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.     

Drug resistance of enteric bacteria. V. High frequency of transduction of R factors with bacteriophage epsilon

Kameda, Mitsuo (Gunma University, Maebashi, Japan), Kenji Harada, Mitsue Suzuki, and Susumu Mitsuhashi. Drug resistance of enteric bacteria. V. High frequency of transduction of R factors with bacteriophage epsilon. J. Bacteriol. 90:1174-1181. 1965.-In the transduction of R factors with phage epsilon(15), a lysate capable of transducing the markers for (TC) or (CM.SM.SA) resistance at high frequency was obtained. The transducing agent is a defective element called epsilon(15)dR(23) which lacks certain functions of phage epsilon(15). After lysogenization with normal epsilon(15) phage and ultraviolet (UV) induction, strains carrying the epsilon(15)dR(23) element produce lysates which have a high frequency of transduction (HFT) on group E(1)Salmonella. Lytic lysates prepared on phage epsilon(15) sensitive strain with the epsilon(15)dR(23) element have a low frequency of transduction (LFT). Lytic growth of phage epsilon(34) on an epsilon(15)dR(23) strain or UV induction of an epsilon(34) lysogenic strain containing epsilon(15)dR(23) results in LFT lysates on group E(2)Salmonella. On UV induction, group E(2)Salmonella (epsilon(15) lysogens) with the epsilon(15)dR(23) element give lysates which are HFT on group E(1)Salmonella but are LFT when tested on group E(2)Salmonella. In all instances, the production of drug-resistant transductants requires infection of the cell with only a single epsilon(15)dR(23) element. It appears that the resistance region of the R factor has replaced that portion of phage genome which is essential for vegetative replication and superinfection immunity. The epsilon(15)dR(23) element does not contain the genetic determinants of the R factor responsible for transmissibility, inhibition of F mating, and interference between two R factors.     

Isolation of Transducing Bacteriophages for the Histidine and Isoleucine-Valine Operons in Escherichia coli K-12

In vitro studies have been of great value in elucidating the mechanism of the regulation of several bacterial operons. To obtain a deoxyribonucleic acid preparation enriched for the histidine (his) and for the isoleucine-valine (ilv) operons, we have isolated bacteriophages carrying the his and the ilv regions of the Escherichia coli chromosome. Transposition of the his operon to a site close to the att80 region of the E. coli chromosome has been carried out selecting for integration of a temperature-sensitive F'his(+) in the tonB locus. This transposed strain has been lysogenized with phi80i(lambda). Upon induction of the lysogen, His(+) transductants have been isolated, which, on further induction give rise to HFT (high frequency of transduction) lysates. Preliminary characterization of the transducing phage is reported. The ilv operon, carried on an F' particle, has been fused to an episome carrying the att80 region. The fused episome has been lysogenized with phi80i lambdat68. Upon induction of the lysogen, Ilv(+) transductants have been isolated which on further induction give rise to HFT lysates.     

Factors that affect transduction in microorganisms]

Data from literature concerning general and specialized transduction in microorganisms are given in the paper. The process of exogenic DNA penetration to the cells of bacteria and participation of protein products of separate phage genes in this process are described. The so-called E-proteins in a set with DNA penetrate through a cell membrane. In phage P22 they are protein products of phage genes 7, 16, 20. In P22 mutants with an altered transducing frequencies (HFT and LFT) the due functions are also coded by the phage genes. It is shown that the process of DNA packing in phages P22, phi 80, lambda and others is genetically determined. The gene transfer frequency depends on UV radiation and the very nature of transducing phages itself. In virulent phages the UV radiation up to inactivation level 95-99% evokes a decrease of their "killer" ability, which is accompanied by an increase of survivability of the formed transductants and, as a result, by enhancement of the transduction transfer frequency. An important role of the transduction analysis for fine mapping of a genome of microorganisms and its significance for practice are shown. A mathematical analysis of the data on cotransduction of linkage markers is presented as such that may be used when determining the value of transduced fragment of a chromosome.     

Mini-D3112 bacteriophage transposable elements for genetic analysis of Pseudomonas aeruginosa.

