Isolation and Characterization of Five Erwinia amylovora Bacteriophages and Assessment of Phage Resistance in Strains of Erwinia amylovora

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages Ea1 and Ea7 and 3 novel phages named Ea100, Ea125, and Ea116C, were identified based on differences in genome size and restriction fragment pattern. Ea1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages Ea100, Ea7, and Ea125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. Ea116C contained an approximately 75-kb genome. Ea1, Ea7, Ea100, Ea125, and Ea116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. Ea116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10⁵ CFU.     

Prophage Origin of a Virulent Phage Appearing on Fermentations of Lactobacillus casei S-1

For protection from the abnormal fermentation of Lactobacillus casei S-1 caused by contamination of a virulent phage, FSV, the origin of this phage was studied. Morphologies, viral structural proteins, and DNA structures of three independent isolates of FSV were compared with those of FSW, which is lysogenized in strain S-1. The results showed (i) that the morphology of FSV phages is indistinguishable from that of FSW and (ii) that all viral structural components found in FSW are present in the particles of FSV's. In addition, restriction endonuclease analyses of viral DNA showed that the HindIII-digested fragments of FSW DNA, the sum of which covered at least 94.7% of this phage genome, were conserved in the FSV DNA digests. Results of Southern filter hybridization of the S-1 and prophage-cured cell (C239) DNAs with FSV DNA as a probe revealed that C239 had lost most of the FSV DNA sequence, whereas S-1 had about one copy of the FSV DNA sequence. These results indicate that virulent phage FSV is derived from the lysogenized phage FSW. Therefore, the appearance of FSV can be eliminated by using the prophage-cured derivative of S-1.     

Characterization of Temperate Bacillus Bacteriophage phi105

Temperate Bacillus phage 105 is serologically unrelated to previously described virulent Bacillus phages. Phage 105 is incapable of generalized transduction. Prophage 105 is inducible with mitomycin C. Phage 105 contains double-stranded deoxyribonucleic acid (DNA) with a molecular weight of about 25 × 10⁷ as determined by band sedimentation and electron microscopy. The per cent guanine plus cytosine of 105 DNA is 43.5 as determined by buoyant density in CsCl and by thermal denaturation. Phage 105 DNA may contain complementary single-stranded ends.     

Common Elements Regulating Gene Expression in Temperate and Lytic Bacteriophages of Lactococcus Species

A phage-inducible middle promoter (P_15A10) from the lytic, lactococcal bacteriophage 31, a member of the P335 species, is located in an 888-base pair fragment near the right cohesive end. Sequence analysis revealed extensive homology (>95%) to the right cohesive ends of two temperate phages of the P335 species, r1t and LC3. Sequencing upstream and downstream of P_15A10 showed that the high degree of homology between 31 and r1t continued beyond the phage promoter. With the exception of one extra open reading frame in 31, the sequences were highly homologous (95 to 98%) between nucleotides 13448 and 16320 of the published r1t sequence. By use of a β-galactosidase (β-Gal) gene under the control of a smaller, more tightly regulated region within the P_15A10 promoter, P_566–888, it was established that mitomycin C induction of a lactococcal strain harboring the prophage r1t induced the P_566–888 promoter, as determined from an increase in β-Gal activity. Hybridization of nine other lactococcal strains with ³²P-labeled P_566–888 showed that the Lactococcus lactis strains C10, ML8, and NCK203 harbored sequences homologous to that of the phage-inducible promoter. Mitomycin C induced the resident prophages in all these strains and concurrently induced the P_566–888 promoter, as determined from an increase in β-Gal activity. DNA restriction analysis revealed that the prophages in C10, ML8, and NCK203 had identical restriction patterns which were different from that of r1t. In addition, DNA sequencing showed that the promoter elements in the three phages were identical to each other and to P_566–888 from the lytic phage 31. These results point to a conserved mechanism in the regulation of gene expression between the lytic phage 31 and at least two temperate bacteriophages and provide further evidence for a link in the evolution of certain temperate phages and lytic phages.     

