Indonesia contained a transferable 216 MDa plasmid, pIN32, exhibiting IncHI2 phenotypic characters, including temperature sensitivity of transfer and the expression of H serotype pili at a repressed level. A derivative plasmid (pIN32-1), which had lost the IncHI2 phenotype, and contained only 60 MDa of the original replicon, was obtained after mating at 37 degrees C. It was IncFII, showed regions of homology with plasmid R100, determined IncFII serotype conjugative pili constitutively and was transfer-derepressed. After overnight growth at 37 degrees C in non-selective medium, pIN32 gave rise to another derivative, pIN32-2 (size 184.3 MDa), which retained the IncHI2 phenotype and several other pIN32 characters.     

Phage inhibition, colicin resistance, and tellurite resistance are encoded by a single cluster of genes on the IncHI2 plasmid R478.

A region of the IncHI2 plasmid R478, encoding the phenotypes of tellurite resistance (Ter), phage inhibition (Phi), and colicin resistance (PacB), was cloned and sequenced. Analysis indicated seven open reading frames (ORFs), whose genes were designated terZ, -A, -B, -C, -D, -E, and -F. Five of these predicted ORFs (A to E) had extensive amino acid homology with the previously reported ORFs of the IncHI2 Ter operon from plasmid pMER610. There were domains of highly conserved amino acid residues within the group TerA, -D, -E, and -F and within TerD, -E, and -Z, but no consensus could be found among all five putative polypeptides. There were also regions of good identity and similarity between individual pairs of ORFs which was not reflected in the multiple alignments. The three phenotypes were expressed in Escherichia coli DH5 alpha by an 8.4-kb EcoRI insert subcloned from a cosmid of R478. The latter insert was clonable only as a double insertion with a 4.5-kb fragment, and forced deletion of the smaller fragment was lethal to cells. This lethality was not dependent on the cloned orientation of either fragment, suggesting that there is a trans-acting element in the 4.5-kb fragment. Tn1000 mutagenesis of one of the double-insert clones, pDT2575, showed that the phenotypes, including multiple colicin resistance, were genetically linked. Transpositions into terD, terC, and terZ reduced or abolished all phenotypes, while inserts into terE and terF had no effect on the phenotypes. Insertions in terA reduced phage inhibition levels only. The presence of the terZ and terF ORFs in pMER610 was confirmed, and derivatives of this plasmid mediated Phi, PacB, and Ter.     

Streptomyces lividans 66 contains a gene for phage resistance which is similar to the phage λea59 endonuclease gene

The DNA of wild-type Streptomyces lividans 66 is degraded during electrophoresis in buffers containing traces of ferrous iron. S. lividans ZX1, a mutant selected for resistance to DNA degradation, simuiltaneously became sensitive to φHAU3, a wide-host-range temperate bacteriophage. A DNA fragment conferring φHAU3 resistance was cloned; it contains a phage resistance gene whose deduced amino acid sequence is similar to the phage λ Ea59 endonuclease. The S. lividansφHAU3 resistance does not seem to be a classical restriction-modification system, because no host-modified phages able to propagate on the wild-type strain could be isolated. The cloned fragment did not make the host DNA prone to degradation during electrophoresis, indicating that the two phenotypes are controlled by different genes which were deleted together from the chromosome of ZX1.     

Phage Resistance in a Phage-Insensitive Strain of Streptococcus lactis: Temperature-Dependent Phage Development and Host-Controlled Phage Replication

Streptococcus lactis ME2 is a dairy starter strain that is insensitive to a variety of phage, including 18. The efficiency of plating of 18 on ME2 and N1 could be increased from <1 × 10⁻⁹ to 5.0 × 10⁻² and from 7.6 × 10⁻⁷ to 2.1 × 10⁻², respectively, when the host strains were subcultured at 40°C before plating the phage and the phage assay plates were incubated at 40°C. Host-dependent replication was demonstrated in N1 at 30°C and in N1 and ME2 at 40°C, suggesting the operation of a temperature-sensitive restriction and modification system in ME2 and N1. The increased sensitivity of ME2 and N1 to 18 at 40°C was also demonstrated by lysis of broth cultures and increased plaque size. ME2 grown at 40°C showed an increased ability to adsorb 18, indicating a second target for temperature-dependent phage sensitivity in ME2. Challenge of N1 with a 18 preparation that had been previously modified for growth on N1 indicated that at 40°C phage development was characterized by a shorter latent period and larger burst size than at 30°C. The evidence presented suggests that the high degree of phage insensitivity expressed by ME2 consists of a variety of temperature-sensitive mechanisms, including (i) the prevention of phage adsorption, (ii) host-controlled restriction of phage, and (iii) suppression of phage development. At 30°C these factors appear to act cooperatively to prevent the successful emergence of lytic phage active against S. lactis ME2.     

Genetic and nucleotide sequence analysis of the gene htdA, which regulates conjugal transfer of IncHI plasmids.

