Interaction between bacteriophage PBS1 and clay minerals and transduction of Bacillus subtilis by clay-phage complexes

Bacteriophage PBS1 of Bacillus subtilis was rapidly adsorbed on montmorillonite (M) and kaolinite (K), and adsorption was maximal after 30 min on both clays. There was no correlation between adsorption and the cation exchange capacity of the clays. Studies with sodium metaphosphate (a polyanion that interacts with positively charged sites on clay) indicated that positively charged sites on K were primarily responsible for the adsorption of the phage, whereas other mechanisms appeared to be involved in adsorption of the phage on M. X-ray diffraction and electron microscopic analyses showed that the phage partially intercalated M. Survival of the phage was increased by adsorption on the clays, and adsorbed phage maintained its ability to transduce bacterial cells for at least 30 days (the longest time studied) after the preparation of the clay–phage complexes. Electron microscopic observations indicated that transduction by the clay–phage complexes was primarily the result of the phage detaching from the clays in the presence of host cells.     

Productive Infection of Bacillus subtilis 168, with Bacteriophage SP-10, Dependent upon Inducing Treatments

Strains of Bacillus that harbor defective phage PBSX were found to be insensitive to SP-10(C), although the phage adsorbed to these insensitive strains. Strains that did not carry the phage were sensitive to SP-10(C). B. subtilis 168 ind(-), which can be tranduced by SP-10(C) but is nonpermissive for the phage, was rendered phage-sensitive after treatment with ultraviolet (UV) light or mitomycin C. After induction with UV light, maximal sensitivity to SP-10(C) was obtained at a multiplicity of infection (MOI) of approximately 14; with mitomycin C induction, an MOI of approximately 1.0 was required. Phage maturation in sensitized cells was followed by plating infected streptomycin-sensitive cells in the presence of streptomycin at various stages during phase development. The latent period was estimated at 60 to 75 min. We suggest that the resistance of B. subtilis 168 to SP-10 is controlled, at least in part, by the presence of a defective prophage.     

Inactivation of clay-associated bacteriophage MS-2 by chlorine.

The model system consisted of bacteriophage MS-2, bentonite clay, and hypochlorous acid (HOC1). Factors that influenced association of the bacterial virus with bentonite were the titer of unadsorbed viruses, clay concentration, cation concentration, temperature, stirring rate, and the presence of soluble organics. Variation of the kinetic adsorption rate constant with stirring speed indicates that phage attachment is a diffusion-limited process; the attachment reaction has an apparent activation energy of 1 kcal/mol. About 18% of clay-associated bacteriophages was recovered by mixing the suspension with an organic eluent. Inactivation data were obtained from batch reactors operated under those conditions in which loss of HOC1 was minimal during the reaction. Bacteriophages attached to clay were more resistant to HOC1 than were freely suspended phages; for equivalent HOC1 concentrations, clay-associated phages required about twice the time that freely suspended phages required for loss of 99% of the initial virus titer.     

Inactivation of clay-associated bacteriophage MS-2 by chlorine.

The model system consisted of bacteriophage MS-2, bentonite clay, and hypochlorous acid (HOC1). Factors that influenced association of the bacterial virus with bentonite were the titer of unadsorbed viruses, clay concentration, cation concentration, temperature, stirring rate, and the presence of soluble organics. Variation of the kinetic adsorption rate constant with stirring speed indicates that phage attachment is a diffusion-limited process; the attachment reaction has an apparent activation energy of 1 kcal/mol. About 18% of clay-associated bacteriophages was recovered by mixing the suspension with an organic eluent. Inactivation data were obtained from batch reactors operated under those conditions in which loss of HOC1 was minimal during the reaction. Bacteriophages attached to clay were more resistant to HOC1 than were freely suspended phages; for equivalent HOC1 concentrations, clay-associated phages required about twice the time that freely suspended phages required for loss of 99% of the initial virus titer.     

Plasmid DNA transduction in Bacillus subtilis

Transduction of Bacillus subtilis pUB110 plasmid by AR9 phage is described. Some aspects of this process are studied. Plasmid transduction depended on multiplicity of infection similar to cases of chromosomal markers transduction, though optimal multiplicity of infection was achieved using low number of phage particles. No cotransduction of plasmid and chromosomal markers was demonstrated. The transduction frequencies of plasmid and chromosomal markers increased after UV irradiation of phage suspensions within the range of definite doses.     

