Tn1545: a conjugative shuttle transposon

Summary Tn1545, from Streptococcus pneumoniae BM4200, confers resistance to kanamycin (aphA-3), erythromycin (ermAM) and tetracycline (tetM). The 25.3 kb element is self-transferable to various Gram-positive bacterial genera where it transposes. Tn1545 was cloned in its entirety in the recombination deficient Escherichia coli HB101 where it was unstable. The three resistance genes aphA-3, ermAM and tetM were expressed but were not transferable to other E. coli cells. Tn1545 transposed from the hybrid plasmid to multiple sites of the chromosome of its new host. The element re-transposed, at a frequency of 5×10^-9, from the chromosome to various sites of a conjugative plasmid where it could be lost by apparently clean excision. The element transformed and transposed to the chromosome of Bacillus subtilis. The properties of the conjugative shuttle transposon Tn1545 may account for the recent emergence of genes from Gram-positive bacteria in Gramnegative organisms.     

The new class 11 transposon Tn163 is plasmid-borne in two unrelated Rhizobium leguminosarum biovar viciae strains

Tn163 is a transposable element identified in Rhizobium leguminosarum bv. viciae by its high insertion rate into positive selection vectors. The 4.6 kb element was found in only one further R. leguminosarum bv. viciae strain out of 70 strains investigated. Both unrelated R. leguminosarum bv. viciae strains contained one copy of the transposable element, which was localized in plasmids native to these strains. DNA sequence analysis revealed three large open reading frames (ORFs) and 38 bp terminal inverted repeats. ORF1 encodes a putative protein of 990 amino acids displaying strong homologies to transposases of class 11 transposons. ORF2, transcribed in the opposite direction, codes for a protein of 213 amino acids which is highly homologous to DNA invertases and resolvases of class II transposons. Homology of ORF1 and ORF2 and the genetic structure of the element indicate that Tn163 can be classified as a class II transposon. It is the first example of a native transposon in the genus Rhizobium. ORF3, which was found not to be involved in the transposition process, encodes a putative protein (256 amino acids) of unknown function. During transposition Tn163 produced direct repeats of 5 bp, which is typical for transposons of the Tn3 family. However, one out of the ten insertion sites sequenced showed a 6 by duplication of the target DNA; all duplicated sequences were A/T rich. Insertion of Tn163 into the sacB gene revealed two hot spots. Chromosomes of different R. leguminosarum bv. viciae strains were found to be highly refractory to the insertion of Tn163.     

Conjugal transfer of enterococcal transposons in Bacillus megaterium

The conjugative enterococcal transposons Tn916 and Tn919 were introduced into Bacillus megaterium by a filtermating technique. The transfer frequencies obtained ranged from 1.3×10^-6 to 6.6×10^-7. The transposons integrated stably into the B. megaterium chromosome. Tn916 could generate auxotrophs and was transferred from B. megaterium Tn916 transconjugants to other species.     

Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium

Summary A simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI-BglII fragment between the kanamycin resistance (Km^r) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Km^r transfer frequency of 10^-6 per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8x10⁶ cpm for a cell density of 10³ colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.     

Isolation of a Tn501 insertion mutant lacking porin protein P of Pseudomonas aeruginosa

Summary In order to demonstrate a role for anion-specific protein P channels in phosphate transport in Pseudomonas aeruginosa PAO, we wished to isolate a transposon insertion mutant deficient in protein P. A number of transposon delivery systems were tested which yielded, for the most part, whole plasmid inserts. Plasmid pMT1000 (Tsuda et al. 1984), a temperature-sensitive R68 plasmid carrying the transposon Tn501, was successfully employed in the isolation of a Tn501 insertion mutant lacking protein P under normally inducing conditions. To identify the mutant deficient in protein P, a protein P-specific polyclonal antiserum was used. This mutant, strain H576, was deficient in high-affinity phosphate transport exhibiting a Km for uptake (3.60±0.64 M) almost ten times greater than that of the wild type strain (Km=0.39 M). There was, however, no change in the V_max for high-affinity phosphate transport as a result of the loss of protein P in this mutant. The protein P-deficiency of the mutant correlated with a growth defect in a phosphate-limited medium resulting in an 18%–35% decrease in growth when compared with the wild type.     

Carbazole/dioxin-degrading car gene cluster is located on the chromosome of Pseudomonas stutzeri strain OM1 in a form different from the simple transposition of Tn4676

The carbazole-degrading (car) operon on the chromosome of Pseudomonas stutzeri strain OM1 showed >99% identity to that in the 72.8 kb catabolic transposon, Tn4676, on plasmid pCAR1. Southern hybridization using probes prepared from the pCAR1 sequence and sequencing analyses showed that the OM1 chromosome contained the 55 kb DNA region, almost all of which was a part of Tn4676, flanked by two copies of novel insertion sequence, ISPst3, and included the car gene.     