IncHI plasmids are naturally repressed for conjugative transfer and do not allow efficient propagation of the IncH pilus-specific phage Hgal. Transposons Tn7, Tn5, and TnlacZ were inserted into IncHI plasmids R478, R477-1, and R27, respectively, leading to the isolation of several plasmid mutants which exhibited increased levels of transfer and also permitted good lysis with phage Hgal. A 4.3-kb HindIII fragment from R478 reversed both phenotypic effects of derepression for the R477-1::Tn5 and the R478::Tn7 derivatives, pKFW99 and pKFW100, respectively. Exonuclease III deletions of this fragment and nucleotide sequence analysis indicated that the gene responsible for transfer repression, named here htdA, encoded a polypeptide of 150 amino acids. Cloning and sequence analysis of pDT2454 (R27::TnlacZ) revealed that the transposon had inserted into an open reading frame (ORF) which had an 83% amino acid identity with the R478 htdA gene. Maxicell analysis showed both the R27 and R478 HtdA products had molecular masses of 19.9 kDa. Conjugation experiments showed that the cloned htdA determinants caused a significant reduction of the transfer frequencies of wild-type R478 and R27 plasmids. Examination of both R478 derepressed mutants, pKFW100 and pKFW101, indicated that both transposon insertions occurred upstream of the htdA ORF. The results suggest that HtdA is a regulatory component of IncH plasmid transfer and also show that the region upstream of the htdA ORF is involved in transfer repression. The locations of the htdA determinants were identified on the plasmid maps of R27 and R478.     

Cloning and characterization of a replicon region of the IncHII plasmid pHH1457

A replicative region of the large conjugative plasmid pHH1457 (incompatibility group HII (IncHII)) was cloned. A 1.4-kbp region, in a stable pSBII14 clone, containing a PolI-independent replicon and determinants for the HII incompatibility phenotype, was selected and characterized. High incompatibility with IncHII plasmids was corroborated. Independent replication of the insert was demonstrated by ligation to an antibiotic resistance cassette. pSBII14 was used as a probe to identify IncHII plasmids from other members of the H complex: IncHI (IncHI1, IncHI2 and IncHI3 subgroups). Hybridization experiments revealed a high homology with the replication region of IncHII plasmids, but not with IncHI1 or IncHI3 plasmid prototypes. Homology with IncHI2 plasmids was observed, suggesting the presence of IncHII-like replicons among this subgroup of plasmids. This is the first report of the characterization of an IncHII plasmid maintenance region.     

Polyvinylamine-graft-TEMPO adsorbs onto, oxidizes, and covalently bonds to wet cellulose

Described is a new, greener approach to increasing adhesion between wet cellulose surfaces. Polyvinylamine (PVAm) with grafted TEMPO spontaneously adsorbs onto cellulose and oxidizes the C6 hydroxyl to aldehyde groups that react to form covalent bonds with primary amines on PVAm. Grafted TEMPO offers two important advantages over solutions of low-molecular-weight water-soluble TEMPO derivatives. First, the oxidation of porous cellulose wood fibers is restricted to the exterior surfaces accessible to high-molecular-weight PVAm. Thus, fibers are not weakened by excessive oxidation of the interior fiber wall surfaces. The second advantage of tethered TEMPO is that the total dose of TEMPO required to oxidize dilute fiber suspensions is much less than that required by water-soluble TEMPO derivatives. PVAm-TEMPO is stable under oxidizing conditions. The oxidation activity of the immobilized TEMPO was demonstrated by the conversion of methylglyoxal to pyruvic acid.     

Phage typing of indigenous soybean-rhizobia and relationship of a phage group strains for their asymbiotic and symbiotic nitrogen fixation

A total of 354 indigenous bradyrhizobia were isolated from soybean nodules collected from five major crop grown regions. Host-specific 12 phages, each active on particular strains were selected. Factors, which influence the interaction between the host and phage, were examined. Four different types of plaques were detected. Nearly 17% of isolates were found resistant to all phages. Phage sensitivity patterns revealed a total of 32 distinct phage genotype groups. Different set of phage combinations expressed variation in specificity for parasitizing against particular group of rhizobia. Distributions of isolates in each phage types differed markedly between regions. Interestingly, nine strains belonging to phage group 16 exhibited high ex planta nitrogenase activity in culture. However, no correlation could be established between high ex planta nitrogenase activity and their symbiotic effectiveness with soybean cultivars. Soybean cv. JS335 showed relatively superior performance than Bragg and Lee with indigenous bradyrhizobial strains. Phage typing revealed the existence of large genetic diversity among native rhizobia and selection of the superior bradyrhizobial strains can also be possible for a given soil-climate-cultivar complex.     

Structural proteins of a lipid-containing bacteriophage which replicates in Escherichia coli: phage PR4.

PR4 is a lipid-containing bacteriophage which is able to replicate in Escherichia coli. The virus was labeled with either [14C]leucine and [14C]threonine or H235SO4 and then purified by several rounds of sucrose gradient centrifugation. Autoradiographs showed the virus to be composed of six major protein species with molecular weights (1) 68 000, (2) 47 500, (3) 38 500, (4) 35 000, (5) 20 700, (6) 16 500 daltons. Electropherograms showed protein No. 2 to be the major protein, comprising about 43% of the total weight of viral protein.     

Phage lytic enzymes: a history

There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of ‘bacteria-eaters' or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal flora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well(Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specifi c disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay(Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes–from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.     

Construction of a human X-chromosome-enriched phage library which facilitates analysis of specific loci

A human X-chromosome-enriched MboI-partial-digest recombinant library in phage lambda Charon30 has been constructed. Twelve out of the thirteen X-chromosome DNA sequences that were tested were present in the library. Most regions were covered in overlapping phage inserts; mean insert size was 13.7 kb. One phage from the library allowed detection of a 225-bp insertion of DNA into a region near the Duchenne muscular dystrophy (DMD) locus. Another recombinant phage represents an expansion of a region which exhibits extensive and varying homology with other human chromosomes, including the Y, as well as with rodent DNA. The present library should have widespread use for examining DNA sequences on the human X chromosome.