Preferential generalized transduction by bacteriophage Mu

Summary The generalized transduction by bacteriophage Mu was found to be preferential for the 0–1 min segment of the E. coli K12 chromosome. This transduction pattern is obtained with phage lysates grown on all F^-, F⁺ and Hfr tested, and is not marker-specific.Phages grown by both lytic infection and by heat induction of prophages at different locations of the host's chromosome show the same transduction pattern, indicating that generation of transducing DNA does not directly depend on excision events. Conjugation of independently obtained Muc ⁺-lysogenic strains of HfrC with a multiauxotrophic F^- recipient strain lysogenic for a Mucts62 prophage, shows that transfer of the temperature-resistance character (Muc ⁺) is not preferentially linked to the 0–1 min segment. The lysogenizing integrations do therefore not take place within the segment preferentially transduced by the phage.A model¹ for the generation of the transducing DNA is proposed, which assumes that for its replication, Mu DNA is integrated close to the 0–1 min segment of the host chromosome, which is then preferentially replicated and packaged into the phage heads.     

Genetic studies of the ribosomal proteins in Escherichia coli. 7. Mapping of several ribosomal protein components by transduction experiments between different strains of Escherichia coli

Summary Two 50s (50-10 and 50-12) and two 30s (30-4 and 30-7) ribosomal proteins could be distinguished between Shigella dysenteriae Sh/s and Escherichia coli K-12 JC411 with CMC column chromatography. On the other hand, E. coli K-12 AT2472 was shown to have a 30s ribosomal protein, 30-6(AT), which is specific to this strain and distinguishable from 30-6 of other E. coli K-12 strains. Transduction experiments by phage Plkc between Sh. dysenteriae Sh/s and E. coli ATSPCO1, a spectinomycin resistant mutant derived from AT2472 in which the 30-4 protein is altered, indicated that the genes specifying the above five ribosomal protein components are located in the streptomycin region on the E. coli chromosome.The gene order for three 50s (50-8, 50-10 and 50-12) and three 30s [str (30-?), 30-4 and 30-6] ribosomal proteins on the chromosome was determined by transduction technique between Sh. dysenteriae Sh/s and E. coli ATSPC01, between E. coli ATSPC01 and E. coli ER05 (an erythromycin resistant strain in which the 50-8 protein is altered), and between Sh. dysenteriae Sh/s and E. coli ERSPC14 (str ^s spc ^r ery ^r), respectively. It was found that these protein genes are arranged on the chromosome in the order of str (30-?)-30-4-30-6-50-8-50-10-50-12.     

Transfer ofF′ fromEscherichia coli K 12 toEscherichia coli B and to strains ofParacolobacter andKlebsiella

The transfer of theF episome fromEscherichia coli K 12 toE. coli B,Paracolobacter andKlebsiella was studied. The frequency of transfer of the episomal markers toE. coli B was very low. The large majority ofE. coli B cells which had received the episomal markerslac ⁺ orgal ⁺ were F^–, which indicates that the episomal markers were stably integrated on the chromosome. Recombinants from K 12 F⁺ × B F^– crosses were mostly F^–. These results suggest that the multiplication of theF-factor ofE. coli K 12 is restricted inE. coli B. The transfer of theF-lac ⁺ Ad ⁺ episome fromE. coli K 12 toParacolobacter andKlebsiella strains was in most cases only possible when donor and acceptor strain were plated together on selective media. Stable incorporation of episomal markers was also found withParacolobacter coliforme. Paracolobacter aerogenoides andKlebsiella aerogenes strains could be infected withF-lac ⁺ Ad ⁺. The episomal markers were not incorporated and the episomes were easily lost, which indicates that these strains contained theF factor in the autonomous state.     