Molecular Characterization of Three Small Isometric-Headed Bacteriophages Which Vary in Their Sensitivity to the Lactococcal Phage Resistance Plasmid pTR2030

Lactococcus lactis LMA12-4 is a pTR2030 transconjugant that has been used as an industrial starter culture because of its resistance to phages predominant in cheese plants. Plasmid pTR2030 interferes with susceptible phages in this host strain via two mechanisms, restriction and modification (R/M) and abortive infection (Hsp). After prolonged use of LMA12-4 transconjugants in the industry, two different bacteriophages, designated nck202.48 (48) and nck202.50 (50), were isolated which could produce plaques on LMA12-4 containing pTR2030. In this study, these two phages were characterized and compared with a third phage, nck202.31 (31), which is susceptible to both the R/M and Hsp activities encoded by pTR2030. Phage 48 was not susceptible to inhibition by Hsp, whereas 50 was unaffected by either the R/M or Hsp mechanisms. All three were small isometric-headed phages, but small differences were noted between the phages in the structural details of the tail base plate, susceptibility to chloroform treatment, and requirements for calcium infectivity. The phage genomes were all between 29.9 and 31.9 kb in length. Phages 31 and 48 harbored cohesive ends, whereas the phage 50 genome was circularly permuted, terminally redundant, and carried a putative packaging initiation site. DNA-DNA hybridization experiments conducted between the phages revealed a common region in 48 and 50 that may correlate with the resistance of the two phages to the Hsp-abortive infection induced by pTR2030. Phage 50 also harbored DNA sequences that shared homology to pTR2030 in the region where R/M activities have been localized on the plasmid. Molecular characterization of the three phages localized regions within the genomes of the pTR2030-resistant phages that may be responsible for circumventing plasmid-encoded Hsp and R/M defense mechanisms in lactococci.     

Effect of Increasing the Copy Number of Bacteriophage Origins of Replication, in trans, on Incoming-Phage Proliferation

Bacteriophage resistance mechanisms which are derived from a bacteriophage genome are termed Per (phage-encoded resistance). When present in trans in Lactococcus lactis NCK203, Per50, the cloned origin of replication from phage 50, interferes with 50 replication. The per50 fragment was found to afford negligible protection to NCK203 against 50 infection when present in a low-copy-number plasmid, pTRK325. A high-copy-number Per50 construct (pTRK323) dramatically affected 50 infection, reducing the efficiency of plaquing (EOP) to 2.5 × 10^-4 and the plaque size to pinhead proportions. This clone also afforded significant protection against other related small isometric phages. Per31 was cloned from phage 31 and demonstrated to function as an origin of replication by enabling replication of per31-containing plasmids, in NCK203, on 31 infection. A low-copy-number Per31 plasmid (pTRK360) reduced the EOP of 31 on NCK203 to 0.3 and the plaque diameter from 1.5 to 0.5 mm. When this plasmid was cloned in high copy number, the EOP was further reduced to 7.2 × 10^-7 but the plaques were large and contained Per31-resistant phages. Characterization of these “new” phages revealed at least two different types that were similar to 31, except that DNA alterations were noted in the region containing the origin. This novel and powerful abortive phage resistance mechanism should prove useful when directed at specific, problematic phages.     

Genomic characterization of Ralstonia solanacearum phage phiRSA1 and its related prophage (phiRSX) in strain GMI1000

