Interspecies transduction of plasmids among Bacillus anthracis, B. cereus, and B. thuringiensis

Bacteriophage CP-51, a generalized transducing phage for Bacillus anthracis, B. cereus, and B. thuringiensis, mediates transduction of plasmid DNA. B. cereus GP7 harbors the 2.8-megadalton multicopy tetracycline resistance plasmid, pBC16. B. thuringiensis 4D11A carries pC194, the 1.8-megadalton multicopy chloramphenicol resistance plasmid. When phage CP-51 was propagated on these strains, it transferred the plasmid-encoded antibiotic resistances to the nonvirulent Weybridge (Sterne) strain of B. anthracis, to B. cereus 569, and to strains of several B. thuringiensis subspecies. The frequency of transfer was as high as 10(-5) transductants per PFU. Tetracycline-resistant and chloramphenicol-resistant transductants contained newly acquired plasmid DNA having the same molecular weight as that contained in the donor strain. Antibiotic-resistant transductants derived from any of the three species were effective donors of plasmids to recipients from all three species.     

Plasmid transduction by Bacillus anthracis bacteriophage CP54

Possibility of plasmid transduction in Bacillus anthracis vaccine strains Sterne and STI-1 by bacteriophage CP54ant having an increased ability of adsorbtion and a shortened period of latent development in Bacillus anthracis cells has been isolated. The main parameters of plasmid transduction by the bacteriophage have been established for the plasmid pTG141 (TcR). They include the effect of multiplicity of infection, the level of UV-inactivation of bacteriophage, the presence of antiphage serum in the incubation medium. Plasmid transduction by the mutant phage CP54ant was found to be more efficient as compared with the one by the parent phage. The isolated transductants served as donors of the transduced plasmid for Bacillus anthracis and Bacillus thuringiensis strains.     

Relationship Between Competence for Transfection and for Transformation

Deoxyribonucleic acid (DNA) extracted from phage SPP1 is highly infectious on Bacillus subtilis competent cells; the efficiency of infection is 5 x 10(3) to 6 x 10(3) phage equivalents per plaque-forming unit. This DNA was used to study the relationship between competence for transfection and for transformation. The experiments were concerned with the frequency of infection and transformation in mutants exhibiting different levels of competence, the effect of periodate on competence for infection and for transformation, the competition between phage and bacterial DNA, the transformation of cells preinfected with phage DNA, and the infection of cells pretreated with bacterial DNA. The data show that B. subtilis cells competent for transformation are also competent for transfection and vice versa; transfection with phage DNA represents, therefore, a simple way to measure the total number of competent cells in a culture. The fraction of competent cells, determined by SPP1 DNA infection, varied from 10(-2) to 7 x 10(-2).     

Transduction of concatemeric plasmids containing the cos site of Lactococcus lactis bacteriophage sk1

Lactococcus lactis bacteriophage sk1 can transduce plasmids containing the phage cos site and surrounding DNA sequences at frequencies as high as 2x10(-3) transductants per PFU. Deletion analysis demonstrated that the presence of phage DNA spanning cos and putative R sites were the most important for efficient plasmid transduction. Inserts of 440 bp containing cos and the R sites were sufficient to induce transduction frequencies of 10(-4) transductants per PFU. The role of the R1 site was investigated by altering 14 of the 19 bases in the site. This resulted in a two-fold decrease in transduction frequency compared to a 26-fold decrease in transduction following deletion of the entire site. It was demonstrated that transducing plasmids were packaged as linear trimeric concatemers commencing at the cos site.     

Transduction of plasmid antibiotic resistance determinants with pseudo-T-even bacteriophages

