A novel transducing phage

Summary Two newly isolated generalized transducing phages, F126 and F130, are in many respects similar to the previously described phage F116 of P. aeruginosa strain PAO. They also share with F116 the property of not responding to host-specific restriction in strain PAO. However, while the transduction ability of phages F116 and F130 is also inert to PAO-specific restriction, transduction mediated by phage F126 is 8–120 fold reduced in restrictive conditions. In experiments designed to explore the conditions necessary to obtain restriction in F126 transduction it was found that it differed from those previously known to specify host-controlled restriction in P. aeruginosa PAO (Rolfe and Holloway, 1968). These observations suggest that more than one independent restriction system operates in strain PAO.     

Reversion and immobilization of phage Mud1 cts (Apr lac) insertion mutations in Salmonella typhimurium

Summary Phage Mud1 cts (Ap^r lac), or Mud1, insertion mutations may be accompanied by adjacent deletion formation which can complicate use of lac fusions generated with this phage for gene regulatory studies. As for phage Mu insertion mutations, phage Mud1 insertions fail to revert at significant frequency (whether or not accompanied by an adjacent deletion). We describe isolation of revertible (X mutant) derivatives of phage Mud1 in Salmonella typhimurium. The X mutant derivatives allow use of reversion as a simple test to determine whether a Mud1 insertion has occurred precisely without an adjacent deletion that may have fused the lac genes to a promoter outside of the gene of interest. In addition, a simple method for stabilizing Mud1 generated lac fusions against subsequent transposition is described.     

Mutagenesis by random cloning of an Escherichia coli kanamycin resistance gene into the genome of the cyanobacterium Synechocystis PCC 6803: Selection of mutants defective in photosynthesis

Summary Photosynthetic mutants of the cyanobacterium Synechocystis PCC 6803 were produced by a random cartridge mutagenesis method leading to gene inactivation. This procedure relies on random ligation of an Escherichia coli kanamycin resistance (Km^r) gene to restriction fragments of genomic DNA from the host. Then recombination occurring during transformation promotes integration of the marker gene into the genome of the recipient cells. Several mutants impaired in photosynthesis were obtained by this procedure. All are partially or totally defective in photosystem II activity and some of them also harbour a functionally modified photosystem I. Restriction and recombination data showed that one mutant (AK1) is best explained as an insertion of the Km^r gene into an AvaII restriction site of the gene psbD-1. All other harbour a deletion, ranging from at least 1.15 kb (AK3) to more than 50 kb (AK9), which partly or fully overlaps the genes psbB and/or psbD-1, depending on the mutant. A genetic-physical map of the more than 60 kb region of the cyanobacterial genome harbouring the genes psbB, psbC and psbD-1 was constructed by combining published sequence data on these genes with the results of recombination and restriction mapping.     

Genetic and physical studies of restriction-deficient mutants of the Inc FIV plasmids R124 and R124/3

Summary R124 and R124/3 are R plasmids that carry the genes for two different restriction and modification systems. The phenotype of strains carrying either of these plasmids along with the F'lac ⁺ plasmid, is restriction-deficient (Res^-). The Res^- phenotype is not due to selection of preexisting mutants but rather to a complex mutational event caused by the F plasmid. Restriction-deficient mutants carry extensive deletions and other DNA rearrangements. Tn7 insertion is used to locate the restriction gene. Many of the Res^- mutants are genetically unstable and revert at exceptionally high frequencies. Reversion is accompanied by DNA rearrangements which result in a net gain of 9 kb of DNA. F derivates of F⁺ which do not cause restriction-deficiency but do cause deletion were used to distinguish between the DNA rearrangements associated with restriction-deficiency and those associated with deletion. From Res⁺ revertants of strains carrying F'lac ⁺ and R124 or R124/3 we have isolated F plasmids that now carry the genes for the R124 or R124/3 restriction and modification systems. It is suggested that interaction between part of the F plasmid and that segment of the R plasmid which controls the switch in Res-Mod specificity which has been observed (Glover et al. 1983) is responsible for the production of restriction-deficiency.     

Transfer of RP4::Mu plasmids to Agrobacterium tumefaciens

Transfers of RP4::Mu plasmids from Escherichia coli to Agrobacterium tumefaciens are very inefficient in contrast to the very efficient transfer of RP4. Apparently, one or more Mu functions prevent RPR::Mu plasmids from establishing in some Gram-negatives other than E. coli. This problem was eliminated by the use of a mutant Mu prophage, Mu cts62r23, in RP4. Moreover, the transfer of RP4::Mu cts62r23 to the Agrobacterium strain C58 was found to be affected by a restriction modification system. The target for this restriction was located on Mu DNA and not on RP4 DNA. The plaque-forming phage production of Mu cts62r23 in Agrobacterium was found to be 10⁶ times lower than in E. coli.     