RSA1 is a wide-host-range bacteriophage isolated from Ralstonia solanacearum. In this study, the complete nucleotide sequence of the RSA1 genomic DNA was determined. The genome was 38,760 bp of double-stranded DNA (65.3% G+C) with 19-bp 5′-extruding cohesive ends (cos) and contained 51 open reading frames (ORFs). Two-thirds of the RSA1 genomic region encodes the phage structural modules, and they are very similar to those reported for coliphage P2 and P2-like phages. A RSA1 minireplicon with an 8.2-kbp early-expressing region was constructed. A late-expression promoter sequence motif was predicted for these RSA1 genes as 5′ TGTTGT-(X)₁₃-ACAACA. The genomic sequence similarity between RSA1 and related phages 52237 and CTX was interrupted by three AT islands, one of which contained an insertion sequence element, suggesting that they were recombinational hot spots. RSA1 was found to be integrated into at least three different strains of R. solanacearum, and the chromosomal integration site (attB) was identified as the 3′ portion of the arginine tRNA(CCG) gene. In the light of the RSA1 gene arrangement, one possible prophage sequence previously detected on the chromosome of R. solanacearum strain GMI1000 was characterized as a RSA1-related prophage (designated RSX). RSX was found to be integrated at the serine tRNA (GGA) gene as an att site, and its size was determined to be 40,713 bp. RSX ORFs shared very high amino acid identity with their RSA1 counterparts. The relationships and evolution of these P2-like phages are discussed.     

Genomic characterization of Ralstonia solanacearum phage phiRSB1, a T7-like wide-host-range phage

RSΒ1 is a wide-host-range, T7-like bacteriophage that infects and efficiently lyses the phytopathogenic bacterium Ralstonia solanacearum. The RSB1 genome comprises 43,079 bp of double-stranded DNA (61.7% G+C) with 325-bp terminal repeats and contains 47 open reading frames. Strong activity of tandem early promoters and wide specificity of phage promoters of RSB1 were demonstrated.     

Genetic Analysis of Chromosomal Regions of Lactococcus lactis Acquired by Recombinant Lytic Phages

Recombinant phages are generated when Lactococcus lactis subsp. lactis harboring plasmids encoding the abortive type (Abi) of phage resistance mechanisms is infected with small isometric phages belonging to the P335 species. These phage variants are likely to be an important source of virulent new phages that appear in dairy fermentations. They are distinguished from their progenitors by resistance to Abi defenses and by altered genome organization, including regions of L. lactis chromosomal DNA. The objective of this study was to characterize four recombinant variants that arose from infection of L. lactis NCK203 (Abi⁺) with phage 31. HindIII restriction maps of the variants (31.1, 31.2, 31.7, and 31.8) were generated, and these maps revealed the regions containing recombinant DNA. The recombinant region of phage 31.1, the variant that occurred most frequently, was sequenced and revealed 7.8 kb of new DNA compared with the parent phage, 31. This region contained numerous instances of homology with various lactococcal temperate phages, as well as homologues of the lambda recombination protein BET and Escherichia coli Holliday junction resolvase Rus, factors which may contribute to efficient recombination processes. A sequence analysis and phenotypic tests revealed a new origin of replication in the 31.1 DNA, which replaced the 31 origin. Three separate HindIII fragments, accounting for most of the recombinant region of 31.1, were separately cloned into gram-positive suicide vector pTRK333 and transformed into NCK203. Chromosomal insertions of each plasmid prevented the appearance of different combinations of recombinant phages. The chromosomal insertions did not affect an inducible prophage present in NCK203. Our results demonstrated that recombinant phages can acquire DNA cassettes from different regions of the chromosome in order to overcome Abi defenses. Disruption of these regions by insertion can alter the types and diversity of new phages that appear during phage-host interactions.     