Small bacteriophage D3112 transposable elements deleted for most of the phage-lytic functions while retaining the sites required for transposition and packaging were constructed to facilitate genetic studies in Pseudomonas aeruginosa. These mini-D derivatives were constructed with the terminal 1.85 kilobases (kb) of the phage left end and 1.4 kb of the phage right end and either the Tn5 kanamycin resistance or the pSC101 (pBR322) tetracycline resistance determinant. Thermally induced lysates of strains lysogenic for both a mini-D element and D3112 cts (temperature-sensitive repressor) transduced P. aeruginosa PAO recipients to drug resistance at frequencies of between 10(-4) and 10(-5)/PFU of the helper phage. As for the parent plaque-forming D3112 phage, the mini-D171 element could insert itself into many different sites in the chromosome but the frequency of insertion into particular genes varied widely. Among 1,000 insertions, none resulted in auxotrophy but 10 resulted in pigment production. Insertions were also selected in a cloning plasmid with a transduction scheme. At least eight different insertion sites were found to have been used among 10 individual insertions. Transductants harboring these mini-D elements were immune to infection by D3112, since they contained the D3112 repressor gene in the left 1.85-kb terminal fragment. Chromosomal genes were transduced in a generalized fashion 100 to 1,000 times more frequently by the mini-D-D3112 cts lysates than by the D3112 cts phage alone. Mini-D171-D3112 cts lysates also yielded some transductants that retained the drug resistance marker of the mini-D element and which were unstable for the chromosomal transduced marker. This is consistent with the miniduction properties of Mu whereby transduced genes are flanked by two mini-D elements in the same orientation.     

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 certain genes by an autonomously replicating Bacillus thuringiensis phage.

A derivative of Bacillus thuringiensis subsp. kurstaki HD1 (HD1-9) released transducing phage (TP21) from late exponential cultures. Three of seven markers tested were transduced into Bacillus cereus, but only two of these (cysC and trpB/F) were transduced at a frequency of more than 100 times the reversion rates. A limited transduction capacity was given further support in that few chromosomal markers were carried in the HD1-9 lysate, as demonstrated by Southern hybridization. Restriction fragments from the phage DNA and from total B. thuringiensis DNA hybridized to an insertion sequence (IS231-like) probe, which may provide a region of homology for transduction. All of the B. cereus transductants contained the phage as a 44-kb plasmid, and each could transduce both the cys and trp genes to other B. cereus auxotrophs, albeit at lower frequencies than those for the B. thuringiensis transducing phage. In some cases, especially for cys, the transduced gene was integrated into the chromosome of the recipient, whereas the trp gene in many cases appeared to be lost with curing of the 44-kb plasmid. In addition, some B. cereus transductants lost prototrophy but retained a 44-kb plasmid, consistent with the presence of TP21 helper phage. These phage may mediate the subsequent transduction from B. cereus phototrophs. TP21 replicates as a plasmid and, at least under the conditions studied, selectively transfers markers to B. cereus.     

Transduction of certain genes by an autonomously replicating Bacillus thuringiensis phage

A derivative of Bacillus thuringiensis subsp. kurstaki HD1 (HD1-9) released transducing phage (TP21) from late exponential cultures. Three of seven markers tested were transduced into Bacillus cereus, but only two of these (cysC and trpB/F) were transduced at a frequency of more than 100 times the reversion rates. A limited transduction capacity was given further support in that few chromosomal markers were carried in the HD1-9 lysate, as demonstrated by Southern hybridization. Restriction fragments from the phage DNA and from total B. thuringiensis DNA hybridized to an insertion sequence (IS231-like) probe, which may provide a region of homology for transduction. All of the B. cereus transductants contained the phage as a 44-kb plasmid, and each could transduce both the cys and trp genes to other B. cereus auxotrophs, albeit at lower frequencies than those for the B. thuringiensis transducing phage. In some cases, especially for cys, the transduced gene was integrated into the chromosome of the recipient, whereas the trp gene in many cases appeared to be lost with curing of the 44-kb plasmid. In addition, some B. cereus transductants lost prototrophy but retained a 44-kb plasmid, consistent with the presence of TP21 helper phage. These phage may mediate the subsequent transduction from B. cereus phototrophs. TP21 replicates as a plasmid and, at least under the conditions studied, selectively transfers markers to B. cereus.     

