Tn2301, a transposon construct carrying the entire transfer region of the F plasmid.

The largest R . BamHI fragment of the plasmid F, which carries the entire F conjugation system, has been cloned into the single R . BamHI site of the ampicillin (Ap) resistance transposon TN1. pDS1106 (ColE1 mob::Tn1) was the vector plasmid, and the resultant conjugative plasmid, pED830, was characterized both genetically and by restriction enzyme analysis. The transposon construct, denoted Tn2301, was transposable at frequencies similar to Tn1 to small nonconjugative plasmids or to the Escherichia coli host chromosome. In the former case, Apr conjugative plasmids were obtained, whereas in the latter case, Hfr strains resulted. Representative Hfr strains were characterized by quantitative and interrupted mating experiments. Extension of this technique for Hfvr formation should aid chromosome mapping both in E. coli and in other bacterial genera.     

The linear plasmid SCP1 of Streptomyces coelicolor A3(2) possesses a centrally located replication origin and shows significant homology to the transposon Tn4811.

The linear plasmid SCP1 of Streptomyces coelicolor A3(2) is one of the genetically more studied linear streptomycete replicons. Although the genetics of SCP1 and its interaction with the host chromosome have been analyzed for nearly three decades no information exists on its replication. With the help of an ordered cosmid contig for the complete 360-kb element, we have localized a 5439-bp fragment from the central region that confers autonomous replication in Streptomyces lividans. The minimal origin contains two overlapping ORFs which are separated from an AT-rich region which might correspond to the replication start point. ORF1 revealed intensive similarity to a class of DNA-primase/helicases of actinophages and archael plasmids. In addition, we have identified a region in both terminal inverted repeats of SCP1 that shows significant homology to the transposable element Tn4811 located near the ends of the S. lividans 66 chromosome.     

Sequence and molecular characterization of a multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri.

A lambda gt11 expression library of Tn5-tagged invasion plasmid pWR110 (from Shigella flexneri serotype 5, strain M90T-W) contained a set of recombinants encoding a 60-kilodalton protein (designated IpaH) recognized by rabbit antisera raised against S. flexneri invasion plasmid antigens (J. M. Buysse, C. K. Stover, E. V. Oaks, M. M. Venkatesan, and D. J. Kopecko, J. Bacteriol. 169:2561-2569, 1987). Southern blot analysis of wild-type S. flexneri serotype 5 invasion plasmid DNA (pWR100) digested with various combinations of five restriction enzymes and hybridized with defined ipaH probes showed complex hybridization patterns resulting from multiple copies of the ipaH gene on pWR100. DNA sequence analysis of a 2.9-kilobase (kb) EcoRI fragment directing IpaH antigen synthesis in plasmid recombinant pWR390 revealed an open reading frame coding for a 532-amino-acid protein (60.8 kilodaltons); this size matched well with the estimated size of IpaH determined by Western blot analysis of M90T-W cells and maxicell analysis of Escherichia coli HB101(pWR390) transformants. Examination of the amino acid sequence of IpaH revealed a hydrophilic protein with six evenly spaced 14-residue (L-X2-L-P-X-L-P-X2-L-X2-L) repeat motifs in the amino-terminal end of the molecule. Southern blot analysis of HindIII-digested pWR100 DNA probed with defined segments of the pWR390 2.9-kb insert demonstrated that the multiple band hybridization pattern resulted from repeats of a significant portion of the ipaH structural gene in five distinct HindIII fragments (9.8, 7.8, 4.5, 2.5, and 1.4 kb). Affinity-purified IpaH antibody, used to monitor the expression of the antigen in M90T-W cells grown at 30 and 37 degrees C, showed that IpaH synthesis was not regulated by growth temperature.     

Identification of an Escherichia coli gene homologous to virR, a regulator of Shigella virulence.

Virulence in Shigella spp., as well as in strains of enteroinvasive Escherichia coli, is regulated by growth temperature. Previously, virR had been identified as the gene controlling the temperature-regulated expression of Shigella virulence. Since Shigella spp. and E. coli are also known to share greater than 90% DNA sequence homology, we sought to determine if nonpathogenic E. coli K-12 C600 contains a gene homologous to the Shigella flexneri 2a gene virR. Through the use of transduction and molecular cloning of strain C600 chromosomal DNA we have shown that E. coli K-12 does indeed contain a gene functionally homologous to the virR of S. flexneri.     

Adhesion and invasion of a mutant Shigella flexneri to an eukaryotic cell line in absence of the 220-kb virulence plasmid

A Shigella flexneri strain, cured of the large 220-kb virulence plasmid, expresses adhering and invading ability in confluent monolayers of HeLa cells similar to its parent strain. Invasion by both the parent and the cured strains resulted in alteration of the monomeric actin (G) in the total actin pool of HeLa cells. Other indicators of invasive characteristics of virulent Shigella strains such as production of keratoconjunctivitis in guinea pig eye in vivo, Congo red binding and expression of contact hemolysin however, indicated loss of invasive properties in the plasmid cured strain. Further, pretreatment of bacterial cells with para-bromophenacyl bromide (p-BPB), a specific chemical inhibitor of phospholipase A, adversely affected adhesion to and invasion of HeLa cells in vitro, irrespective of the presence of the 220-kb plasmid indicating the possible involvement of the enzyme phospholipase A in the invasion process. Adherence of both the strains to guinea pig colonic epithelial cells (CECs) in vitro was reduced significantly on pretreatment of bacteria or CECs with p-BPB. Expression of exocellular enzymes viz. protease, elastase, phospholipase A and phospholipase C were not related to the large plasmid.     