Orientation of the genetic and physical map of Rhizobium meliloti temperate phage 16-3

Summary The attachment site, the C cistron of Rhizobium meliloti temperate phage 16-3, and the insertion of the host cys46 ⁺ gene in the phage genome were localized on the HindIII and EcoRJ restriction endonuclease maps, as well as mapped genetically. The strategy employed included restriction analysis and Southern in situ hybridization of plasmid pGY1, which carries the bacterial chromosome region containing the integration site of 16-3, plasmid pGY2, which carries the 16-3 prophage, deletion and inversion mutants, and the cys46 ⁺ transducing 16-3 particles. The colinear array of genetic and physical data was possible. The possibility of isolation of a replacement phage vector for Rhizobium is discussed.     

Transposition of a DNA sequence determining kanamycin resistance into the single-stranded genome of bacteriophage fd

Summary Derivatives of bacteriophages fd which transduce kanamycin resistance were selected after growth of the phage in an E. coli strain that carried transposon 5 (Tn5). Different clones of transducing phage and their DNAs were characterized by gel electrophoresis, electron microscopy, and by their ability to multiply in the absence of helper phage. Integration of the intact transposon into the full size phage genome was correlated with an increase in size of the phage particle from 0.95 to 1.7 , and with the appearance in the phage DNA of the stem loop structure characteristic for single-stranded Tn5 DNA. In nondefective phages the site of insertion was mapped by heteroduplex analysis within the intergenic region of the phage genome. Defective transducing phages were characterized as an insertion of Tn5 into a phage gene, and/or as a partial deletion or duplication of phage and transposon DNA. The size of the transducting phage from different defective clones varied from 0.6 to 3.0 and was directly proportional to the DNA content. These results demonstrate that filamentous bacteriophage are highly capable to replicate and package very different amounts of foreign DNA.This work was presented at the EMBO Workshop on single-stranded DNA viruses, October 1976, Harpert, The Netherlands     

Cloning of sporulation gene spoIVC in Bacillus subtilis

Summary Sporulation gene spoIVC of Bacillus subtilis was cloned by the prophage transformation method in temperate phage 105. The specialized transducing phage, 105spoIVC-1, restored the sporulation of the asporogenous mutant of B. subtilis strain 1S47 (spoIVC133). Transformation experiments showed that the spoIVC gene resides on a 7.3 kb HindIII restriction fragment. Subsequent analysis of the 7.3 kb HindIII fragment with restriction endonuclease EcoRI showed that the spoIVC gene resides on a 3.6 kb EcoRI fragment within the 7.3 kb fragment. The 3.6 kb fragment was recloned into the unique EcoRI site of plasmid pUB110 and deletion derivatives having a deletion within the 3.6 kb insert were constructed. The plasmid carrying the entire spoIVC gene restored the sporulation of strain HU1214 (spoIVC133, recE4) at a frequency of 10⁷ spores/ml, and reduced the sporulation of strain HU1018 (spo ⁺, recE4) to 10⁷ spores/ml.     

Characterisation of transfer-deficient mutants of the R100-1TcS plasmid pDU202, caused by insertion of Tn10

Summary The transposition of Tn10 from the E. coli chromosome to pDU202 (a Tc^S deletion mutant of R100-1) was selected by a mating technique: it took place at a frequency of 10^-7 in both rec ⁺ and recA hosts. Seventeen transfer deficient mutants of pDU202 caused by the insertion of Tn10 into the R factor's transfer genes were analysed. Insertion of Tn10 occurred at at least eight sites, with some apparent clustering in the second part of the transfer operon, and with equal numbers in each orientation. Both strongly and weakly polar insertions were observed, and the strength of the polarity was correlated with the Tn10 orientation. The map order of the second part of the transfer operon was confirmed as traC traF traH traG traS traD, analogous to that of F. The strongly polar insertion mutants still expressed traI at wild-type levels, providing further evidence that this gene does not belong to the transfer operon of R100.     