Effect of ultraviolet C irradiation on folate and free amino acid contents in leaves of Pisum sativum

The effect of ultraviolet radiation of the C region on the content of general folates and free amino acids in leaves of pea (Pisum sativum, variety Neistoshchimyi) was studied. It was found that one of rapidly detectable consequences of the UV irradiation is the photolysis of general folates and pteridines. It was shown that the photolysis of folates results in the formation of the tolerant compound pterin-6-carboxylic acid. It was assumed that this compound sensitizes the formation of singlet molecular oxygen. Pterin-6-carboxylic acid strongly fluoresces by the action of UV light. The relative quantum fluorescence yield of pterin-6-carboxylic acid at 293 K is approximately 2.0 (absolute valuer approximately 0.58). The kinetics of changes in the content of free amino acids 0.5, 1.0, 10, and 40 min after the exposure to UV light was studied. Changes in qualitative composition and quantitative content of free amino acids in leaves of irradiated plants, occurring due to glycolytic processes are discussed.     

Repair and recombination of nonreplicating UV-irradiated phage DNA in E. coli III. Enhancement of excision repair in UV-treated bacteria

Summary The question of whether induction of the SOS response in Escherichia coli increases the efficiency of excision repair was addressed by measuring repair of UV-damaged nonreplicating lambda phage DNA in previously irradiated bacteria. Prior UV irradiation of lex ⁺ bacteria enhanced both the rate of regeneration of infective phage DNA (about 10-fold) and the rate of cyclobutane dimer removal early in repressed infections. Indirect induction of SOS-regulated repair activities by the nonreplicating irradiated phage DNA itself seemed negligible. Prior bacterial irradiation reduced the frequency of recombination (loss of a tandem chromosomal duplication) of nonreplicating UV-irradiated DNA. In this respect UV-stimulated recombination of nonreplicating DNA differs from RecF-dependent recombination processes that are stimulated by increased SOS expression.Surprisingly, prior UV irradiation of lexA3 bacteria caused a small but reproducible increase in the regeneration of infective phage DNA.     

Effect of Several Clay Minerals and Humic Acid on the Survival of Klebsiella aerogenes Exposed to Ultraviolet Irradiation

The effect of various clay minerals and humic acid on the survival of Klebsiella aerogenes exposed to ultraviolet (UV) irradiation was investigated. A protective effect was observed and found to depend on the specific light absorption and light scattering properties of the clay minerals and the humic acid used. The higher the specific absorption, the better was the survival of K. aerogenes after UV irradiation. Bacterial survival was lower in clays saturated with divalent cations (Ca, Zn) than in those homoionic to monovalent cations (K).     

Bacillus subtilis mutants defective in bacteriophage phi 29 head assembly.

Virus assembly mutants of asporogenous Bacillus subtilis defective in bacteriophage phi 29 head assembly were detected by the use of antibodies that reacted strongly with the free dodecameric phi 29 portal vertex composed of gene product 10 (gp10) but weakly with the portal vertex assembled into proheads or phage. Phage adsorption and the synthesis of phage proteins, DNA-gene product 3, and prohead RNA were normal in these mutants, but prohead and phage production was greatly reduced. The assembly defect was transferred to competent B. subtilis by transformation and transduction. PBS1 transduction showed that the vam locus was linked to Tn917 located at 317 degrees on the B. subtilis chromosome.     

Clay-Nucleic Acid Complexes: Characteristics and Implications for the Preservation of Genetic Material in Primeval Habitats

The equilibrium adsorption of three nucleic acids: chromosomal DNA, supercoiled plasmid DNA, and 25S rRNA, on the clay minerals, montmorillonite (M) and kaolinite (K), were studied. Adsorption of the nucleic acid on the clays was rapid and maximal after 90 min of contact time. Chromosomal DNA was adsorbed to a greater extent than plasmid DNA and RNA, and the adsorption was also greater on M than on K. Adsorption isotherms were of the L type, and a plateau was reached with all the complexes, with the exception of chromosomal DNA adsorbed on M. To determine where nucleic acids are adsorbed on clay minerals and the nature of the interaction, complexes were studied by X-ray diffraction (X-RD), electron microscopy, and Fourier transform infrared (FT-IR) spectroscopy. X-RD showed that nucleic acids did not penetrate the clay, indicating that the adsorption occurred primarily on the external surfaces of clay particles, as also suggested by electron microscopy observations. FT-IR spectra of clay-tightly bound nucleic acid complexes showed absorption bands that indicate a variation of the nucleic acids status as a consequence of their adsorption on clay. Data obtained suggested that the formation of clay-nucleic acid complex could have an important role in the preservation of genetic material in primeval habitats.     