Genomic and functional analysis of the IncP-9 naphthalene-catabolic plasmid NAH7 and its transposon Tn4655 suggests catabolic gene spread by a tyrosine recombinase

The naphthalene-catabolic (nah) genes on the incompatibility group P-9 (IncP-9) self-transmissible plasmid NAH7 from Pseudomonas putida G7 are some of the most extensively characterized genetic determinants for bacterial aerobic catabolism of aromatic hydrocarbons. In contrast to the detailed studies of its catabolic cascade and enzymatic functions, the biological characteristics of plasmid NAH7 have remained unclear. Our sequence determination in this study together with the previously deposited sequences revealed the entire structure of NAH7 (82,232 bp). Comparison of NAH7 with two other completely sequenced IncP-9 catabolic plasmids, pDTG1 and pWW0, revealed that the three plasmids share very high nucleotide similarities in a 39-kb region encoding the basic plasmid functions (the IncP-9 backbone). The backbone of NAH7 is phylogenetically more related to that of pDTG1 than that of pWW0. These three plasmids carry their catabolic gene clusters at different positions on the IncP-9 backbone. All of the NAH7-specified nah genes are located on a class II transposon, Tn4655. Our analysis of the Tn4655-encoded site-specific recombination system revealed that (i) a novel tyrosine recombinase, TnpI, catalyzed both the intra- and intermolecular recombination between two copies of the attI site, (ii) the functional attI site was located within a 119-bp segment, and (iii) the site-specific strand exchange occurred within a 30-bp segment in the 41-bp CORE site. Our results and the sequence data of other naphthalene-catabolic plasmids, pDTG1 and pND6-1, suggest a potential role of the TnpI-attI recombination system in the establishment of these catabolic plasmids.     

The identification of a tetracycline resistance gene tet(M), on a Tn916-like transposon,in the Bacillus cereus group

In order to investigate whether resistance genes present in bacteria in manure could transfer to indigenous soil bacteria, resistant isolates belonging to the Bacillus cereus group (Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis) were isolated from farm soil (72 isolates) and manure (12 isolates) samples. These isolates were screened for tetracycline resistance genes (tet(K), tet(L), tet(M), tet(O), tet(S) and tet(T)). Of 88 isolates examined, three (3.4%) isolates carried both tet(M) and tet(L) genes, while four (4.5%) isolates carried the tet(L) gene. Eighty-one (92.1%) isolates did not contain any of the tested genes. All tet(M) positive isolates carried transposon Tn916 and could transfer this mobile DNA element to other Gram-positive bacteria.     

The cloning and transposon Tn5 mutagenesis of the glnA region of Klebsiella pneumoniae: identification of glnR, a gene involved in the regulation of the nif and hut operons

Summary A 15 kilobase HindIII fragment of Klebsiella pneumoniae DNA containing the glnA gene was cloned into the plasmid vector pACYC184. The resulting plasmid, pFB51, complements glnA ^- mutations in Escherichia coli and K. pneumoniae. pFB51 also complements the GlnR phenotype of a Klebsiella pneumoniae gln regulatory mutant (KP5060) defined by the restoration of Hut⁺ and Nif⁺ (histidine utilization and nitrogen fixation) phenotypes to this strain. Three recombinant plasmids containing subsegments of the 15 kb HindIII fragment were derived from pFB51. Plasmid pFB514 which contains a spontaneous 4 kb delection of K. pneumoniae DNA from pFB51 is more stable than pFB51 and is still able to complement glnA ^- mutations and the GlnR^- phenotype of KP5060. Plasmids pFB53 and pFB54, which contain a 6.5 kb SalI DNA fragment from pFB51 recloned into pACYC184 in opposite orientations, complement glnA ^- mutations but not the GlnR^- phenotype of KP5060. Plasmids pFB514 and pFB53 were mutagenized by transposon Tn5 resulting in a total of 92 Tn5 insertions in the cloned fragments. Utilizing these insertion mutations, a correlated physical and genetic map was constructed by determining the physical location of each Tn5 insertion and by analyzing the ability of each Tn5 mutated plasmid to complement a glnA ^- strain of E. coli and a glnA ^- GlnR^- strain of K. pneumoniae. Two classes of Tn5 insertions with an altered Gln phenotpye were obtained. One cluster of insertions spanning a 1.3 kb region abolished complementation of the glnA ^- mutations. A second 2 kb cluster of Tn5 insertions, immediately adjacent to the first cluster, abolished the ability of pFB514 plasmid to complement the GlnR^- phenotype, while glnA ^- complementation was unaffected. We propose that the second cluster of Tn5 insertions define a DNA region coding for a positive regulatory factor for nitrogen fixation (nif) and histidine utilization (hut) genes (glnR).     

Construction and characterization of Tn5virB, a transposon that generates nonpolar mutations, and its use to define virB8 as an essential virulence gene in Agrobacterium tumefaciens.

Transfer of the T-DNA from the Ti plasmid of Agrobacterium tumefaciens into plant cells involves movement of a single-stranded DNA-protein intermediate across several membrane and cell wall barriers. The 11 VirB proteins encoded by the Ti plasmid are hypothesized to form at least part of a membrane-localized T-DNA transport apparatus. Although available genetic and biochemical analyses support this hypothesis, detailed study of this transport apparatus is hindered by the fact that most available mutations in the virB operon are in the form of transposon insertions that have polar effects. In this study we constructed a transposon, Tn5virB, that can be used to generate nonpolar insertions in operons of A. tumefaciens and used it to demonstrate that virB8 is an essential virulence gene.