A derivative of Tn5 with direct terminal repeats can transpose

The 5.7 kb⁴ transposable kanamycin resistance determinant Tn5 contains 1.5 kb terminal inverted repeats which we here call arms. Tn5's arms contain the genes and sites necessary for Tn5 transposition, and are not homologous to previously described transposable elements. To determine whether one or both arms is a transposable (IS) element, we transposed Tn5 to pBR322 and used restriction endonuclease digestion and ligation in vitro to generate plasmid derivatives designated pTn5-DR1 and pTn5-DR2 in which Tn5's arms were present in direct rather than in inverted orientation. Analysis of transposition products from dimeric forms of the pTn5-DR1 plasmid to phage λ showed that the outside and inside termini of right and of left arms could function in transposition. We conclude that both of Tn5's arms are transposable elements and name them IS50L (left) and IS50R (right). IS50R, which encodes transposase, was used several-fold more frequently than IS50L, which contain an ochre mutant allele of transposase: this implies that Tn5's transposase acts preferentially on the DNA segment which encodes it. Analysis of transpositions of the amp^rkan^r element Tn5-DR2 to the lac operon showed that Tn5-DR2, like Tn5 wild-type, exhibits regional preference without strict site specificity in the choice of insertion sites.     

Virulence genes are carried by a megaplasmid of the plant pathogen Pseudomonas solanacearum

Summary A class of avirulent mutants of the plant pathogenic bacterium Pseudomonas solanacearum, strain GMI1000, resistant to acridine orange (Acr^r), harbour a deletion of over 85 kb in their genome. This deletion affects, a1,000 kb megaplasmid which has previously been shown to be present in most of the strains of this species. In addition at least 11 out of 13 independent Tn5 insertions, leading to loss of virulence, are located on the megaplasmid. Nine of them are present in the region which is deleted from the Acr^r mutants. These results suggest that the majority of virulence genes identified so far are plasmid borne.     

Restriction endonuclease mapping of R27 (TP117), an incompatibility group HI subgroup 1 plasmid from Salmonella typhimurium

A circular map of the IncHI plasmid R27 corresponding to a genome size of 182 kb was established using the restriction endonucleases ApaI, XbaI, and PstI. The map was derived from the results obtained by hybridizing individual ApaI and XbaI fragments to blotted digests of the plasmid, as well as from complete and partial digests. Analysis of a deletion mutant derived by in vitro digestion with PstI and of transfer-defective and tetracycline-sensitive deletion mutants of R27 derived by Tn5 insertion were instrumental in determining the positions of some fragments.     

Packaging of an oversize transducing genome by Salmonella phage P22

The DNA in specialized transducing particles of the Salmonella phage P22 was examined by electron microscopy. The transducing particles of P22Tc-10 (which transduce tetracycline-resistance) are shown to contain DNA molecules that are incomplete permuted fragments of an oversize genome, as predicted by the genetic results of Chan et al. (1972). The oversize transducing genome differs from the P22 wild-type genome by a large (mol. wt 2.5 × 10⁶) insertion of foreign DNA. The insertion, as seen in heteroduplexes, has an unusual lariat-like structure, which suggests that the insertion contains a non-tandem reverse duplication.By comparing wild-type P22 with P22Tc-10 and deletion revertants of P22Tc-10, we show by direct physical means, that the amount of terminal repetition in P22 phage DNA is a direct function of the genome size, as predicted from the model for circular permutation and terminal repetition suggested by Streisinger et al. (1967).     

Use of pulsed-field-gradient gel electrophoresis to construct a physical map of the Caulobacter crescentus genome

The restriction enzyme DraI cleaves the Caulobacter crescentus genome into at least 35 fragments which have been resolved in agarose gels using pulsed-field-gradient gel electrophoresis (PFGE). When digests were performed using DNA from strains containing Tn5 insertion mutations, altered band migrations were observed. Using PFGE with the appropriate pulse times, size differences as small as 2% could be resolved in large fragments. Using this approach, we have constructed a partial physical map of the genome which correlates well with the C. crescentus genetic map and have shown the size of the genome to be approx. 3800 kb. Using hybridization with cloned genes, we have determined the map locations of five previously unmapped genes. In addition, we have shown that PFGE can be used to rapidly determine the map locations of new insertion mutations or the sizes of deletion mutations.     