Dot Blot Enzyme-Linked Immunosorbent Assay for Monitoring the Fate of Insecticidal Toxins from Bacillus thuringiensis in Soil

The release of transgenic plants and microorganisms expressing truncated genes from Bacillus thuringiensis that code for active insecticidal toxins rather than for the inactive protoxins could result in the accumulation of these active proteins in soil, especially when bound on clay minerals and other soil particles. To monitor the fate of these toxins in soil, a dot blot enzyme-linked immunosorbent assay (ELISA) that detects free and particle-bound toxins from B. thuringiensis subsp. kurstaki and subsp. tenebrionis was developed. The lower limit of detection of the toxins, either free or adsorbed or bound on the clay minerals montmorillonite (M) or kaolinite (K) or on the clay-particle-size fraction separated from soil (by sedimentation according to Stokes' Law), was approximately 3 ng. Antibodies (Ab) to the toxins from B. thuringiensis subsp. kurstaki and from B. thuringiensis subsp. thuringiensis were raised in goats and rabbits, respectively, and each Ab was rendered specific by adsorption onto CNBr-activated Sepharose coupled with the other toxin. The preadsorbed Ab were specific for the toxins from both subspecies, both free and bound on M, K, or the clay-particle-size fraction of soil. The toxins that were added to sterile and nonsterile soil amended with M or K or not amended were detected on the clay-particle-size fraction of the soil after various periods of incubation by the dot blot ELISA. No toxins were detected on the silt- and sand-particle-size fractions. Each dot blot, containing various amounts of toxins and/or clays, was applied to a polyvinylidene difluoride membrane in a dot blot vacuum system. The toxins were still detectable on the clay-particle-size fraction of nonsterile soil after 40 days. This agreed with preliminary results of other studies in this laboratory that when these toxins bind on clay minerals, they become resistant to utilization by microorganisms.     

W-reactivation of phage lambda in X-irradiated mutants ofEscherichia coli K-12

Summary The survival of UV irradiated phage lambda was increased on X-irradiatedE. coli K-12 host cells over that on unirradiated cells. The frequency of c mutants among the surviving phages was to a similar extent increased by the X-ray exposure of the host cells as by UV light. This W-reactivation of phage lambda occurred inuvrA, polA, andrecB mutants besides the wild type at about equal X-ray doses, however, at a reduced reactivation efficiency compared with the wild type. W-reactivation was undetectable inrecA mutants. While maximal UV induced W-reactivation occurred 30 min after irradiation, the maximal X-ray induced reactivation was found immediately after irradiation. Chloramphenicol (100 µg/ml) and nitrofurantoin (50 µg/ml) inhibited W-reactivation of phage lambda if added before irradiation of the host cells, indicating the necessity of protein synthesis for W-reactivation.     

Study of MFD in Bacillus subtilis.

The frequency of UV-induced extragenic suppressor reversions to leucine independence in B. subtilis carrying a leu8 mutation decreased when irradiated cells were temporarily incubated in medium deprived of nitrogen sources. This mutation frequency decline (MFD) was inhibited by acriflavine and was poorly expressed in a uvr1 mutant. Consequently, MFD may be considered as the manifestation of an anti-mutagenic activity of excision repair. MFD was decelerated and even vanished in cells subjected to prolonged starvation of nitrogen sources before irradiation. MFD was accelerated in bacteria that were first irradiated and incubated in nutritional medium for at least 30 min. The stimulation of MFD by UV exposure was observed only in the uvr+ strain and depended on protein synthesis after irradiation. It is assumed that different rates of MFD in cells of various pre-radiation histories reflect different levels of the excision-repair activity inherent in these cells.     