The Prophage of SPβc2DcitK1, a Defective Specialized Transducing Phage of BACILLUS SUBTILIS

The defective specialized transducing phage SP beta c2dcitK1 carries two known bacterial genes, kauA and citK, as well as SP beta hage markers including the heat-sensitive repressor allele, c2. Some phage genes (including essential ones) are missing. When SP beta c2dcitK1 transduces SP beta-sensitive cells of Bacillus subtilis, the defective prophage is inserted into sites in the homologous bacterial DNA of the attSP beta-kauA-citK region of the recipient chromosome. During the growth of these transductants, occasional excisions occur that result in the loss of the phage genes and of the heterogenotic state. These excisions increase greatly in frequency during growth at repressor-inactivating temperatures. The kinds of insertions and excisions seen suggest that a Campbell-type (CAMPBELL 1962) circular phage genome may occur transiently. If the transductants are superinfected by SP beta c2 or by the clear-plaque mutant SP beta c1, the resulting double lysogen can be heat induced to release high-frequency-of-transduction (HFT) lysates for kauA and citK.     

Construction and characterization of recombinant phage phi 105 d(Cmrmet) for cloning in Bacillus subtilis

A 1.6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage phi 105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted phi 105 d(Cmrmet), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.     

The variation in frequency with which markers are transduced by phage P1 is primarily a result of discrimination during recombination

Summary The efficiency of recovery of P1 transductants is marker dependent and normally varies over a 25-fold range. UV irradiation of either transducing lysates or recipient cells results in a selective stimulation of the transduction of markers which are normally transduced poorly. As a result the range in frequency of transduction is reduced to about 3-fold and resembles the gene frequency distribution expected in the donor cells. We conclude that P1 transducing lysates are likely to contain a random sample of donor DNA but that the recombination system of the recipient cell exhibits a preference for the DNA of some regions over that of others. Damage to DNA presumably overrides this specificity.     

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.     

Conditionally transposition-defective derivative of Mu d1(Amp Lac).

A Mu d1 derivative is described which is useful for genetic manipulation of Mu-lac fusion insertions. A double mutant of the specialized transducing phage Mu d1(Amp Lac c62ts) was isolated which is conditionally defective in transposition ability. The Mu d1 derivative, designated Mu d1-8(Tpn[Am] Amp Lac c62ts), carries mutations which virtually eliminate transposition in strains lacking an amber suppressor. In such strains, the Mu d1-8 prophage behaves like a standard transposon. It can be moved from one strain of Salmonella typhimurium to another by the general transducing phage P22 with almost 100% inheritance of the donor insertion mutation. When introduced into a recipient carrying supD, supE, or supF, 89 to 94% of the Ampr transductants were transpositions of the donor Mu d1-8, from the transduced fragment into new sites. The stability of Mu d1-8 in a wild-type, suppressor-free background was sufficient to permit use of the fusion to select constitutive mutations without prior isolation of deletions to stabilize the fusion. Fusion strains could be grown at elevated temperature without induction of the Mu d prophage. The transposition defect of Mu d1-8 was corrected by a plasmid carrying the Mu A and B genes.     

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).     

Isolation and characterization of transducing coliphage fd carrying a kanamycin resistance gene.

The DNA segment (Tn903) with a size of 3100 nucleotide pairs which carries a gene specifying kanamycin resistance derived from a chimeric plasmid pML21 (Hershfield et al., 1976) was transposed to various sites on the filamentous phage fd DNA. Wild type fd can be restored by excision of Tn903 from the resulting hybrid DNA molecule. The fd DNA carrying Tn903 when converted to the mature phage particle, was capable of transducing the kanamycin marker, and its replicative form DNA could be maintained in a bacterial cell like a plasmid.     