Tetracycline-resistance inEscherichia coli; a genetic contribution

A non-transmissible tetracycline-resistance plasmid inE. coli was found to be transmissible by transduction and by conjugation with the aid of theE. coli K12 sex-factor. Transfer of the tetracycline-resistance plasmid (R-tet) by transduction or conjugation to anE. coli K12 Hfr strain revealed that the plasmid was incompatible with the integrated F-factor. Selection for tetracycline-resistance after conjugation or transduction yielded Hfr colonies which carried the tetracycline-resistance determinant as a chromosomal marker. The tetracycline-resistance determinant was integrated at the 1 min region of theE. coli chromosome map (Taylor and Trotter, 1967) between the markersara andleu. Apart from Hfr colonies with a chromosomal tetracycline-resistance determinant, F-gal⁺-mediated transfer of R-tet to strain Hfr R4 gave some colonies in which the tetracycline-resistance determinant was carried on a fused plasmid that, besides the resistance determinant, contained thegal ⁺ marker of the original F-gal ⁺. This fused plasmid is transmissible and confers to an F⁻ cell male-specific phage-sensitivity, like an F-factor does. It is suggested that this fused plasmid, which is compatible with the integrated F-factor in the Hfr R4 cells, arose by recombination between F-gal ⁺ and R-tet.     

Generalized transduction in Rhizobium meliloti

Summary The phage 11 of R. meliloti performs generalized transduction. This was confirmed by the variety of single markers transferred and by separating transducing particles containing BUdR-labelled bacterial DNA. The transduction frequencies depended on the marker. Linked alleles were mapped by cotransduction on fragments of bacterial DNA equal in size to the phage DNA. With crosses between antibiotic resistancy and auxotrophic markers a partial map was constructed with str, cml, pur-19, and leu-44 sites. With a few multi-auxotrophic mutants linkage data of conjugation were compared with the linkage by cotransduction.     

A Simple Method for Multiple Modification of the Escherichia coli K-12 Chromosome

We developed a simple method of generating markerless deletions in the Escherichia coli chromosome. The method consists of two recombination events stimulated by λ Red recombinase. The first recombination replaced a target region with a marker cassette and the second then eliminated the marker cassette. The marker cassette included an antibiotic resistant gene and a negative selection marker (Bacillus subtilis sacB). Since sacB makes E. coli sensitive to sucrose, a markerless deletion strain was successfully selected using its sucrose-resistant phenotype. To stimulate these recombination events, 1-kbp homologous sequences adjacent to the target region were connected to both ends of the marker cassette or connected to each other by PCR. The average efficiency of the recombinations was 24% and 93% respectively. Eliminating the marker cassette with a fragment including an additional sequence, insertion was also possible. This markerless deletion method should be useful in creating a highly modified E. coli chromosome.     

Integration and Intramolecular Transposition of the TnBP3 Transposon ofBordetella pertussis in the Escherichia coliK12 Cells Mutant for the Hpr Protein of the Phosphoenolpyruvate-Dependent Phosphotransferase System

Integration of a plasmid carrying the TnBP3 transposon of Bordetella pertussisinto the chromosome of Escherichia coliand transpositions of the integrated structure within a chromosome in the wild-type and mutant cells ptsHdevoid of the major Hpr protein of the phosphoenolpyruvate-dependent phosphotransferase system were studied. When transposed to a new chromosome site, the integrated structure was precisely (or almost precisely) excised from the metYgene sequence, which resulted in restoration of the Met⁺phenotype. The integration and transposition events were only observed in the E. colicells carrying the ptsH ⁺allele. The ptsHmutations inhibited integration and intramolecular transposition, which were restored after phenotypic or genetic suppression of the ptsHmutation. The intensity of the processes studied were suggested to depend on the integrity of a chain that ensures transferring of the phosphoryl residue by proteins of the phosphotransferase system in E. coliK12.     

Chromosome transfer and R-prime formation by an RP4::mini-Mu derivative in Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Proteus mirabilis

We have introduced into the wide host range conjugative plasmid RP4, a mini-Mu derivative which was known to be able to transpose spontaneously in E. coli K-12, and to induce in such a host several kinds of chromosomal rearrangements including replicon fusions. Unlike RP4, RP4::mini-Mu can mediate the transfer of the host chromosome to a recipient bacterium and generate R primes at high frequencies (10^?4 for the transfer of a given marker, 10^?5 for the formation of R primes carrying a given marker). Two such RP4::mini-Mu plasmids were introduced into one Salmonella typhimurium strain, one Klebsiella pneumoniae strain, and one Proteus mirabilis strain. Each of these three strains were mated with an E. coli K-12 recipient and transconjugants carrying R primes were recovered in all three cases at frequencies ranging from 5 × 10^?6 to 10^?7.     

A stochastic model of recombination during conjugation in Escherichia coli K-12.