Transduction of antibiotic resistance determinants of the plasmid pBR322 with pseudoT-even bacteriophages RB42, RB43, and RB49 was studied. It is established that antibiotic resistance determinants of plasmid pBR322 from Escherichia coli recA(+)- and recA(-)-donor strains do not differ significantly in respect to the efficiency of transduction. Amber mutants RB43-21, RB43-33, and a double amber mutant RB43am21am33 were obtained. These mutants facilitated transduction experiments in some cases. Transduction of antibiotic resistance markers of the vector plasmid pBR325 and recombinant plasmid pVT123, containing a DNA fragment with hoc segE uvsW genes of phage T4, was studied. The frequency of appearance of transductants resistant to pseudoT-even bacteriophages used in transduction was determined, and the sensitivity of resistant transductants to 32 RB bacteriophages and also to phages lambda, T2, T4, T5, T6, T7, and BF23 was estimated. The efficiency of plating pseudoT-even bacteriophages RB42 and RB43 on strain E. coli 802 himA hip carrying mutations in genes that encode subunits of the Integration Host Factor (IHF) was shown to be higher than on isogenic strain E. coli 802. The growth of pseudoT-even bacteriophages limited in vivo by modification-restriction systems of chromosomal (EcoKI, EcoBI), phage (EcoP1I), and plasmid (EcoRI, EcoR124I, and EcoR124II) localization was analyzed. It was shown that these phages were only slightly restricted by the type I modification-restriction systems EcoBI, EcoR124I, and EcoR124II. Phage RB42 was restricted by systems EcoKI, EcoP1I, and EcoRI; phage RB43, by systems EcoKI and EcoRI; and phage RB49, by the EcoRI modification-restriction system.     

Plasmid R68.45 and S-a transduction by the temperate bacteriophage 59 of Erwinia carotovora 268

Temperature bacteriophage 59 of Erwinia carotovera 268 had transduced extrachromosomal DNA: plasmids of R68.45 and S-a. Before plasmid transduction experiments the suitable donor strains of indicator culture Erwinia horticola 450 harbouring R68.45 and S-a were created. The frequency of plasmid R68.45 transfer from Pseudomonas putida to E. horticola 450-8 by conjugation was equal to 5 x 10(-8) per a donor cell and in the case of S-a--from E. coli C600 for the same recipient cells--was 2 x 10(-6). Bacteriophage 59 has transduced only separate markers of plasmid R68.45, since plasmid S-a is probably transduced by the phage as an intact unit.     

Production of Bacteriophage by Temperature-Sensitive Sporulation Mutants of Bacillus cereus T

Five temperature-sensitive sporulation mutants of Bacillus cereus T have been isolated. These mutants are blocked at stage 0 of sporulation at the restrictive temperature (37 C) but are able to sporulate at nearly normal frequencies at the permissive temperature (26 C). A bacteriophage that forms a stable lysogen in the parent strain is induced at increased frequencies in the mutants. This induction is accompanied, in some of the mutants, by a reduction in immunity to the phage. Revertants, selected for their ability to sporulate normally at both temperatures, lose their ability to produce high titers of the phage. In addition to this lytic phage, an apparently defective phage has been found in lysates of the mutants. Strains cured of the plaque-forming phage still carry the defective phage. Comparisons of physical and biological properties of the plaque-forming phage with those of the two Bacillus cereus phages most similar to it have shown that this phage is not identical to either of them. The maximal titer of phage produced in cultures of the parent strain is about 10(3) plaque-forming units (PFU) per ml at both temperatures. The maximal titers of phage produced by the mutant are 4 x 10(9) PFU/ml at 37 C and 7 x 10(8) PFU/ml at 26 C. Both mutant and parent strains release over 90% of the phage they produce after the onset of stationary phase.     

Role of lysogenizing phages in the spread of drug-resistance plasmids in a staphylococcal population

The frequency of the transduction of plasmids rms5, rms7, pT127, pC194, pS194 and pUB101 by phages belonging to serological group B (80, 52, 52A, 53, 85, phi 11, S2) in two systems was compared. In system 1 phages for transduction were obtained from plasmid-containing lysogenic donors in the process of induction with mitomycin C; in system 2 phages for transduction were obtained by their multiplication in plasmid-containing nonlysogenic donors. In system 1 the transduction of plasmids rms5, rms7, pT127, pS194 by phage 52A was found to occur with a greater (by 3-5 orders) frequency than in system 2 (the frequency of transduction was 10(-2) to 10(-4), and 10(-6) to 10(-8) respectively). A similar situation was observed with plasmids rms5 and rms7 and phage 52; plasmid pT127 and phage 53; but not observed with plasmids rms5 and rms7 and phages 80, phi 11 and S2; plasmids pC194 and pS194 and phage 53; plasmid pUB101 and phages 52A, 80 and phi 11; plasmids pC194, pS194 and pT127 and phage 85.     