Chitinase-mediated destructive antagonistic potential of Pseudomonas aeruginosa GRC1 against Sclerotinia sclerotiorum causing stem rot of peanut

Pseudomonas aeruginosa GRC₁ exhibited strong antagonistic activity against Sclerotinia sclerotiorum, in vitro and in vivo. Scanning electron microscopic (SEM) studies showed morphological abnormalities such as perforation, lysis and fragmentation of hyphae of S. sclerotiorum caused by P. aeruginosa GRC₁. This strain produced extracellular chitinase enzyme, the role of which was clearly demonstrated through Tn5 mutagenesis. Bacterization of peanut seeds with GRC₁ resulted in increased seed germination and reduced stem-rot of peanut in S. sclerotiorum-infested soil by 97%. Other vegetative and yield plant parameters such as nodules per plant, pods and grain yield per plant were enhanced with a statistical significance in comparison to control. Neomycin resistant (GRC₁^neo+) bacterium was a good root colonizer and frequently isolated from rhizosphere of peanut plants. These findings showed P. aeruginosa GRC₁ as a potential biocontrol agent against S. sclerotiorum.     

The sequence and function of the recA gene and its protein in Pseudomonas aeruginosa PAO

Summary The recA gene of Pseudomonas aeruginosa has been isolated and its nucleotide sequence has been determined. The coding region of the recA gene has 1038 bp specifying 346 amino acids. The recA protein of P. aeruginosa showed a striking homology with that of Escherichia coli except for the carboxy-terminal region both at the nucleotide and amino acid level. The recA ⁺-carrying plasmids restored the UV sensitivity and recombination ability of several rec mutants of P. aeruginosa. The precise location of the recA gene on the chromosome was deduced from the analysis of R plasmids.     

IS21 insertion in the trfA replication control gene of chromosomally integrated plasmid RP1: a property of stable Pseudomonas aeruginosa Hfr strains

Summary Broad host range IncP-1 plasmids are able to integrate into the chromosome of gram-negative bacteria. Strains carrying an integrated plasmid can be obtained when the markers of a temperature-sensitive (ts) plasmid derivative are selected at non-permissive temperature; in this way Hfr (high frequency) donor strains can be formed. The integrated plasmids, however, tend to be unstable in the absence of continuous selective pressure. In order to obtain stable Hfr donor strains of Pseudomonas aeruginosa PAO, we constructed a derivative of an RP1 (ts) plasmid, pME134, which was defective in the resolvase gene (tnpR) of transposon Tn801. Chromosomal integration of pME134 was selected in a recombination-deficient (rec-102) PAO strain at 43°C. Plasmid integration occurred at different sites resulting in a useful set of Hfr strains that transferred chromosomal markers unidirectionally. The tnpR and rec-102 mutations prevented plasmid excision from the chromosome. In several (but not all) Hfr strains that grew well and retained the integrated plasmid at temperatures below 43°C, the insertion element IS21 of RP1 was found to be inserted into the trfA locus (specifying an essential trans-acting replication funtion) of the integrated plasmid. One such Hfr strain was rendered rec ⁺; from its chromosome the pME134::IS21 plasmid (=pME14) was excised and transferred by conjugation to Escherichia coli where pME14 could replicate autonomously only when a helper plasmid provided the trfA ⁺ function in trans. Thus, it appears that trfA inactivation favours the stability of chromosomally integrated RP1 in P. aeruginosa.     

Characterization of Tn10d-Cam: a transposition-defective Tn10 specifying chloramphenicol resistance

Summary We have constructed a small, transposition-defective derivative of the transposon Tn10 that carries the chloramphenicol acetyltransferase gene of pACYC184. This new genetic element, Tn10d-Cam, transposes when Tn10 transposase is provided from a multi-copy plasmid. Transposon insertion mutagenesis of Salmonella typhimurium was performed by using a strain carrying a Tn10d-Cam insertion in an Escherichia coli F' episome as the donor in transductional crosses into recipients that carried a plasmid expressing Tn10 transposase. Tn10d-Cam insertion mutations were also generated by complementation in cis of Tn10d-Cam by a cotransducible Tn10 element that overproduces transposase. Here, transposase was provided only transiently, and the Tn10d-Cam insertion mutations were recovered in a transposase-free strain. Cis complementation was used for mutagenesis of a plasmid target. The site specificity of insertion and the effect of insertions on expression of a downstream gene were investigated, using Tn10d-Cam insertions in a plasmid carrying a segment of the histidine operon.     