A mathematical model is constructed in which recombination in E. coli K-12 is considered as a stochastic Markov process. The model takes into account the possibility of inclusion in the recombinant structure of the origin of the donor chromosome and makes it possible to correctly describe results of irradiation of the donor Hfr before crossing. Formulae are deduced for frequencies of unselected markers for cases when the counter-selected maternal marker lies in the distal or proximal region of the chromosome.     

P1 transduction map spanning the replication terminus of Escherichia coli K12

Summary The region of the E. coli chromosome that contains the replication terminus has not previously been spanned by P1 cotransduction. We have used Tn5, Tn9 and Tn10 transposons inserted in this region as genetic markers, and have constructed a genetic map that extends from fnr (min 29.3) to manA (min 35.7). The relevant transposons that have been mapped in this region and which are described in this report are trgl::Tn5 (min 31.1), zdc-235::Tn10 (min 32.3), zdd-230::Tn9 (min 33.3), and zde-234::Tn10 (min 34.2). The size of this region as determined by P1 cotransduction is very similar to previous estimates obtained by bacterial conjugation.     

Transformation in E. coli K12: Relation of linkage to distance between markers

Summary E. coli K12 transformants, selected as leu ⁺ or pyrA ⁺ transformants, were analysed for inheritance of some closely linked unselected markers. Based on the observation that the number of recombinants which require, besides an integration event, one or more crossing-over events was negligible, a simple mapping function was deduced. The function L=e ^–kd , which directly relates observed linkage of an unselected marker and the relative distance of that unselected marker to the selected marker, gave a consistent interpretation of the experimental results.     

Allele-specific CAPS markers based on point mutations in resistance alleles at the pvr1 locus encoding eIF4E in Capsicum

Marker-assisted selection has been widely implemented in crop breeding and can be especially useful in cases where the traits of interest show recessive or polygenic inheritance and/or are difficult or impossible to select directly. Most indirect selection is based on DNA polymorphism linked to the target trait, resulting in error when the polymorphism recombines away from the mutation responsible for the trait and/or when the linkage between the mutation and the polymorphism is not conserved in all relevant genetic backgrounds. In this paper, we report the generation and use of molecular markers that define loci for selection using cleaved amplified polymorphic sequences (CAPS). These CAPS markers are based on nucleotide polymorphisms in the resistance gene that are perfectly correlated with disease resistance, the trait of interest. As a consequence, the possibility that the marker will not be linked to the trait in all backgrounds or that the marker will recombine away from the trait is eliminated. We have generated CAPS markers for three recessive viral resistance alleles used widely in pepper breeding, pvr1, pvr1 ¹, and pvr1 ². These markers are based on single nucleotide polymorphisms (SNPs) within the coding region of the pvr1 locus encoding an eIF4E homolog on chromosome 3. These three markers define a system of indirect selection for potyvirus resistance in Capsicum based on genomic sequence. We demonstrate the utility of this marker system using commercially significant germplasm representing two Capsicum species. Application of these markers to Capsicum improvement is discussed.     

Gross Map Distances and Hfr Transfer Times in Escherichia coli K-12

Hfr strains B4 and B8 transfer the Escherichia coli chromosome in opposite directions, each transferring lac⁺ as the last known marker. They were mated in concurrent crosses with the proA leu metE lys trp purE lac strain χ462. Analysis of the time of entry values for these markers showed that Hfr strain B8 transfers the whole chromosome more rapidly than does Hfr strain B4. In both crosses, the rate of transfer observed decelerates. If deceleration occurs as a function of the amount of chromosome transferred, the data are consistent with the markers examined being very accurately placed on the Taylor-Trotter map of the E. coli K-12 genome.     