Characterisation of a lambda transducing phage carrying the F conjugation gene traG

Summary A lambda transducing phage carrying the traGSTD genes of the E. coli K12 sex factor F was isolated by an in vivo technique, and characterized in ra complementation tests, by determining its restriction endonuclease fragment sizes, and by measuring heteroduplex molecules. The size and location on the F physical map of the tra transducing segment was thereby determined.Comparison of the proteins synthesized in UV-irradiated cells by this phage and by a derivative carrying the amber traG79 mutation, allowed the traG product to be identified as a protein of molecular weight 100,000. In the same experiments, the sizes of the traT and traD products made by the phage were also measured, being 25,000 and 85,000 daltons respectively.     

A restriction endonuclease map of Streptomyces phage VWB

Summary A physical map of the actinophage VWB has been constructed using the restriction endonucleases BglII, ClaI, EcoRI, EcoRV, HindIII, KpnI and SphI. Phage VWB, genome size 47.3 kb, propagates on Streptomyces venezuelae, and it can also lysogenise this species. The three BglII-generated fragments of VWB DNA were cloned in pBR322, and subsequently mapped. In this manner the restriction map of the VWB phage genome was constructed.Abbreviations dam DNA adenine methylase activity - kb kilobase pairs - :: novel joint     

Physical mapping of the BglI, BglII, PstI and EcoRI restriction fragments of staphylococcal phage phi 11 DNA

Summary A cleavage map of the generalized transducing staphylococcal phage 11 DNA has been constructed by reciprocal double digestion. All three BglI, the six BglII, the three PstI, and 11 out of 15 EcoRI sites have been mapped. The map is circular, with a total length of 42 kb, and has been divided into 100 map units. The phage DNA is cyclically permuted and has a terminal redundancy of about 11 kb. The preferential starting point and direction for packaging DNA lies at map unit 79 and proceeds towards higher map units.     

Isolation and characterization of a new ruminal bacteriophage lytic toStreptococcus bovis

A new bacteriophage, designated F4, was isolated from the ruminal fluid of a calf. The host range of F4 phage was limited to five strains ofStreptococcus bovis out of ten tested on which clear plaques 0.6–1.2 mm in diameter were found. Bacteriophage F4 had an elongated head 75 nm long and 33 nm wide with a noncontractile flexible tail 100 nm in length on average. This phage is defective in the generation of plaques at low multiplicities of infection. Its genome consists of double-stranded linear DNA of 60.38 kb lacking cohesive ends. The F4 DNA was analyzed with 13 restriction enzymes. The restriction enzymes that did not cleave it wereBamHI,EcoRI,PvuI, andSmaI. The circular restriction map was constructed with four restriction endonucleases (XbaI,EcoI,SalI, andBglI).     

The chromosome of Salmonella paratyphi A is inverted by recombination between rrnH and rrnG.

Salmonella paratyphi A, a human-adapted bacterial pathogen, causes paratyphoid enteric fever. We established the genome map of strain ATCC 9150 by the use of four endonucleases, XbaI, I-CeuI, AvrII (= BlnI), and SpeI, which generated 27, 7, 19, and 38 fragments, respectively; the sum of the fragments in each case indicates a genome size of ca. 4,600 kb. With phage P22, we transduced Tn10 insertions in known genes from Salmonella typhimurium LT2 to S. paratyphi A ATCC 9150 and located these insertions on the S. paratyphi A chromosome through the XbaI and AvrII sites in Tn10 and through the increased size of the SpeI fragment bearing a Tn10. Compared with the maps of other Salmonella species, the S. paratyphi A genomic map showed two major differences: (i) an insertion of about 100 kb of DNA between rrnH/G and proB and (ii) an inversion of half the genome between rrnH and rrnG, postulated to be due to homologous recombination between the rrn genes. We propose that during the evolution of S. paratyphi A, the first rearrangement event was the 100-kb insertion, which disrupted the chromosomal balance between oriC and the termination of replication, forcing the rrnH/G inversion to restore the balance. The insertion and the inversion are both present in all 10 independent wild-type S. paratyphi A strains tested.     

Restriction map of Tn7

Tn7, a transposon of 14 kb, encodes resistance to trimethoprim (Tp) and streptomycin (Sm). A cleavage site map of this transposon for twenty-two different restriction enzymes as determined by comparison of restriction enzyme cleavage patterns of the plasmids ColE1 and ColE1::Tn7 is presented. The precise localization of these sites was facilitated by the use of two deletion derivatives of ColE1::Tn7: pGB2 and ColE1::Tn7Δ6, and by the use of pOB14 and pOB15 which contain a part of Tn7 cloned into the plasmid pBR322. This map should aid in the study of the structural and genetic organization of this transposon.