Differential functional behavior of viral phi29, Nf and GA-1 SSB proteins

DNA replication of 29 and related phages takes place via a strand displacement mechanism, a process that generates large amounts of single-stranded DNA (ssDNA). Consequently, phage-encoded ssDNA-binding proteins (SSBs) are essential proteins during phage 29-like DNA replication. In the present work we analyze the helix-destabilizing activity of the SSBs of 29 and the related phages Nf and GA-1, their ability to eliminate non-productive binding of 29 DNA polymerase to ssDNA and their stimulatory effect on replication by 29 DNA polymerase in primed M13 ssDNA replication, a situation that resembles type II replicative intermediates that occur during 29-like DNA replication. Significant differences have been appreciated in the functional behavior of the three SSBs. First, the GA-1 SSB is able to display helix-destabilizing activity and to stimulate dNTP incorporation by 29 DNA polymerase in the M13 DNA replication assay, even at SSB concentrations at which the 29 and Nf SSBs do not show any effect. On the other hand, the 29 SSB is the only one of the three SSBs able to increase the replication rate of 29 DNA polymerase in primed M13 ssDNA replication. From the fact that the 29 SSB, but not the Nf SSB, stimulates the replication rate of Nf DNA polymerase we conclude that the different behaviors of the SSBs on stimulation of the replication rate of 29 and Nf DNA polymerases is most likely due to formation of different nucleoprotein complexes of the SSBs with the ssDNA rather than to a specific interaction between the SSB and the corresponding DNA polymerase. A model that correlates the thermodynamic parameters that define SSB–ssDNA nucleoprotein complex formation with the functional stimulatory effect of the SSB on 29-like DNA replication has been proposed.     

Pasteurella pestis Bacteriophage H and Escherichia coli Bacteriophage φII Are Nearly Identical

Electron microscopy examination of II-H deoxyribonucleic acid heteroduplexes, together with polyacrylamide gel electrophoretic analyses of phage ribonucleic acid species and proteins labeled in II or H-infected cells, demonstrates that Pasteurella pestis phage H is nearly identical to coliphage II.     

Bacteriophage phi6: a Lipid-Containing Virus of Pseudomonas phaseolicola

The purification and properties of a lipid-containing bacteriophage, 6, are described. The phage contains a lipid envelope which is probably essential for infection. Infectivity of 6 was lost in the presence of organic solvents, sodium deoxycholate, and phospholipase A. The fatty acid composition of the phage lipid was similar to that of the Pseudomonas phaseolicola host cells. The phage was composed of about 25% lipid, 13% RNA, and 62% protein. The buoyant density of 6 was 1.27 g/ml in cesium chloride. The morphology of 6 was unusual; it had a polyhedral head of about 60 nm surrounded by a membranous, compressible envelope which appeared to assume an elongated configuration upon attachment to pili. The adsorption rate constant was 3.3 × 10⁻¹⁰ ml/min in a semi-synthetic medium and 3.8 × 10⁻¹⁰ ml/min in a nutrient broth-yeast extract medium. The latent period was shorter in the former medium (80-115 min compared with 120-160 min), and the average burst size was larger (250-400 compared with 125-150). The eclipse period coincided with the latent period.     

DNA-Mediated Prophage Induction in Bacillus subtilis Lysogenic for φ105c4

Prophage was induced when strains of Bacillus subtilis 168 lysogenic for 105c4 were grown to competence and exposed to specific bacterial DNAs. The time course of phage production was similar to that observed for mitomycin C induction of wild-type prophage. Induction was directly dependent upon DNA concentration up to levels which were saturating for the transformation of bacterial auxotrophic markers. The extent of induction varied with the source of DNA. The burst of phage induced by DNA isolated from a W23 strain of B. subtilis was fivefold less than that induced by DNA from B. subtilis 168 strains, while B. licheniformis DNA was completely inactive. This order of inducing activity was correlated with the ability of the respective DNAs to transform auxotrophic markers carried by one of the 105c4 lysogens. Differences in inducing activity also were observed for different forms of 105 DNA. The DNAs isolated from 105 phage particles and 105c4 lysogens were inactive, whereas DNA from cells lysogenized by wild-type 105 induced a burst of phage. When tested for transforming activity, however, both 105c4 and 105 lysogen DNAs were equally effective. An induction mechanism which involves recombination at the prophage insertion site is proposed to explain these differences.     