Site-specific recombination in Escherichia coli between the att sites of plasmid pSE211 from Saccharopolyspora erythraea

Summary pSE211 fromSaccharopolyspora erythraea integrates site-specifically into the chromosome through conservative recombination betweenattP andattB, the plasmid and chromosomal attachment sites. Integration depends on the presence ofint, an open reading frame (ORF) that lies adjacent toattP and encodes the putative integrase. Immediately upstream ofint liesxis (formerly calledorf2) which encodes a basic protein that is thought to exhibit DNA binding.xis andint were cloned in various combinations in pUC18 and expressed constitutively inEscherichia coli from thelac promoter.attP andattB were cloned inStreptomyces orE. coli plasmids containing kanamycin resistance (Km^R) or chloramphenicol resistance (Cm^R) markers. Stable Km^R Cm^R cointegrates formed byattP ×attB orattP ×attP recombination (integration) were obtained inE. coli hosts that expressedint. Co-integrates were not found in hosts expressingint+xis. Excision (intraplasmidatt site recombination) was examined by constructing plasmids carryingattL andattR or twoattP sites separating Cm^R from Km^R and by following segregation of the markers in various hosts. BothattL ×attR andattP ×attP excision depended on bothxis andint inE. coli. pSE211att site integration and excision were not affected by a deletion inhimA, the gene encoding a subunit of integration host factor.     

Use of RP4-prime plasmids constructed in vitro to promote a polarized transfer of the chromosome in Escherichia coli and Rhizobium meliloti.

Summary RP4-prime plasmids containing chromosomal fragments of either Escherichia coli or Rhizobium meliloti were constructed in vitro. When introduced into E. coli or R. meliloti respectively, they promoted a polarized transfer of the chromosome as demonstrated either by the gradient of transfer of various markers or by the study of the genetic constitution of recombinants. In E. coli, mobilization was shown to be dependent upon the presence of a functional rec A system. Inheritance of markers was due to their integration into the chromosome of the recipient as shown by the need for a functional rec A system in the recipient E. coli or by mobilization of recessive markers in R. meliloti. The system described could be applied to genetic mapping in any Gram negative bacteria.     

Mapping of the <Emphasis Type="Italic">S. demissum</Emphasis> late blight resistance gene <Emphasis Type="Italic">R8</Emphasis> to a new locus on chromosome IX

The use of resistant varieties is an important tool in the management of late blight, which threatens potato production worldwide. Clone MaR8 from the Mastenbroek differential set has strong resistance to Phytophthora infestans, the causal agent of late blight. The F1 progeny of a cross between the susceptible cultivar Concurrent and MaR8 were assessed for late blight resistance in field trials inoculated with an incompatible P. infestans isolate. A 1:1 segregation of resistance and susceptibility was observed, indicating that the resistance gene referred to as R8, is present in simplex in the tetraploid MaR8 clone. NBS profiling and successive marker sequence comparison to the potato and tomato genome draft sequences, suggested that the R8 gene is located on the long arm of chromosome IX and not on the short arm of chromosome XI as was suggested previously. Analysis of SSR, CAPS and SCAR markers confirmed that R8 was on the distal end of the long arm of chromosome IX. R gene cluster directed profiling markers CDP^Sw54 and CDP^Sw55 flanked the R8 gene at the distal end (1 cM). CDP^Tm21-1, CDP^Tm21-2 and CDP^Tm22 flanked the R8 gene on the proximal side (2 cM). An additional co-segregating marker (CDP^Hero3) was found, which will be useful for marker assisted breeding and map based cloning of R8.     

Plasmid replication and Hfr formation in strains of Escherichia coli carrying seg mutations.

Summary Several conditional-lethal mutations that do not permit the replication of F-factors ofEscherichia coli K-12 are located at a site calledseg. This gene is located on theE. coli chromosome betweenserB andthr. It is unrelated to other known genes involved in DNA replication. Strains carryingseg mutations were unable to replicate F-lac⁺, several F-gal⁺s, F-his⁺ and bacteriophage at 42°. However, neither phage T4, ColE1, nor any of the R factors tested were prevented from replicating at 42°C.When the kinetics of the loss of F-primes is studied inseg strains, it is found that the rate of curing depends on the size of the plasmid, larger F factors curing faster than smaller ones, and that Hfrs are formed at high frequencies. The Hfrs showed both F-genote enlargement and normal transfer of chromosomal markers. The F-genotes are unstable and segregate chromosomal markers at high frequencies. Some orthodox Hfrs were examined, and two that were known to revert to the F⁺ condition relatively frequently were found to generate enlarged F-genotes on mating, whereas two strains that were very stable with respect to reversion to the F⁺ state did not show F-genote formation.F-genote formation fromseg Hfr strains is dependent of a functionalrecA gene, as F-genote formation was not seen with aseg-2, recA-1 Hfr. This is in contrast to F-genote enlargement shown by both orthodox Hfrs and an Hfr strain constructed by integration of a temperature-sensitive F-gal⁺, whose F-genote enlargement is Rec-independent. Thus there may be more than one mechanism for the formation of enlarged F-genotes.     

