Bacillus pumilus plasmid pPL10: properties and insertion into Bacillus subtilis 168 by transformation.

Bacillus pumilus ATCC 12140 harbors 10 or more copies per chromosome of each two small plasmids. Variants of this strain were isolated which were sensitive to a killing activity produced by the plasmid-containing parent. Each of 24 such sensitive (S) variants tested lacked detectable levels of supercoiled deoxyribonucleic acid. Transduction of S variants to the Kill+ phenotype was performed using phage PBP1 propagated on a mutant of ATCC 12140, designated strain L10, that remained Kill+ but retained only a single plasmid species (plasmid pPL10; molecular weight, approximately 4.4 X 10(6), approximately 20 copies per chromosome; RHO = 1.698). Resulting Kill+ transductants of S variants contained a single plasmid species having a size and copy number comparable to that of pPL10. Transfer of pPL10 from strain L10 TO B. pumilus strain NRS 576 was accomplished by transduction with selection for the Kill+ phenotype. Strain NRS 576 naturally harbors about two copies per chromosome of a 28-million-dalton plasmid, pPL576. In Kill + transductants of NRS 576, plasmids pPL10 and pPL576 stably coexisted at a ratio of about 11 molecules of pPL10 to 1 molecule of pPL576. Therefore, pPL576 and pPL10 are compatible plasmids. B. subtilis 168 is naturally resistant to pPl10- determined killing activity. Plasmid pPl10 was therefore inserted into B-subtilis 168 by transformation, using an indirect selection procedure and a spoB mutant as recipient. The plasmid is stably maintained at an estimated 10 copies per chromosome in the spore- recipient and in spore+ transformants. pPL10 is sensitive to cleavage by the endonucleases Hind III and EcoR1.     

Lambda phagemids and their transducing properties

Two recombinant lambda DNAs, lambda gt::pMB9 and lambda NM::pBR322, containing, respectively, the pMB9 and pBR322 replicon were constructed and characterized. Both constructs (phagemid DNAs) transfect Escherichia coli cells, producing mature infectious phage progenies. Alternatively, drug-resistant colonies of transductants can be selected upon infection with these phages (phagemid particles) that maintain phagemid DNA in the cell in the form of covalently closed circular plasmids. The efficiency of transduction for nonlysogenic E. coli strains with lambda gt::pMB9 phage producing lambda repressor cIts ranges from 10(-7) to 10(-2) transductant colonies per input phage, depending on the temperature and strain used, while lambda NM::pBR322 phage carrying imm21 transduces with a frequency of up to 1. This means that each lambda NM::pBR322 phagemid particle is capable of establishing itself in the cell as a nonlethal plasmid, permitting formation of a resistant bacterial colony. The maximal level of transduction with lambda gt::pMB9 was obtained when E. coli cells lysogenic for lambda were used. Thus, we believe that the efficiency of transduction is determined by the turn-on of the phage repressor in the transductant. In addition, we have found that all lambda gt::pMB9-containing transductants under certain conditions harbor precisely excised pMB9; excision of pBR322 from lambda NM::pBR322 has not been observed.     

High-Frequency Plasmid Transduction by Lactobacillus gasseri Bacteriophage phiadh

The temperate bacteriophage phiadh mediates plasmid DNA transduction in Lactobacillus gasseri ADH at frequencies in the range of 10 to 10 transductants per PFU. BglII-generated DNA fragments from phage phiadh were cloned into the BclI site of the transducible plasmid vector pGK12 (4.4 kb). Phage phiadh 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 10- to 10-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 phiadh. DNA hybridization analysis of pTRK170-transducing phage particles revealed that pTRK170 had integrated into the phiadh 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::phiadh molecule. In addition to strain ADH, pTRK170 could be transduced via phiadh to eight different L. gasseri strains, including the neotype strain, F. Gasser 63 AM (ATCC 33323).     

Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome.

An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.     

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.     

Drug Resistance of Staphylococci VII. Genetic Determinants Responsible for the Resistance to Tetracycline, Streptomycin, Sulfanilamide, and Penicillin

A genetic analysis of resistance to tetracycline (TC), streptomycin (SM), sulfanilamide (SA), and penicillin G was carried out through transduction with phage lysates obtained from a multiply resistant strain of Staphylococcus aureus by ultraviolet irradiation. All transductants acquired resistance to both TC and SA, even when singly selected for either SA or TC resistance. The locus responsible for TC resistance could not be separated genetically from that for SA resistance. On the other hand, in transduction of SM resistance, about 30% of the transductants jointly acquired resistance to both TC and SA. These observations suggest that the loci governing resistance to TC, SA, and SM exist close together on a single genetic unit, this probably being the chromosome.     

Specialized transduction of kanamycin resistance in a Providence strain.

