Inversion of the metE-oriC-rpsE chromosome segment in Escherichia coli K12

We have described recently a large inversion of the Escherichia coli chromosome (designated udpPf1), including region of the chromosomal replication region (oriC). The udpPf1 inversion was induced by Tn10 transposon (metE::Tn10). It results in increased expression of the uridine phosphorylase gene (udp) which is closely linked to the metE gene. The data of conjugational and transductional experiments presented in this report demonstrate that the udpPf1 inversion covers a chromosomal segment extending over 12 min of the E. coli genetic map and including the rpsE, crp and metE::Tn5 markers. The results are presented indicating that the increased uridine phosphorylase activity is due to fusion of the udp gene to a more strong promoter located, probably, in the operon for ribosomal proteins cluster, near 73 min on the E. coli chromosome.     

Tn10-mediated inversions fuse uridine phosphorylase (udp) and rRNA genes of Escherichia coli.

Two strains carrying metE::Tn10 insertions (upstream of the udp gene) were used to isolate mutants of Escherichia coli overexpressing udp. These strains differ in their gene order; one contains an inversion between the rrnD and rrnE rRNA operons. Selection was based on the ability of overexpressed Udp to complement thymine auxotrophy. Chromosomal rearrangements that connect the udp gene and promoters of different rrn operons were obtained by this selection. Seven of 14 independent mutants selected in one of the initial strains contained similar inversions of the metE-rrnD segment of the chromosome (about 12% of its length). Another mutant contained traces of a more complicated event, inversion between rrnB and rrnG operons, which was followed by reinversion of the segment between metE and the hybrid rrnG/B operon. Similar inversions (udp-rrn) in a strain already carrying an rrnE-rrnD inversion flip the chromosomal segment between metE and rrnD/E in the opposite direction. In this case, inversions are also accompanied by duplications of the chromosomal region between the rrnA and hybrid udp-rrnD/E operons. PCR amplification with a set of oligonucleotides from the rrn, Tn5, and met genes was used for more detailed mapping. Amplified fragments of the rearranged chromosomes connecting rrnD sequences and insertion elements were sequenced, and inversion endpoints were established.     

Chromosomal inversion accompanied by an enhancement of uridine phosphorylase gene expression in Escherichia coli K-12

In thymine requiring auxotrophs of Escherichia coli the uridine phosphorylase enzyme (udp gene) can catalyze nonspecifically conversion of thymine to thymidine. By selection for effective utilization of exogenous thymine, it is possible to isolate forms with increased expression of the udp gene. Mutants with increased gene expression were isolated from the strain with transposon Tn10 within the metE gene closely linked to udp. Some mutants (designated udpPf) losing Tn10 but retaining the Met- phenotype are characterized by disturbance of recombination in the metE-udp region: they do not form Met+ transductants in P1 transduction with the wild-type donor strain. However, recovery of homology in the chromosomal metE-udp region takes place with low frequency in P1 transduction using the strain with Tn10 insertion in metE as a donor. Data obtained in transductional and conjugational experiments demonstrate that the udpPf1 mutant studied is an inversion extending about 3 min of the E. coli chromosome and including the region of chromosomal replication origin (oriC).     

Cloning the uridine phosphorylase (udp) gene of Escherichia coli and its expression in recombinant plasmids

On the basis of Escherichia coli DNA and vectors pBR322, pUC19, hybrid plasmids restoring Udp+ phenotype in the E. coli deletion (delta udp) mutant have been obtained. The udp gene is carried by a 8 kb PstI fragment (on the pUD2) and by a smaller 2.87 kb PstI-SalGI fragment from the PstI fragment (pUD7). The uridine phosphorylase level was 30 times higher in the cells containing hybrid plasmid as compared to the strain with chromosomal location of the udp gene. On the other hand, the measurements of uridine phosphorylase activity in the cytR- and cya- background indicate that expression of the cloned udp gene escapes partially negative control of the CytR repressor and positive control of cAMP--CRP complex. These data suggest that the 2.87 kb PstI--SalGI-fragment contains the intact udp gene which is transcribed from its own promoter. Increase in the activity of beta-galactosidase encoded by udp-lacZ fusion has been observed in the presence of pUD2 or pUD7, which was suggested to be the consequence of titration of CytR repressor molecules in the operator region of the cloned udp.     

Insertions of the Transposon Tn1 into the PSEUDOMONAS AERUGINOSA Chromosome

The transposon Tn1 has been translocated to the chromosome of Pseudomonas aeruginosa from plasmid R18, following hydroxylamine mutagenesis of the plasmid. Twelve insertions were mapped to six distinct sites distal to 55 min of the origin of chromosome transfer by the plasmid FP2. These map locations were confirmed by host chromosome mobilization tests mediated by plasmids R18 or R91-5, due to Tn1 homology between plasmid and host chromosome. All the Tn1 chromosomal inserts were retransposable to other plasmids (Sa, R931 and R38). The behavior of Tn1 in P. aeruginosa was very similar to its behavior in Escherichia coli with respect to regional specificity, orientation of insertion and in serving as regions of homology for host chromosome mobilization by plasmids. This last property has permitted the demonstration that Tn1 on R18 and R91-5 is in opposite orientation with respect to the origin of transfer (oriT) of the two plasmids.     