High-Frequency Plasmid Transduction by Lactobacillus gasseri Bacteriophage φadh

The temperate bacteriophage adh mediates plasmid DNA transduction in Lactobacillus gasseri ADH at frequencies in the range of 10^-8 to 10^-10 transductants per PFU. BglII-generated DNA fragments from phage adh were cloned into the BclI site of the transducible plasmid vector pGK12 (4.4 kb). Phage adh lysates induced from Lactobacillus lysogens harboring pGK12 or the recombinant plasmids were used to transduce strain ADH to chloramphenicol resistance. The transduction frequencies of recombinant plasmids were 10²- to 10⁵-fold higher than that of native pGK12. The increase in frequency generally correlated with the extent of DNA-DNA homology between plasmid and phage DNAs. The highest transduction frequency was obtained with plasmid pTRK170 (6.6 kb), a pGK12 derivative containing the 1.4- and 0.8-kb BglII DNA fragments of adh. DNA hybridization analysis of pTRK170-transducing phage particles revealed that pTRK170 had integrated into the adh genome, suggesting that recombination between homologous sequences present in phage and plasmid DNAs was responsible for the formation of high-frequency transducing phage particles. Plasmid DNA analysis of 13 transductants containing pTRK170 showed that each had acquired intact plasmids, indicating that in the process of transduction a further recombination step was involved in the resolution of plasmid DNA monomers from the recombinant pTRK170::adh molecule. In addition to strain ADH, pTRK170 could be transduced via adh to eight different L. gasseri strains, including the neotype strain, F. Gasser 63 AM (ATCC 33323).     

Interaction between Light Quality and Light Quantity in the Photoregulation of Anthocyanin Production

The interaction between phytochrome photoequilibrium () and photon flux in the photoregulation of anthocyanin production under prolonged irradiation was studied in seedlings of Brassica oleracea L. and Lycopersicon esculentum Mill. In cabbage, anthocyanin production increases with decreasing , reaching a maximum at the lowest value ( = 0.13) used in this study; in tomato, the extent of the response is higher at intermediate values, reaching a maximum at = 0.46. In cabbage, the response increases with increasing photon flux at all values; however, the response to changes in photon flux is minimal at = 0.85, and, at = 0.13, minimal at photon fluxes higher than 5 micromolar per square meter per second. In tomato, the response increases with increasing photon flux at = 0.46, 0.65, and 0.85, the response to changes in photon fluxes being minimal at = 0.85; at = 0.13 and 0.29 the response first increases (significantly at = 0.29 and minimally at = 0.13) and then decreases with increasing photon fluxes, the transition occurring at about 1 micromolar per square meter per second at = 0.13, and at 5 micromolar per square meter per second at = 0.29. The patterns of light quality-quantity interaction in the photoregulation of anthocyanin production are significantly different in cabbage and tomato and are also significantly different than those observed for other photomorphogenic responses to prolonged irradiations.     

Cell wall yield properties of growing tissue : evaluation by in vivo stress relaxation

Growing pea stem tissue, when isolated from an external supply of water, undegoes stress relaxation because of continued loosening of the cell wall. A theoretical analysis is presented to show that such stress relaxation should result in an exponential decrease in turgor pressure down to the yield threshold (Y), with a rate constant given by ε where is the metabolically maintained irreversible extensibility of the cell wall and ε is the volumetric elastic modulus of the cell. This theory represents a new method to determine in growing tissues.

Stress relaxation was measured in pea (Pisum sativus L.) stem segments using the pressure microprobe technique. From the rate of stress relaxation, of segments pretreated with water was calculated to be 0.08 per megapascal per hour while that of auxin-pretreated tissue was 0.24 per megapascal per hour. These values agreed closely with estimates of made by a steady-state technique. The yield threshold (0.29 megapascal) was not affected by auxin. Technical difficulties with measuring by stress relaxation may arise due to an internal water reserve or due to changes in subsequent to excision. These difficulties are discussed and evaluated.