Properties of a transducing system with a phage able to transduce a kanamycin-resistance marker of the T compatibility group plasmid R394 at a frequency of 2 times 10(-2)/plaque-forming unit adsorbed are described. The phage was detected in Providence strain P29 transduced to kanamycin resistance by Providence phage PL25 grown on this strain harbouring the R factor. Four P29 transductants, specially selected at the lowest multiplicities of infection of the high frequency transducing (HFT) phage, were defective lysogens. They plated PL25 with an efficiency of I and only one liberated low-titre phage spontaneously or on u.v. induction. The defect in maturation function could be corrected by introduction of a wild PL25 prophage. The transducing phage was serologically frequency was increased by the simultaneous presence of homologous non-transducing phage. Transductants did not transfer the kanamycin-resistance marker by conjugation, and produced kanamycin-sensitive segregants at a moderate rate. These segregants could be transduced to kanamycin resistance by the HFT phage. Irradiation of HFT lysates by u.v. produced an exponential fall in transduction frequency. It was concluded that the defective phage transduced by lysogenization. Kanamycin-resistant transductants could themselves be transduced by streptomycin resistance by PL25 reared on a streptomycin-resistant mutant. Lysogenic transductants produced by the HFT phage did not always liberate HFT phage on u.v. induction. Possible explantations are considered.     

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.     

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.     

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.     

Frequency of F116-mediated transduction of Pseudomonas aeruginosa in a freshwater environment.

Transduction of Pseudomonas aeruginosa streptomycin resistance by a generalized transducing phage, F116, was shown to occur during a 10-day incubation in a flow-through environmental test chamber suspended in a freshwater reservoir. Mean F116 transduction frequencies ranged from 1.4 X 10(-5) to 8.3 X 10(-2) transductants per recipient during the in situ incubation. These transduction frequencies were comparable to transduction frequencies determined in preliminary laboratory transduction experiments. The results demonstrate the potential for naturally occurring transduction in aquatic environments and concurrent environmental and ecological ramifications.     

STAPHYLOCOCCAL TRANSDUCING PARTICLE.

Dowell, C. E. (The University of Texas, Dallas) and E. D. Rosenblum. Staphylococcal transducing particle. J. Bacteriol. 84:1076-1079. 1962.-When novobiocin-resistant transductants were isolated under conditions that permitted superinfection, almost all the clones were lysogenic for the transducing phage. If superinfection was prevented, then the transductants isolated were nonlysogenic, suggesting the defective nature of the transducing particle. It was noted that the transducing and plaque-forming particles showed no appreciable difference in buoyant density. No difference was found in transduction rates when either sensitive or lysogenic cells were used as recipients. Transduction rates as high as one transductant per 7 x 10(4) phage particles were obtained for novobiocin resistance.     

吸水链霉菌Streptomyces hygroscopicus 17997中噬菌体基因转移系统的建立及其应用

由吸水链霉菌Streptomyces hygroscopicus 17997产生的格尔德霉素geldanamycin(GA)属安莎类抗生素,具有良好的抗肿瘤和抗病毒活性。本文应用链霉菌温和噬菌体ΦC31衍生的KC515载体,在吸水链霉菌S．hygroscopicus 17997中建立并优化了S．hygroscopicus 17997的基因转染体系。利用所建立的基因转染体系,以基因阻断技术从S．hygroscopicus 17997基因文库含有多组PKS基因柯斯质粒中,鉴定了与GA PKS生物合成相关基因的柯斯质粒,该工作为GA生物合成基因簇的克隆奠定了基础。     

Frequency of F116-mediated transduction of Pseudomonas aeruginosa in a freshwater environment.

Transduction of Pseudomonas aeruginosa streptomycin resistance by a generalized transducing phage, F116, was shown to occur during a 10-day incubation in a flow-through environmental test chamber suspended in a freshwater reservoir. Mean F116 transduction frequencies ranged from 1.4 X 10(-5) to 8.3 X 10(-2) transductants per recipient during the in situ incubation. These transduction frequencies were comparable to transduction frequencies determined in preliminary laboratory transduction experiments. The results demonstrate the potential for naturally occurring transduction in aquatic environments and concurrent environmental and ecological ramifications.     

A generalised transducing bacteriophage for Rhodococcus erythropolis

Summary Q4, a bacteriophage isolated from soil, mediated the transduction of a number of unlinked markers in Rhodococcus erythropolis. Highest numbers of transductants were obtained at multiplicities of infection of over 100, transductants only being obtained because of the temperate nature of the phage. Under optimal conditions, transduction to prototrophy of auxotrophic markers was over 50 times the spontaneous reversion rate and transduction of some antibitic resistance markers was over 10 times the spontaneous mutation rate. Segregation of unselected, but linked, markers was observed and the phage was used to order loci in a three factor cross. The virus required magnesium ions. Highest phage titres and greatest transduction frequency were obtained with stationary phase cultures.     