Characterization of the transposon carrying the STII gene of enterotoxigenic Escherichia coli

Summary The Escherichia coli enterotoxin STII gene is carried by Tn4521. The terminal repeats of Tn4521 are composed of IS2 sequences; however, neither repeat is a complete IS2. In order to determine how this seemingly defective transposon could transpose, mutations were generated within Tn4521 to determine the regions essential for transposition. The left terminal repeat region was found to be non-essential, but the right terminal repeat area was demonstrated to be crucial for transposition. Within the right terminal repeat area is an open reading frame (ORF), capable of encoding a 159 amino acid protein, which was shown by frameshift mutation analysis to be required for transposition. This protein may be the transposase of Tn4521. A pair of 11 bp repeat sequences flanking the ORF was also found to be important. The right 11 bp repeat is part of the left IS2 terminal sequence, and the left 11 bp repeat is located about 300 bp upstream from the right IS2 terminal sequence located within the right terminal repeat region. The results of this study suggest that Tn4521 is a functional transposon and that the sequence including this pair of 11 bp sequences plus the intervening sequence is a transposable element which may be responsible for Tn4521 transposition.     

Characterization of the gene for the Fe-protein of the vanadium dependent alternative nitrogenase of Azotobacter vinelandii and construction of a Tn5 mutant

Summary A sequence homologous to the conventional nifH gene has been cloned from a different region of the Azotobacter vinelandii genome. Tn5 insertions were obtained in this clone and the mutagenized plasmid was used for marker exchange with A. vinelandii strain CA12 (nifHDK) to obtain Tn5 mutants. These mutants exhibited a Nif^- phenotype in the presence of vanadium, unlike CA12 which was Nif⁺ on vanadium-containing medium. The gene in the cloned nifH-like region is therefore apparently involved in the vanadium dependent alternative pathway of nitrogen fixation. This gene, nifH2, has been sequenced and encodes a protein of 289 amino acids that is similar to nifH in nucleotide sequence, deduced amino acid sequence, predicted secondary structure and hydrophobicity profile. A second open reading frame downstream of nifH2 codes for a protein of 64 amino acids, similar to the ferredoxin (Fd)-like protein encoded downstream of nifH ^* in A. chroococcum. Sequence analysis suggests that the nifH2 and Fd-like genes are in a single operon.     

Evidence for stable transformation of Pseudomonas aeruginosa with a pUC-based plasmid

Pseudomonas aeruginosa was transformed with pUC8:16, a pUC-based plasmid bearing the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). Transformation was initially indicated by an increase in ampicillin resistance from 1500 to 2500 mg l^–1. Presence of the plasmid in P. aeruginosa was confirmed by amplification of a portion of vgb from and detection of VHb in the transformant but not the untransformed host. Southern blot analysis further indicated that pUC8:16 existed as an autonomous plasmid rather than integrated into the chromosome of the P. aeruginosa transformant.     

Purification and biochemical characterization of a native invertase from the hydrogen-producing Thermotoga neapolitana (DSM 4359)

This is the first report describing the purification and enzymatic properties of a native invertase (β-D-fructosidase) in Thermotogales. The invertase of the hydrogen-producing thermophilic bacterium Thermotoga neapolitana DSM 4359 (hereby named Tni) was a monomer of about 47 kDa having an amino acid sequence quite different from other invertases studied up to now. Its properties and substrates specificity let us classify this protein as a solute-binding protein with invertase activity. Tni was specific for the fructose moiety and the enzyme released fructose from sucrose and raffinose and the fructose polymer inulin was hydrolyzed in an endo-type fashion. Tni had an optimum temperature of 85°C at pH 6.0. At temperatures of 80–85°C, the enzyme retained at least 50% of its initial activity during a 6 h preincubation period. Tni had a K _m and k _cat /K _m values (at 85°C and pH 6.0) of about 14 mM and 5.2 × 10⁸ M⁻¹ s⁻¹, respectively. Dedicated to the memory of Prof. R. A. Nicolaus, founder of the Institute (1968).     

Transposition and transduction of plasmid DNA inStreptomyces spp.

Summary To expand the application of molecular genetics to many different streptomycete species, we have been developing two potentially widely applicable methodologies: transposon mutagenesis and plasmid transduction. We constructed three transposons from theStreptomyces lividans insertion sequence IS493. Tn5096 and Tn5097 contain an apramycin resistance gene inserted in different orientations between the two open reading frames of IS493. These transposons transpose from different plasmids into many different sites in theStreptomyces griseofuscus chromosome and into its resident linear plasmids. Tn5099 contains a promoterlessxylE gene and a hygromycin-resistance gene inserted in IS493 close to one end. Tn5099 transposes inS. griseofuscus giving operon fusions in some cases that drive expression of thexylE gene product, catechol deoxygenase, giving yellow colonies in the presence of catechol. We have also developed plasmid vectors that can be transduced into many streptomycete species by bacteriophage FP43. We describe the characterization of FP43 and mapping of several bacteriophage functions. The region of cloned FP43 DNA essential for plasmid transduction includes the origin for headful packaging.     