A generalized transducing phage of Pseudomonas cepacia

A generalized transducing phage, named CP75, was derived from a lysogenic strain of Pseudomonas cepacia. The frequency of transduction per phage particle ranged from 1.0 X 10(-6) to 2.0 X 10(-6) for a given marker. About half of the 105 P. cepacia strains tested were sensitive to the phage. The molecular size of the CP75 genome was approximately 52 kb.     

Transfection of Escherichia coli Spheroplasts I. General Facilitation of Double-Stranded Deoxyribonucleic Acid Infectivity by Protamine Sulfate

The addition of 25 mug of protamine sulfate per ml to lysozyme-ethylenediamine-tetraacetic acid spheroplasts of Escherichia coli stimulates transfection not only for T1 phage deoxyribonucleic acid (DNA; Hotz and Mauser, 1969) but also for the following phage DNA species: lambda, 10,000-fold to an efficiency of 10(-3) infective centers per DNA molecule; phiX174 replicative form, 300-fold to an efficiency of 5 x 10(-2); fd replicative form, 300-fold to 10(-6); T7, 300-fold to 3 x 10(-7). Three native phage DNA species were not infective at all in the absence of protamine sulfate but were infective in the presence of protamine sulfate with the following efficiencies: T4, 10(-5); T5, 3 x 10(-6); and P22, 3 x 10(-9). The effect of protamine sulfate is specific for double-stranded DNA. The application of infectivity assays to the study of phage DNA replication, recombination, prophage integration, prophage excision, and interspecies transfection are discussed.     

Transduction in Streptomyces hygroscopicus mediated by the temperate bacteriophage SH10

Summary The temperate actinophage SH10 mediates generalized transduction in Streptomyces hygroscopicus at low frequency. The efficiency of transduction depends on the average phage input, age of outgrowing spores of the recipient and on the selective marker. The highest EOT was found for the auxotrophic mutants 21 (phe^-) and 5(try^-)(4.1x10^-6 and 2.7x10^-6, respectively). Transduction of the thermosensitive mutant NG14-216 ts 35 was two orders of magnitude lower (2.5x10^-8). The transductant colonies segregated into stable and unstable clones. Stable transductants were never found to be lysogenic for phage SH10.     

Concurrent changes in transducing efficiency and content of transforming deoxyribonucleic acid in Bacillus subtilis bacteriophage SP-10

Taylor, Martha J. (Fort Detrick, Frederick, Md.), and Curtis B. Thorne. Concurrent changes in transducing efficiency and content of transforming deoxyribonucleic acid in Bacillus subtilis bacteriophage SP-10. J. Bacteriol. 91:81-88. 1966.-Spores of Bacillus subtilis W-23-S(r) infected with transducing phage SP-10 served as convenient inocula for broth cultures from which transducing phage was harvested. Methods are described for producing highly infected spores. The inoculum level of infected spores in nutrient broth-yeast extract-glucose medium affected the transducing efficiency of SP-10 in lysates of these cultures. Phage in lysates of cultures inoculated with about 10(5) or fewer spores per milliliter transduced 20- to 350-fold more efficiently than did phage in lysates from cultures inoculated with 10(6) to 10(7) spores per milliliter. Transduction frequencies in the order of 10(-5) per plaque-forming unit were obtained routinely, and some infected-spore preparations yielded phage that gave frequencies as high as 10(-4). The combination of inoculum level and incubation time required to produce the best transducing phage had to be determined empirically for each batch of infected spores. Several possible explanations for the difference between lysates having high (HTE) and those having low (LTE) transducing efficiency were ruled out by special experiments. The hypothesis is presented that some cultural condition resulting from a relatively low inoculum of phage-infected spores favors the incorporation by phage particles of bacterial deoxyribonucleic acid (DNA) in the manner required for the production of transducing phage. Support for this hypothesis is a demonstration, through transformation experiments with DNA extracted from HTE and LTE phage particles, that populations of HTE phage particles yielded significantly more (7 to 27 times) transforming activity per microgram of DNA than did populations of LTE phage.     