New class of mutations in Escherichia coli (uup) that affect precise excision of insertion elements and bacteriophage Mu growth

We have used a papillation screening technique to isolate mutations that increase the precise excision of insertion elements. The three mutations isolated stimulated precise excision of Tn5, Tn10, and the IS elements. They had a large, 20- to 600-fold, effect on excision of Tn5 at various chromosomal sites. The varied stimulation for different Tn5 insertions showed that the mutations altered the relationship between a precise excision activity and the chromosomal sequence flanking an inserted Tn5. A much smaller stimulation was observed for insertions on the plasmid F'128. The stimulation was recA independent. The mutations also reduced the rate of production of bacteriophage Mu progeny. The mutations were mapped by two- and three-factor crosses with closely linked Tn10 insertions. They defined the uup locus, located at 21.3 min on the Escherichia coli map, next to pyrD.     

Transfer and properties of some natural and suicide replicons in Pasteurella multocida

We tested the transfer of several plasmids and transposons from Escherichia coli to Pasteurella multocida by filter mating. Two plasmids, pRKTV5 (pRK2013::Tn7) and pUW964 (pRKTV5::Tn5), were derived from pRK2013--a narrow-host-range plasmid with the broad-host-range IncP conjugation genes. Most P. multocida transconjugants obtained with pRKTV5 had Tn7 insertions in the chromosome but some had insertions of the whole plasmid. By contrast, all the transconjugants obtained with pUW964 had insertions of this plasmid or a deleted variant. pUW964 mediated low-frequency transfer of Tn7 or chromosomal markers between P. multocida strains. Broad-host-range IncP plasmid RP4 (RK2) did not yield selectable transconjugants in P. multocida but two plasmids derived by Tn5 insertion into a kanamycin-sensitive derivative of RP4 did yield transconjugants. pSUP1011, a narrow-host-range p15A replicon with the RP4 mob region allowing mobilization by the IncP conjugation genes also yielded transconjugants while several other plasmids tested did not transfer markers to P. multocida.     

Specificity of Tn9 insertion into the genome of bacteriophage lambda att80 depending on its preceding location

It was shown that the site of previous integration (the donor site) of Tn9 affects the specificity of its next integration into the target molecule--phage lambda att80 DNA. The transposon integration sites were mapped by restriction and heteroduplex analysis following Tn9 transposition from chromosomal sites of Escherichia coli K-12 differing in location and Tn9 stability. When transposed from chromosomal galT::IS1 gene, Tn9 inserted into the site with coordinates 44,5 +/- 2 kb of lambda att80; when transposed from chromosomal attTn9A site, the transposon inserted into the sites with coordinates 31 +/- 0,7 kb or 33,3 +/- 0,5 kb. In the course of transposition of Tn9 from chromosomal attTn9N site the transposon inserted into the lambda att80 site with coordinates 26,5 +/- 5 kb. In the latter case, the increase of Tn9 single-stranded loop and the appearance of two new HindIII cleavage sites were observed in heteroduplex experiments. The data were interpreted as indicating structural rearrangements of Tn9 or linked sequences in the course of transposition.     

Cloning and expression of the metE gene in Escherichia coli

A lambda-transducing phage was isolated that contains the metE gene. This gene codes for N5-methyl-H4-folate:homocysteine methyltransferase (EC 2.1.1.14), an enzyme that catalyzes the terminal reaction in methionine biosynthesis. A 9.1-kb EcoR1 fragment of this phage, containing the metE gene, was then cloned into pBR325. This plasmid, pJ19, was used to transform Escherichia coli strain 2276, a metE mutant, and restore the MetE+ phenotype. Although the transformed cells produced large amounts of the metE protein in vivo, in vitro studies using pJ19 as template showed low synthesis of the metE protein.     

Two-step method for curing Escherichia coli of ColE1-derived plasmids

To cure Escherichia coli for plasmids derived from the ColE1 replicon advantage is taken of the fact that maintenance of this replicon requires a wild-type allele of polA, encoding DNA polymerase I. Curing is achieved by cotransduction of a mutant polA allele with metE::Tn10, fadAB::Tn10 or other transposon insertions near polA. Reciprocal transduction to Met(+) Pol(+) or to Fad(+) Pol(+) ensures reestablishment of the original genotype except for loss of the plasmid. A set of useful bacterial strains is provided.     

Identification of regions of the Streptococcus faecalis plasmid pCF-10 that encode antibiotic resistance and pheromone response functions

The conjugative plasmid pCF-10 (58 kb) of Streptococcus faecalis has been mapped with restriction enzymes. By restriction mapping and Southern hybridization analysis, a 16-kb segment of the plasmid was shown to resemble closely the conjugative tetracycline resistance transposon, Tn916. Mutagenesis of the plasmid with the erythromycin resistance transposon Tn917 was used to localize a tetracycline resistance determinant and several regions involved in conjugal transfer. Fifty Tn917 insertions (outside the region of the plasmid homologous to Tn916) affecting mating behavior and the ability of donor cells to respond to the sex pheromone cCF-10 were mapped to nine distinct segments, or tra regions. Insertions into tra regions 1-3 and 7-9 led to an enhanced transfer ability of mutant plasmids relative to the transfer frequency obtained for the wild-type plasmid. Cells carrying these mutant plasmids differed in colony morphology or growth in broth culture from cells carrying pCF-10. Insertions into tra regions 4-6 resulted in reduced plasmid transfer, or completely eliminated the mating potential of donor cells. Insertions generating transfer-defective plasmids could be grouped further according to the ability of strains harboring the mutant plasmids to respond to cCF-10. HindIII fragments of pCF-10 coding for transfer functions have been cloned into Escherichia coli.