A theoretical analysis is also presented to show that the tissue hydraulic conductance may be estimated from the T_½ of tissue swelling. Experimentally, pea stems had a swelling T_½ of 2.0 minutes, corresponding to a relative hydraulic conductance of about 2.0 per megapascal per hour. This value is at least 8 times larger than . From these data and from computer modeling, it appears that the radial gradient in water potential which sustains water uptake in growing pea segments is small (0.04 megapascal). This means that hydraulic conductance does not substantially restrict growth. The results also demonstrate that the stimulation of growth by auxin can be entirely accounted for by the change in .

     

Biochemical and biophysical properties of hyphomicrobium bacteriophage hyphi30

Hyphomicrobium bacteriophage Hy30 and its nucleic acid were studied to determine some of their biochemical and biophysical properties. The molecular weight of the phage is 55.4 × 10⁶, and its buoyant density is 1.508 g/ml. The nucleic acid of Hy30 is linear, double-stranded DNA with a molecular weight of 29.7 × 10⁶. The guanine-plus-cytosine content of the DNA was 62% as determined from its melting temperature and buoyant density.     

Comparative Genomic Analyses of the Vibrio Pathogenicity Island and Cholera Toxin Prophage Regions in Nonepidemic Serogroup Strains of Vibrio cholerae

Two major virulence factors are associated with epidemic strains (O1 and O139 serogroups) of Vibrio cholerae: cholera toxin encoded by the ctxAB genes and toxin-coregulated pilus encoded by the tcpA gene. The ctx genes reside in the genome of a filamentous phage (CTX), and the tcpA gene resides in a vibrio pathogenicity island (VPI) which has also been proposed to be a filamentous phage designated VPI. In order to determine the prevalence of horizontal transfer of VPI and CTX among nonepidemic (non-O1 and non-O139 serogroups) V. cholerae, 300 strains of both clinical and environmental origin were screened for the presence of tcpA and ctxAB. In this paper, we present the comparative genetic analyses of 11 nonepidemic serogroup strains which carry the VPI cluster. Seven of the 11 VPI⁺ strains have also acquired the CTX. Multilocus sequence typing and restriction fragment length polymorphism analyses of the VPI and CTX prophage regions revealed that the non-O1 and non-O139 strains were genetically diverse and clustered in lineages distinct from that of the epidemic strains. The left end of the VPI in the non-O1 and non-O139 strains exhibited extensive DNA rearrangements. In addition, several CTX prophage types characterized by novel repressor (rstR) and ctxAB genes and VPIs with novel tcpA genes were found in these strains. These data suggest that the potentially pathogenic, nonepidemic, non-O1 and non-O139 strains identified in our study most likely evolved by sequential horizontal acquisition of the VPI and CTX independently rather than by exchange of O-antigen biosynthesis regions in an existing epidemic strain.     

Isolation and Characterization of Temperate Bacteriophages of Clostridium difficile

The lack of information on bacteriophages of Clostridium difficile prompted this study. Three of 56 clinical C. difficile isolates yielded double-stranded DNA phages C2, C5, C6, and C8 upon induction. Superinfection and DNA analyses revealed relatedness between the phages, while partial sequencing of C2 showed nucleotide homology to the sequenced C. difficile strain CD630.     

Photocontrol of Hypocotyl Elongation in Light-Grown Cucumis sativus L. : Responses to Phytochrome Photostationary State and Fluence Rate

The effects of the calculated photostationary state of phytochrome (_c) and the photon fluence rate on the elongation growth of the hypocotyl of light-grown seedlings of Cucumis sativus L. are examined. Two threshold responses to _c are found at values of 0.06 and 0.43. At _c = 0.06, there is no response at any fluence rate. In the _c range 0.1 to 0.43, elongation growth does not respond to changes in _c. Above the second threshold (_c = 0.43), there is a strong response to changes in _c. At all values of _c at and above 0.1, there is a response to fluence rate. A linear relationship can be demonstrated between a factor comprised of the logarithm of phytochrome cycling rate (a fluence-rate-dependent process) and _c, and the growth response.