Isolation of F' plasmids carrying a portion of the Salmonella typhimurium histidine transport operon.

The transposable drug resistance element Tn10 was employed as a region of homology to direct the insertion of Tn10-containing derivatives of F'ts114 lac into the chromosome of a Salmonella typhimurium strain that carries a Tn10 insertion in the histidine transport operon. Based on the direction of transfer of the resulting Hfr strains, the chromosomal Tn10 insertion was determined to be in orientation "A." New F' plasmids were selectively generated from one of the Hfr strains. The F' factors carry an intact dhuA hisJ portion of the histidine transport operon. A Southern hybridization revealed that one of the F' plasmids was formed by a type II excision event.     

Transposon mutagenesis analysis of meta-cleavage pathway operon genes of the TOL plasmid of Pseudomonas putida mt-2.

Hybrid plasmids containing the regulated meta-cleavage pathway operon of TOL plasmid pWWO were mutagenized with transposon Tn1000 or Tn5. The resulting insertion mutant plasmids were examined for their ability to express eight of the catabolic enzymes in Escherichia coli. The physical locations of the insertions in each of 28 Tn1000 and 5 Tn5 derivative plasmids were determined by restriction endonuclease cleavage analysis. This information permitted the construction of a precise physical and genetic map of the meta-cleavage pathway operon. The gene order xylD (toluate dioxygenase), L (dihydroxycyclohexidiene carboxylate dehydrogenase), E (catechol 2,3-dioxygenase), G (hydroxymuconic semialdehyde dehydrogenase), F (hydroxymuconic semialdehyde hydrolase), J (2-oxopent-4-enoate hydratase), I (4-oxalocrotonate decarboxylase), and H (4-oxalocrotonate tautomerase) was established, and gene sizes were estimated. Tn1000 insertions within catabolic genes exerted polar effects on distal structural genes of the operon, but not on an adjacent regulatory gene xylS.     

The transposable element Tn3 promotes general recombination at the neighboring regions

Transposon Tn3 was inserted into a tRNA operon of the amber suppressor Su+2 on a transducing phage (lambda hcI857nin5pSu+2) by selecting phages with ampicillin resistance and Su- phenotypes. In a strain thus obtained, Tn3 was inserted between the promoter and the first tRNA gene of the operon, which was determined by DNA sequencing. The Su+2 tRNA operon on the transducing phage consisted of two tRNA genes for tRNA(Met) and Su+2 tRNA(2Gln), which was a deletion derivative of the supB-E tRNA operon of E. coli containing seven tRNA genes in the order of promoter-Met-Leu-Gln1-Gln1-Met-Gln2-Gln2. Proliferating the lambda hcI857nin5pSu+2::Tn3 in E. coli cells, a number of phages which had lost Tn3 were isolated, and their tRNA gene compositions as well as the DNA structures of the tRNA operon were analyzed. In many cases the tRNA genes which had been deleted from the original transducing phage were regained from the chromosomal supB-E operon. Thus the loss of Tn3 from the phages was not due to excision of the transposon but due to the replacement of a portion of the tRNA operon, including Tn3, with the host homologous region that did not contain Tn3. This type of replacement takes place rather efficiently as a consequence of Tn3 insertion, owing to the general recombination occurring between homologous tRNA genes of phage and host chromosomes in the presence of either host recA or phage red. No such enhanced recombination in a similar cross between phage and host chromosomes was observed with the Tn3 present in the trans position on an independent plasmid. We conclude that inserting Tn3 in cis promotes general recombination in the neighboring regions. Possible mechanisms for this new type of genetic effect of Tn3 are discussed. During the course of this study, a natural defective mutation (T11) was also detected in one of the duplicated tRNA(2Gln) genes in an E. coli K12 strain we used.     

Variation in the genome of CTXphi prophage of Vibrio cholerae biovar El Tor caused by Tn5-Mob transposon