Formation of recombinants inEscherichia coli and a gene controlling the expression of a donor marker

In our experiments we tried to explain some anomalies in the formation of recombinants in anEscherichia coli mutant strain which, compared with the control, yields a lower number of recombinants. Following the transfer of C¹⁴-thymidine-labelled donor chromosome it was found that the low recombinant frequency is not due to a lower effectiveness of DNA transfer into the recipient cell; similarly, using the technique of interrupted mating between the cells we were able to detect that the rate of chromosome transfer is the same as in the control. The low frequency of recombinants may be explained by some restriction processes which take place in the recipient cell following the chromosome transfer. In addition, a relation between the localization of a marker and its subsequent expression in the recombinants was observed. Analyzing this phenomenon we were able to find a gene located near thethr marker which might modify the expression of integrated donor marker in the zygote. The probable mode of action of this gene is discussed.     

Selection againstdam methylation sites in the genomes of DNA of enterobacteriophages

Summary Postreplicative methylation of adenine inEscherichia coli DNA to produce G^6m ATC (where^6mA is 6-methyladenine) has been associated with preferential daughter-strand repair and possibly regulation of replication. An analysis was undertaken to determine if these, or other, as yet unknown roles of GATC, have had an effect on the frequency of GATC inE. coli or bacteriophage DNA. It was first ascertained that the most accurate predictions of GATC frequency were based on the observed frequencies of GAT and ATC, which would be expected since these predictors take into account preferences in codon usage. The predicted frequencies were compared with observed GATC frequencies in all available bacterial and phage nucleotide sequences. The frequency of GATC was close to the predicted frequency in most genes ofE. coli and its RNA bacteriophages and in the genes of nonenteric bacteria and their bacteriophages. However, for DNA enterobacteriophages the observed frequency of DNA enterobacteriophages the observed frequency of GATC was generally significantly lower than predicted when assessed by the chi square test. No elevation in the rate of mutation of^6mA in GATC relative to other bases was found when pairs of DNA sequences from closely related phages or pairs of homologous genes from enterobacteria were compared, nor was any preferred pathway for mutation of^6mA evident in theE. coli DNA bacteriophages. This situation contrasts with that of 5-methylcytosine, which is hypermutable, with a preferred pathway to thymine. Thus, the low level of GATC in enterobacteriophages is probably due not to^6mA hypermutability, but to selection against GATC in order to bypass a GATC-mediated host function.     

The genetic map of the mitochondrial genome in yeast

Summary An approach for the screening of mit ^- mutants, the isolation and preliminary classification of a series of such mutants is reported. Loss and retention of 8 mit ^- and 6 drug ^r markers in mitDNA was analyzed in populations of rho^- clones derived from four yeast strains. The populations studied constitute a representative fraction of the rho^- petites formed during growth at 35° C under the influence of mutation tsp-25 which is in common to the four strains. The majority of the rho^- clones retained several of the markers studied. Depending on the marker regarded retention frequencies between 15% (oxi3) and 45% (oli1, cob) were observed. Loss of one and retention of the other of a pair of markers was determined in all rho^- clones of the four populations. The frequencies of marker separation by rho^- deletion thus obtained are assumed to reflect the distance between markers on the mitochondrial genome: the higher the frequency of separation the longer the distance between two markers. Based on these frequencies a unique order of markers on a circular map was determined. Positions of markers on a scale from 0 to 100 were found to be: cap/ery (0) — olil (16) — cob1-1354 (21) — ana101 (22) — cob2-1625 (24) — oli2 (35) — pho1 (40) — oxi3-2501 (44) — oxi3-3771 (47) — par (65) — oxi2 (79) — oxil (87) tms8 (93) —cap (100). The relevance of this map as to the faithful representation of the topology of gene loci on mitDNA is discussed. Correlation of retention frequencies of markers to their map positions reveals a pronounced polarity: mitDNA segments carrying the cob-oli1 segment prevail whereas segments retaining oxi3 are the least frequent.