Protoplast transformation of Bacillus stearothermophilus NUB36 by plasmid DNA

An efficient protoplast transformation system was established for Bacillus stearothermophilus NUB3621 using thermophilic plasmid pTHT15 Tcr (4.5 kb) and mesophilic plasmid pLW05 Cmr (3 kb), a spontaneous deletion derivative of pPL401 Cmr Kmr. The efficiency of transformation of NUB3621 with pLW05 and pTHT15 was 2 x 10(7) to 4 x 10(8) transformants per micrograms DNA. The transformation frequency (transformants per regenerant) was 0.5 to 1.0. Chloramphenicol-resistant and tetracycline-resistant transformants were obtained when competent cells of Bacillus subtilis were transformed with pLW05 [2.5 x 10(5) transformants (microgram DNA)-1] and pTHT15 [1.8 x 10(5) transformants (micrograms DNA)-1], respectively. Thus, these plasmids are shuttle vectors for mesophilic and thermophilic bacilli. Plasmid pLW05 Cmr was not stably maintained in cultures growing at temperatures between 50 and 65 degrees C but the thermostable chloramphenicol acetyltransferase was active in vivo at temperatures up to 70 degrees C. In contrast, thermophilic plasmid pTHT15 Tcr was stable in cultures growing at temperatures up to 60 degrees C but the tetracycline resistance protein was relatively thermolabile at higher temperatures. The estimated copy number of pLW05 in cells of NUB3621 growing at 50, 60, and 65 degrees C was 69, 18, and 1 per chromosome equivalent, respectively. The estimated copy number of pTHT15 in cells of NUB3621 growing at 50 or 60 degrees C was about 41 to 45 per chromosome equivalent and 12 in cells growing at 65 degrees C.     

High-frequency transduction of pBR322 by cytosine-substituted T4 bacteriophage: evidence for encapsulation and transfer of head-to-tail plasmid concatemers

Transduction of plasmid pBR322 by cytosine-substituted T4 phages has been studied. Three T4 phage mutants which substitute cytosine for all of hydroxymethylcytosine residues in the DNA, were shown to transduce pBR322 at frequencies of 2 × 10^?2 to 4 × 10^?3 transductants per singly infected cell. Also, three T4 phage strains which partially substitute cytosine for hydroxymethylcytosine, transduced pBR322 at frequencies of 2 × 10^?3 to 2 × 10^?4. The transduction frequencies of pBR322 we attained are at least 10-fold higher than those reported by G. G. Wilson, K. Young, and G. J. Edlin (1979, Nature (London)280, 80–82). We found that multiplicity of infection in preparation of the transducing phage is the most important factor affecting the frequency of pBR322 transduction. When a lysate made at a multiplicity of infection ranging from 0.5 to 0.05 was used as the donor phage, transduction frequency of pBR322 was 10- to 40-fold higher than that of high-m.o.i. lysate. The transduction frequency was not affected by either restriction systems or amber suppressors of the recipient cells. However, no pBR322-containing transductant was obtained when either recA or polA mutants were used as the recipients. DNA from T4dC phage containing pBR322-transducing particles was analyzed on agarose gel electrophoresis after cleavage with restriction endonucleases. It was suggested that the pBR322 DNA in the T4dC phage particles exists as head-to-tail concatemers.     

Transformation of the archaebacterium Halobacterium volcanii with genomic DNA.

We describe optimization of a transformation system for the halophilic archaebacterium Halobacterium volcanii. Transformation of spheroplasts in the presence of polyethylene glycol permits the uptake and expression of high-molecular-weight linear fragments of genomic DNA as well as plasmid or bacteriophage DNA. Transformations can be performed with either fresh or frozen cell preparations. Auxotrophic mutants were transformed to prototrophy with genomic DNA from wild-type cells with efficiencies of 5 x 10(4)/micrograms of DNA and frequencies of 8 x 10(-5) per regenerated spheroplast. The overall efficiency of transformation with genomic DNA implies that genetic recombination is an efficient process in H. volcanii.     