A key pathogenicity factor of the cholera etiologic agent is cholera toxin (CT) whose synthesis is encoded by the ctxAB operon forming apart of the CTXphi ptophage. Alterations in the virulent properties of the cholera vibrios are based on the variability of the CTXphi prophage containing the genes for ctxAB, zot, ace, cep, orfU, and psh in its core region. At the same time, the mechanism of the porophage genome reorganization needs further and more profound analysis. The goal of this work was to demonstrate that transposon Tn5-Mob (Kmr), when introduced into the chromosome of the V. cholera model strain MAK757 El Tor biovar containing two copies of the CTXphi prophage provoked a reorganization in the CTXphi prophage consisting in the deletion of zot, ace, cep, orfU genes. The level of the CT biosynthesis in the insertion mutants MAK757 chr::Tn5-Mob still retaining only the ctxAB operon, increased more than 2000 times as compared to that of the original strain. The enhanced CT production was shown to be associated with the altered structure of the chromosomal DNA region containing one copy of the ctxAB operon encoding this protein biosynthesis. The mutation in the CTXphi genome induced by Tn5-Mob was unstable. Among 600 isolated colonies obtained after dissemination of the MAK757 chr::Tn5-Mob transposant capable of CT overproduction in the full medium with no antibiotics, 5.8% gave clones that in parallel to the loss of Kmr marker, appeared to be deprived of the ctxAB operon thus becoming non-toxinogenic. The observed formation of the V. cholerae insertion mutants both capable of CT overproduction and non-toxinogenic ones, may be indicative of an important role played in the evolution of the cholera pathogen by the CTXphi genome variability induced by Tn elements. The plasmidless V. cholerae El Tor strain characterized by type II CT hyperproduction thus obtained in our experiments could be used for the production of this protein routinely applied to construct efficient cholera diagnostic and prophylactic preparations.     

Polarity of Tn5 insertion mutations in Escherichia coli.

We assessed the effect of insertions of the kanamycin resistance transposon Tn5 in the lac operon of Escherichia coli on the expression of distal genes lacY and lacA (melibiose fermentation at 41 degrees C and thiogalactoside transacetylase synthesis, respectively). Every insertion mutation tested (41 in lacZ and 23 in lacY) was strongly polar. However, approximately one-third of the insertion mutants expressed distal genes at low levels due to a promoter associated with Tn5. To localize this promoter, we (i) reversed the orientation of Tn5 at several sites and (ii) replaced wild-type Tn5 with several substitution derivatives which lack Tn5's central region. Neither alteration changed the expression of distal genes. Thus, in contrast to transposons IS2 and TnA. Tn5's ability to turn on distal gene expression is not due to a promoter in its central region and therefore is not dependent on the overall orientation of Tn5 in the operon. Our results suggest that the promoter is within 186 base pairs of the ends of Tn5. It is possible that the promoter is detected in only a fraction of insertions because it overlaps Tn5-target sequence boundary.     

Transposon Tn21, Flagship of the Floating Genome

The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.     

Transposon Tn21, flagship of the floating genome.

The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.     

Mutations of plasmid pBS286 blocking the initial stages of naphthalene oxidation induced by Tn5

A collection of Tn5 transposon Nah- mutants of the plasmid pBS286 was obtained. The insertion sites were localized and orientation of Tn5 determined. The mutants obtained were biochemically analyzed, the nah-region map of the plasmid being elaborated. Structural genes of the nah operon were shown to be organized similarly to those of the nah1 operon of the NAH7 plasmid discussed in the literature. The data obtained are in favour of the previously published information on the presence of elements operating the pBS286 plasmid. The results are given indicating a possibility of regulating the expression of catechol splitting meta-pathway genes with participation of products on early stages of naphthalene oxidation.     

Glycerol facilitator of Escherichia coli: cloning of glpF and identification of the glpF product.

The glycerol facilitator is known as the only example of a transport protein that catalyzes facilitated diffusion across the Escherichia coli inner membrane. Here we show that the gene encoding the facilitator, glpF, is the first gene in an operon with glpK, encoding glycerol kinase, at 88 min of the E. coli chromosome. The operon is transcribed counterclockwise. We cloned the glpF gene, demonstrated that it complemented a chromosomal glycerol transport-minus mutation, and identified the gene product. The GlpF protein appeared in the membrane fraction of plasmid-bearing strains and had an apparent Mr of 25,000.     

Evidence for a common mechanism for the insertion of the Tn10 transposon and for the generation of Tn10-stimulated deletions

Summary Mutations in and near the Salmonella typhimurium histidine transport operon were generated by insertion of the translocatable tetracycline-resistance element Tn10. Deletion mutants affecting histidine transport genes were subsequently isolated in several of the Tn10-containing strains. Tn10 insertions in hisJ occurred preferentially at one site, designated site A. This same site was also the preferential endpoint of deletions originating from Tn10 insertions at two neighboring sites. Thus, Tn10 insertion and Tn10-stimulated deletion formation appear to involve a common DNA-recogition step.     

Mechanism of F factor-enhanced excision of transposon Tn5

The reversion of lac:: Tn5 insertion mutations was used to examine the control of excision of the kanamycin-resistance transposon Tn5 in Escherichia coli. Earlier work which showed that the fertility factor F enhances Tn5 excision had led another group to suggest that this is due to the product of a putative transposable element-specific "recombination" gene in the F factor which can act on Tn5 located anywhere in the genome. We show, however, that Tn5 is excised from sites in the lac operon of F'lac plasmids several orders of magnitude more efficiently than from the same sites in the chromosomes of F-, F+ or homozygous lac:: Tn5[F'lac:: Tn5] strains. Thus F enhances Tn5 excision, but only if F and Tn5 are in cis in the same DNA molecule. Bacterial crosses showed that transfer of F'lac:: Tn5 plasmids by conjugation stimulates Tn5 excision, and that transfer is frequent even within F' populations. These results suggest that the ability of F to enhance excision is the consequence of DNA transfer in conjugation.     