Some features of the reaction of intracellular bacteriophages T1 to UV irradiation.

The reaction of complexes pf phage T1-cells of E. coli B or E. coli Bs-1 to UV irradiation was investigated. The complexes were irradiated at various stage of infection, and their survival, extent of Hcr and Phr, were evaluated. It was found that the UV resistance of phage DNA in the second half of the latent period fluctuates. Hcr after UV exposure at these stages of infection operates in a small volume. The ability of intracellular phage to photoreactivate when cells of E. coli B were infected is constant after irradiation at many stages of infection, except the early ones. In the complexes of phage T1-bacteria of E. coli Bs-1 this ability declines while infection is promoted. The daughter phage particles released from UV irradiated complexes undergo Phr and Hcr only after irradiation at the late stages of infection. This was not the cases when complexes of phage-bacteria were irradiated during the first half of the latent period. A possible tole of UV-damaged phage DNA in propagation of infection and in maturation of phage particles is discussed.     

Discovery, purification, and characterization of a temperate transducing bacteriophage for Bordetella avium

We discovered and characterized a temperate transducing bacteriophage (Ba1) for the avian respiratory pathogen Bordetella avium. Ba1 was initially identified along with one other phage (Ba2) following screening of four strains of B. avium for lysogeny. Of the two phage, only Ba1 showed the ability to transduce via an allelic replacement mechanism and was studied further. With regard to host range, Ba1 grew on six of nine clinical isolates of B. avium but failed to grow on any tested strains of Bordetella bronchiseptica, Bordetella hinzii, Bordetella pertussis, or Bordetella parapertussis. Ba1 was purified by CsCl gradient centrifugation and was found to have an icosahedral head that contained a linear genome of approximately 46.5 kb (contour length) of double-stranded DNA and a contractile, sheathed tail. Ba1 readily lysogenized our laboratory B. avium strain (197N), and the prophage state was stable for at least 25 generations in the absence of external infection. DNA hybridization studies indicated the prophage was integrated at a preferred site on both the host and phage replicons. Ba1 transduced five distinctly different insertion mutations, suggesting that transduction was generalized. Transduction frequencies ranged from approximately 2 x 10(-7) to 1 x 10(-8) transductants/PFU depending upon the marker being transduced. UV irradiation of transducing lysates markedly improved transduction frequency and reduced the number of transductants that were lysogenized during the transduction process. Ba1 may prove to be a useful genetic tool for studying B. avium virulence factors.     

The interactive effect of ultraviolet irradiation and 5-bromouracil at the rib-1 locus in Neurospora crassa.

5-Bromouracil (5-BU) was shown to enhance reverse mutation rates when added to conidia of a rib-1 strain of Neurospora crassa previously irradiated with ultraviolet light. A comparison is presented between various incubation intervals in the presence and absence of 5-BU while varying the UV dose. An increase in mutation frequency ranging from 0.028 - 2.05 X 10(2) times greater than the spontaneous frequency, UV frequency alone or 5-BU frequency alone was observed when UV irradiation and 5-BU were combined.     

Transduction of Lactose Metabolism in Streptococcus lactis C2

Ultraviolet (UV)-induced phage lysates, from lactose-positive (lac(+)) Streptococcus lactis C2, transduced lactose fermenting ability to lac(-) recipient cells of this organism. Although the phage titer could not be determined due to the absence of an appropriate indicator strain, the number of transductants was proportional to the amount of phage lysate added. Treatment of the lysate with deoxyribonuclease had no effect on this conversion, indicating the observed genetic change was not mediated by free deoxyribonucleic acid. When the lac(+) transductants were isolated and exposed to UV irradiation, lysates with higher transducing ability were obtained. The transducing ability of this lysate was about 100-fold higher than that observed in the original lysates. The lac(+) transductants were unstable since lac(-) segregants occurred at high frequency. The phage lysate from S. lactis C2 also transduced maltose and mannose metabolism to the respective negative recipient cells. The results demonstrate the transduction of carbohydrate markers by a streptococcal phage and establish a genetic transfer system in group N streptococci.     