Characterization of a Generalized Transducing Phage of Poly-(gamma)-Glutamic Acid-Producing Bacillus subtilis and Its Application for Analysis of Tn917-LTV1 Insertional Mutants Defective in Poly-(gamma)-Glutamic Acid Production

A generalized transducing phage, (phi)BN100, was isolated from a Bacillus subtilis (natto) strain producing poly-(gamma)-glutamic acid ((gamma)PGA). Transduction frequencies for a given marker ranged from 3.8 x 10(sup-8) to 1.6 x 10(sup-6) per phage particle. The genome size of the phage was approximately 42 kb. (phi)BN100 was used successfully to identify bona fide Tn917-LTV1 transpositional mutants defective in (gamma)PGA production.     

Interaction between phage G13 and its oligosaccharide receptor studied by equilibrium dialysis

The reversible binding of phage G13, a phi X174-like single-strand DNA phage, to a 3H-labelled nonasaccharide from the lipopolysaccharide of its natural host Escherichia coli C was studied with equilibrium dialysis. The binding constant (Ka) was determined to 1.3 x 10(7) M-1 in Scatchard and Lineweaver-Burk plots. Approximately one saccharide bound per G13 phage particle which suggests that only one of the 12 spikes in each G13 virion was engaged in the phage/receptor saccharide interaction. Equilibrium dialysis inhibition experiments with saccharides from lipopolysaccharides of an isogenic series of Salmonella typhimurium mutants showed that hepta- and pentasaccharides from two G13-sensitive bacteria, i.e., with efficiencies of plating of 0.1-1.0 compared to E. coli C, were efficient inhibitors with Ka-values greater than or equal to 1.2 x 10(7) M-1. The octa- and hexasaccharides from two G13 resistant strains, with efficiency of plating less than or equal to x 10(-4), were either greater than 1000-fold or greater than 15-fold less efficient as inhibitors with Ka-values less than or equal to 8.8 x 10(5) M-1. The results show that phage G13 binds in a specific and reversible way to penta-, hepta-, and nonasaccharides from G13 sensitive bacteria with the specificity residing in the hexose and heptose region of the core lipopolysaccharide.     

Transduction of Pseudomonas aeruginosa with a mutant of bacteriophage E79.

A mutant of the virulent bacteriophage E79 was isolated which mediated generalized transduction in Pseudomonas aeruginosa. Variable recovery of transductants as a result of phage killing was avoided by the use of recipients carrying the IncP-2 plasmid R38, and transduction frequencies of 4 X 10(-6) to 1 X 10(-5) per plaque-forming unit were obtained. Linkage studies have indicated that the coinheritance frequencies are less than would be expected from the published molecular weight of E79 deoxyribonucleic acid (120 X 10(6). By using recipients carrying R38, low-frequency transduction by wild-type E79 and two other virulent phages, F8 and phi 16, was demonstrated.     

Transposition of the phage D3112 genome in Escherichia coli cells

It has been demonstrated that the genome of phage D3112 of Preudomonas aeruginosa can be transposed into Escherichia coli chromosome as a component of the hybrid plasmid RP4 TcrKms::D3112. Also, transposition of D3112 from E. coli (D3112) chromosome into RP4 plasmid occurs. The phage stimulates the chromosome mobilizing activity of RP4 plasmid, similar to other transposons. E. coli (RP4::D3112) cells were previously shown to form no colonies at 30 degrees C. Auxotrophic mutants and mutants incapable of utilizing different carbohydrates were found among E. coli clones survived after a long incubation at 30 degrees C (at frequencies approximately 10(-3) - 10(-4). These mutants inherited stably the capability to produce D3112 phage. E. coli auxotrophic mutants have arisen indeed as a consequence of phage integration into the E. coli chromosome, since prototrophic transductants derived from these mutants after their treatment with generalized transducing P1 phage have lost the ability to produce D3112 phage. Clones with mutations in Km or Tc genes of RP4 plasmid, occurring at high frequencies (about 3%) were found after introduction of RP4 into E. coli (D3112). These mutant RP4 plasmids carry insertions of D3112 genomes. Clones of E. coli which lost mutant plasmids still produce D3112 and retain their initial auxotrophic mutations.