Importance of Facilitated Diffusion for Effective Utilization of Glycerol by Escherichia coli

Wild-type Escherichia coli possesses an inducible permeation system which catalyzes facilitated diffusion of glycerol into the cell. A spectrophotometric method can be used to assess the presence of this mechanism. The structural gene for the facilitator (glpF) and the structural gene for glycerol kinase (glpK) apparently belong to a single operon. The glpF(+) allele permits effective glycerol utilization by the cells, and, at millimolar concentrations of glycerol, cells carrying the glpF(+) allele grow much faster than glpF genotypes. Although the glycerol-scavenging power of the cell depends both on the facilitated entry of the substrate and its subsequent trapping by an adenosine triphosphate-dependent phosphorylation, the two gene products, the facilitator and kinase, function independently. Wild-type Shigella flexneri appears to be glpK(+) but glpF. This organism grows slowly in media at low concentrations of glycerol. When the glpF(+) and glpK(+) alleles of E. coli are inserted into the S. flexneri genome by transduction, the hybrid strain grows rapidly in low glycerol medium. Vice versa, when the glpF and glpK(+) alleles of S. flexneri are incorporated into E. coli, the hybrid strain grows slowly in low glycerol medium.     

Surveying a supercoil domain by using the gamma delta resolution system in Salmonella typhimurium.

A genetic system was developed to investigate the supercoil structure of bacterial chromosomes. New res-carrying transposons were derived from MudI1734 (MudJr1 and MudJr2) and Tn10 (Tn10dGn). The MudJr1 and MudJr2 elements each have a res site in opposite orientation so that when paired with a Tn10dGn element in the same chromosome, one MudJr res site will be ordered as a direct repeat. Deletion formation was studied in a nonessential region (approximately 100 kb) that extends from the his operon through the cob operon. Strains with a MudJr insertion in the cobT gene at the 5' end of the cob operon plus a Tn10dGn insertion positioned either clockwise or counterclockwise from cobT were exposed to a burst of RES protein. Following a pulse of resolvase expression, deletion formation was monitored by scoring the loss of the Lac+ phenotype or by loss of tetracycline resistance. In exponentially growing populations, deletion products appeared quickly in some cells (in 10 min) but also occurred more than an hour after RES induction. The frequency of deletion (y) diminished with increasing distance (x) between res sites. Results from 15 deletion intervals fit the exponential equation y = 120 . 10(-0.02x). We found that res sites can be plectonemically interwound over long distances ( > 100 kb) and that barriers to supercoil diffusion are placed stochastically within the 43- to 45-min region of the chromosome.     

Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway

Exoenzyme S is an extracellular ADP-ribosyltransferase of Pseudomonas aeruginosa . Transposon mutagenesis of P. aeruginosa 388 was used to identify genes required for exoenzyme S production. Five Tn 5  Tc insertion mutants were isolated which exhibited an exoenzyme S-deficient phenotype (388::Tn 5  Tc 469, 550, 3453, 4885, and 5590). Mapping experiments demonstrated that 388::Tn 5  Tc 3453, 4885, and 5590 possessed insertions within a 5.0 kb Eco RI fragment that is not contiguous with the exoenzyme S trans -regulatory operon. 388::Tn 5  Tc 469 and 550 mapped to a region downstream of the trans -regulatory operon which has been previously shown to contain a promoter region that is co-ordinately regulated with exoenzyme S synthesis. Nucleotide sequence analysis of a 7.2 kb region flanking the 388::Tn 5  Tc 469 and 550 insertions, identified 12 contiguous open reading frames (ORFs). Database searches indicated that the first ORF, ExsD, is unique. The other 11 ORFs demonstrated high homology to the YscB–L proteins of the yersiniae Yop type III export apparatus. RNase-protection analysis of wild-type and mutant strains indicated that exsD and pscB–L form an operon. To determine whether ExoS was exported by a type III mechanism, derivatives consisting of internal deletions or lacking amino- or carboxy-terminal residues were expressed in P. aeruginosa . Deletion analyses indicated that the amino-terminal nine residues are required for ExoS export. Combined data from mutagenesis, regulatory, expression, and sequence analyses provide strong evidence that P. aeruginosa possesses a type III secretion apparatus which is required for the export of exoenzyme S and potentially other co-ordinately regulated proteins.