Identification of Escherichia coli genes whose expression increases as a function of external pH

Summary Using transposon TnphoA and a plate screening method, we have isolated a set of Escherichia coli strains carrying phoA fusions with genes whose expression is modulated as a function of external pH. Besides fusions with the ompF gene and the malB locus, thirteen independent fusions were analysed whose expression is maximal during growth at pHs ranging from 7.0 to 8.5 and minimal during growth at pH 5.0. Six different genetic loci, called phmA, phmB, phmC, phmD, phmE and phmF (for pH modulated) were characterized and localized on the E. coli chromosome at approx. 12, 18, 41, 45, 75 and 84 min, respectively. Expression of phmA: :phoA fusions is also influenced when internal pH or environmental conditions such as osmolarity or anaerobiosis are modified. EnvZ protein is not involved in the regulation of phm : :phoA fusions.     

Multiple copies of functional, Tet(M)-encoding Tn916-like elements in a clinical Enterococcus faecium isolate

Tn916 and similar elements are very common in clinical enterococcal isolates, and are responsible for transmission of a variety of resistance determinants. It is commonly assumed that clinical strains carrying Tn916 have a single copy, although the actual number of copies in clinical isolates has never been systematically studied. We report a clinical isolate of Enterococcus faecium in which three distinct and excision-proficient copies of Tn916-like elements are present in the genome. All of the elements contain tet(M) genes, at least one of which confers resistance to tetracycline and minocycline. Two elements (Tn6085a, Tn6085b) are indistinguishable, containing an inserted 2758 bp Group II intron at the start of open reading frame Tn916ORF_06. The third (Tn6084) also contains the intron, but also has an ISEfa11 integrated upstream of tet(M). All three copies are able to excise from plasmid vectors when cloned in E. coli, and at least two of the elements can transfer to an E. faecium recipient strain. These data indicate that nearly identical Tn916-like elements encoding Tet(M)-mediated tetracycline/minocycline resistance can coexist in clinical E. faecium isolates.     

Analysis of the Complete Nucleotide Sequence of the Tetracycline-Resistance Transposon Tn10

An analysis of the complete nucleotide sequence of the composite tetracycline-resistance transposon Tn10 (9147 bp) from the Salmonella typhi conjugative plasmid R27 is presented. A comparison of the protein sequences from IS10-right and IS10-left transposases has identified four amino acid differences. These residues appear to play an important role in normal transposase function and may account for the differences in exhibited transposition activities. The tetracycline determinants encoded by this version of Tn10 share >99% identity with those of Tn10^R100, demonstrating the conservation that exists between these transposons. A previously uncharacterized 3000-bp region of Tn10 contains four putative open reading frames. One of these open reading frames shares 55% identity with the glutamate permease protein sequence from Haemophilus influenzae although it was unable to complement an Escherichia coli glutamate permease mutant, with which it shares 51% identity. The three remaining putative open reading frames are arranged as a discrete genetic unit adjacent to the glutamate permease homolog and are transcribed in the opposite direction. Two of these open reading frames are homologous with Bacillus subtilis proteins of unknown functions while the other has no homologs in the database. The presence of an aminoacyl-tRNA synthetase class II motif in one of these open reading frames in combination with the glutamate permease homolog allows us to postulate that this region of Tn10 could once have played a role in amino acid metabolism.     

Transposable multiple antibiotic resistance in Streptococcus pneumoniae

Summary A mobile genetic element, designated Tn1545, was detected in the chromosome of Streptococcus pneumoniae BM4200, a clinical isolate multiply resistant to antibiotics. The 25.3 kb element conferred resistance to kanamycin and structurally related aminoglycosides by synthesis of a 3-aminoglycoside phosphotranferase type III (aphA-3), to macrolide-lincosamide-streptogramin B-type antibiotics (ermAM), and to tetracycline (tetM). Tn1545 was self-transferable to a recombination deficient S. faecalis strain where it was able to transpose to various sites, induce insertional mutations and was apparently cleanly excised. The element also conjugated to and transposed to the chromosome of S. faecalis, S. lactis, S. diacetylactis, S. cremoris, S. sanguis, Staphylococcus aureus, and Listeria monocytogenes. The properties of the conjugative transposon Tn1545 could account for the sudden emergence, rapid dissemination, and stabilisation of multiple resistance to antibiotics in S. pneumoniae in the absence of plasmids.