Inducible reactivation of UV-irradiated cholera phage e5 in Vibrio cholerae MAK757

Summary The survival of UV-irradiated cholera phage e5 was found to increase when the host cells, Vibrio cholerae MAK757, were exposed to a low dose of UV irradiation before phage infection (Weigle reactivation), indicating the existence of a UV-inducible DNA repair pathway (SOS repair) in V. cholerae MAK757. The induction signal generated by UV irradiation was transient in nature and lasted about 20–30 min at 37°C. Maximal weigle reactivation of the phage was obtained when the host cells were irradiated with a UV dose of 16 J/m². V. cholerae MAK757 was also found to possess efficient photoreactivation and host cell reactivation of UV-damaged DNA in phage e5.     

Recombination-deficient mutants of Bacillus subtilis.

Two mutant strains of Bacillus subtilis Marburg, NIG43 and NIG45, were isolated. They showed high sensitivities to gamma rays, ultraviolet light (UV), and chemicals. Deficiencies in genetic recombination of these two mutants were shown by the experiments on their capacity in transformation. SPO2 transfection, and PBS1 phage transduction, as well as on their radiation and drug sensitivities and their Hcr+ capacity for UV-exposed phage M2. Some of these characteristics were compared with those of the known strains possessing the recA1 or recB2 alleles. Mapping studies revealed that the mutation rec-43 of strain NIG43 lies in the region of chromosome replication origin. The order was purA dna-8132 rec-43. Another mutation, rec-45, of strain NIG45 was found to be tightly linked to recA1. The mutation rec-43 reduced mainly the frequency of PBS1 transduction. On the other hand, the mutation rec-45 reduced the frequency of recombination involved both in transformation and PBS1 transduction. The mutation rec-43 of strain NIG43 is conditional, but rec-45 of strain NIG45 is not. The UV impairment in cellular survival of strain NIG43 was gradually reverted at higher salt or sucrose concentrations, suggesting cellular possession of a mutated gene produce whose function is conditional. In contrast to several other recombination-deficient strains, SPO2 lysogens of strain NIG43 and NIG45 were not inducible, indicating involvement of rec-43+ or rec-45+ gene product in the development of SPO2 prophage to a vegetative form. The UV-induced deoxyribonucleic acid degradation in vegetative cells was higher in rec-43 and rec-45 strains.     

Stability and photochemical properties of vanadate-trapped nucleotide complexes of gizzard myosin in the 6S and 10S conformations: identification of an active-site serine.

The properties of divalent metal.ADP.vanadate (V(i)) complexes of the 6S extended and 10S folded conformations of gizzard myosin before and after UV irradiation have been studied. The half-lives of both 6S and 10S myosin.MgADP.V(i) complexes in the dark at 0 degrees C are on the order of 2 weeks. Brief irradiation with UV light, however, photomodified the enzyme as suggested by changes in the NH(4+)-, K(+)-, and Ca(2+)-ATPase activities, and destabilized the complexes. The 6S complex, when irradiated, released ADP and V(i) rapidly (t1/2 less than or equal to 1 min) as has been observed in comparable experiments with skeletal myosin subfragment 1 (S1) [Grammer et al. (1988) Biochemistry 27, 8408-8415]. The irradiated 10S complex released approximately 20% of the ADP and V(i) rapidly (t1/2 less than or equal to 1 min), but the remainder stayed trapped, possibly as the vanadyl (VO2+).ADP complex, for much longer times (t1/2 approximately 8 h). The site of photomodification was sought by reducing both photomodified 6S and 10S myosin with NaB3H4. Amino acid composition analyses identified [3H]serine as the only labeled residue(s), suggesting that the hydroxymethyl group of serine had been oxidized to an aldehyde as shown previously for photomodified skeletal myosin S1 [Cremo et al. (1989) J. Biol. Chem. 264, 6608-6611]. The 29-kDa NH2-terminal tryptic peptide from the heavy chain was found to contain essentially all of the [3H]serine. Preparations of 6S and 10S [3H]myosin were digested exhaustively with trypsin. An identical [3H]peptide was purified from each preparation and its sequence determined to be Glu169-Asp-Gln-Ser-Ile-Leu-(Cys)-Thr-Gly-[3H]Ser-Gly-Ala-Gly-Ly s183.(ABSTRACT TRUNCATED AT 250 WORDS)