Behavior of coliphage lambda in hybrids between Escherichia coli and Salmonella

Salmonella typhosa hybrids able to adsorb lambda were obtained by mating S. typhosa recipients with Escherichia coli K-12 donors. After adsorption of wild-type lambda to these S. typhosa hybrids, no plaques or infective centers could be detected. E. coli K-12 gal(+) genes carried by the defective phage lambdadg were transduced to S. typhosa hybrids with HFT lysates derived from E. coli heterogenotes. The lysogenic state which resulted in the S. typhosa hybrids after gal(+) transduction differed from that of E. coli. Ability to produce lambda, initially present, was permanently segregated by transductants of the S. typhosa hybrid. S. typhosa lysogens did not lyse upon treatment for phage induction with mitomycin C, ultraviolet light, or heat in the case of thermoinducible lambda. A further difference in the behavior of lambda in Salmonella hybrids was the absence of zygotic induction of the prophage when transferred from E. coli K-12 donors to S. typhosa. A new lambda mutant class, capable of forming plaques on S. typhosa hybrids refractory to wild-type lambda, was isolated at low frequency by plating lambda on S. typhosa hybrid WR4254. Such mutants have been designated as lambdasx, and a mutant allele of lambdasx was located between the P and Q genes of the lambda chromosome. Plaques were formed also on the S. typhosa hybrid host with a series of lambda(i21) hybrid phages which contain the N gene of phage 21. The significance of these results in terms of Salmonella species as hosts for lambda is discussed.     

Specialized transduction of the ilvD-thyB-ilvA region mediated by Bacillus subtilis bacteriophage SP beta

Specialized transducing SP beta particles were found that carried the Bacillus subtilis genes lying to the left of the prophage attachment site. Three classes of transducing particles were differentiated, depending upon whether they carried ilvA only, thyB and ilvA, or ilvD, thyB, and ilvA. Lysates prepared by the induction of strains that carried both a transducing phage and a plaque-forming phage contained the two particles in a ratio of about 1:3,000. When the transducing particles were used to transduce a phage-sensitive auxotrophic strain to prototrophy, some of the transductants carried only the transducing phage genomes which, by themselves, were defective. One putative nondefective transducing phage (for ilvA only) is also described. SP beta can mediate specialized transduction even in the absence of the major (recE) bacterial recombination system.     

Specialized transduction with lambda plac5: dependence on recB.

Genetically disabled lambda plac5 transducing phage derivatives were used to study the recB dependence of recombination during specialized transduction. The frequency of transduction was normalized to colony-forming units, and the end product of recombination was monitored by scoring for addition and substitution transductants. When a chromosomal lac gene was the recipient DNA substrate molecule, both the normalized transduction frequency and the proportion of addition and substitution transductants showed essentially no recB dependence. There was a pronounced recB dependence for both normalized transduction frequency and recombination end product formation when F42 lac was the recipient DNA substrate. recB appears to have no significant role in the recombination that occurs between the two lac regions in an addition transductant. UV irradiation of the transducing phages increased the absolute level of both addition and substitution transductants obtained with a chromosomal lac gene but resulted in a considerable change in the relative frequency of addition versus substitution transductants.     

Transduction of R plasmids by bacteriophages P1 and P22

Summary We demonstrated that bacteriophages P1 and P22 were able to form various types of hybrids with six out of seven different R plasmids tested. When the same R plasmid was used for isolation, P1-R hybrids usually carried more resistance markers than P22-R. Several genetical observations suggest that the hybrid prophages carried the resistance markers transposed to the phage genomes without loss of essential phage genes. Upon UV-irradiation the prophages produced phage lysates that transduced the relevant resistance markers at high frequencies by lysogenic conversion. The insertion of the resistance markers was even acquired by the P1 or P22 genomes during one-step growth in R⁺ cells. Some lytically prepared lysates grown on R⁺ cells contained the hybrids at a frequency of 10^-7 to 10^-6/plaqueforming unit. Analysis of P1-transductants for resistance markers of the R plasmids revealed in some cases more specialized transductants than generalized transductants. These results strongly indicate that a precise genetic map of an R plasmid can not be established only on the basis of co-transduction frequencies of the resistance markers of the R plasmid.     

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.