Transduction by bacteriophage P22 in nonsmooth mutants of Salmonella typhimurium

The general transducing phage P22 attacks only smooth (S) Salmonella with O antigen 12, determined by the oligosaccharide repeating unit constituting the distal part of the somatic lipopolysaccharide (LPS) side chain; non-S mutants, whose LPS contain few or no O repeating units, appear to be resistant. Auxotrophic non-S mutants of Salmonella typhimurium LT2 were tested as transductional recipients. Some transductants (0.5 to 5% as many as from S recipients) were obtained from most semirough recipients, either of class D (presumed leaky rouA mutants) or of a class due to mutation near his (presumed leaky rouB mutants), and from recipients lacking uridine diphosphogalactose epimerase or phosphomannose isomerase. Transductants were not obtained from several rouA, rouB, "heptose-negative," and glucose-1-transferase mutants, nor from most semirough class C mutants, whose LPS side chains each bear a single O oligosaccharide unit. Most transductants evoked from non-S recipients by temperate (c(+)) phage P22 were nonlysogenic, and virulent P22.c2 phage was about as effective as P22.c(+) in transduction to non-S recipients; probably all P22 transducing particles neither lysogenize nor kill. The extended-host-range mutant P22h gave qualitatively similar results,but evoked 5- to 30-fold more transductants from some non-S recipients than did P22. Probably, the LPS of non-S mutants susceptible to transduction contains a few O-specific oligosaccharide units, conferring a slight ability to adsorb P22 and a greater ability to adsorb P22h.     

Mechanism of pBR322 transduction mediated by cytosine-substituting T4 bacteriophage

Summary A cytosine-substitution type mutant of bacteriophage T4 (T4dC phage) has been shown to mediate the transfer of plasmid pBR322. The transduction frequency was around 10^-2 per singly infected cell at low multiplicity of infection. The transductants contained either a monomer or multimers of pBR322. The transducing capacity of T4dC phage was resistant to methylmethanesulfonate treatment. The results of Southern blotting experiments have indicated that the pBR322 DNA exists as head-to-tail concatemers in the transducing particles. The mechanism of transfer of pBR322 mediated by T4dC phages is discussed     

Phage A25-mediated transfer induction of a prophage in Streptococcus pyogenes

Summary The group A streptococcal strain 56188 used as standard donor in transduction with the virulent phage A25 is lysogenic for a phage called P56188. By using specific antiphage sera it is shown that A25 lysates obtained from 56188 contain a fraction of about 10^-4 phenotypically A25 but genotypically P56188 particles. A25-mediated transduction of prophage P56188 is measured by scoring plaques produced by transfer induction on 5004, a lysogenic strain unable to support the growth of A25. Data are obtained suggesting that A25 can also transduce a prophage carried by strain T25₃.Prophage P5004 present in 5004 is found to interfere with the propagation of A25 but does not seem to exert its action by directing extensive degradation of A25 DNA. Lysogenization of SM27 with P5004 leads to dramatically decreased burst sizes of A25, associated with the loss of its ability to plaque on this strain. Furthermore, P5004 lysogens of SM27 yield fewer streptomycin resistant transductants than their parent but gain the ability to serve as donors in A25-mediated transduction. A comparison of the burst size and the yield of transducing particles of A25 on various lysogenic and nonlysogenic hosts suggests that interfering with A25 growth is a widespread property of streptococcal prophages, which might favour processes leading to the formation of transducing A25 particles.     

Transduction of penicillinase production and other antibiotic-resistance markers in Staphylococcus epidermidis.

Transduction of resistance from a multiply antibiotic-resistant strain of Staphylococcus epidermidis sub-group II was studied using the typing phage 108. The effect of increasing doses of ultraviolet radiation on the transducing phage was used to indicate the chromosomal or plasmid nature of the genes. Tetracycline and chloramphenicol resistance behaved as plasmid genes and streptomycin resistance as a chromosomal marker. It was also possible to transduce penicillin resistance (Pc) due to penicillinase production (bla+) using a low level of benzylpenicillin (0.03 microgram ml-1) for recovery. Approximately 10(-5) transductant colonies per phage input were obtained and ultraviolet kinetics indicated that Pc was plasmid carried. Pc transductants fell into two categories. In one group PC was stable as in the donor strain and transductants had the same phage sensitivity as the recipient. In the other, Pc was unstable at 37 degrees C and the instability was enhanced by growth at approximately 43.5 degrees C; these transductants also gained genes for restriction and modification of certain phages. Transductants that subsequently lost bla+ also lost the restriction and modification characters.     

Construction of variants of Yersinia pestis EV76 (RIEG line) vaccine strain differing in antibiotic resistance spectra with stage-by-stage transduction of R-transposons]

Mono- and polyresistant variants of the plague vaccinal strain Yersinia pestis EV76 (RIEG line) were constructed with stage-by-stage transduction of transposons Tn5, T, Tn10 and Tn9 located in the phage vectors. Methods for stage-by-stage selection of the transductants that preserved intact determinants of surviving, antiphagocytic activity and immunogenic properties were designed for their further testing as model vaccinal strains in combined specific and emergent medicamentous prophylaxis of plague.