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.     

Tn2001, a transposon encoding chloramphenicol resistance in Pseudomonas aeruginosa.

We isolated a new transposon, Tn2001, from the group P-2 plasmid Rms159-1 in Pseudomonas aeruginosa. Tn2001-encoded chloramphenicol resistance did not result from the formation of chloramphenicol acetyltransferase. Tn2001 was transposable between temperate phages and conjugative and nonconjugative plasmids belonging to various incompatibility groups, including P-1, P-3, P-4, P-5, P-7, and P-8 in P. aeruginosa. Transposition occurred independently of the general recombination ability of the Pseudomonas host, and its frequency varied between 10(-1) and 10(-8), depending upon the donor and recipient replicons. Tn2001 transposition also occurred in a recombination-deficient strain of Escherichia coli. Agarose gel electrophoresis and electron microscopic observations revealed that Tn2001 could transpose to different sites in the RP4 replicon and that the transposed deoxyribonucleic acid fragment was 2.1 kilobases long.     

Tn5-rpsL: a new derivative of transposon Tn5 useful in plasmid curing

The rpsL gene of Escherichia coli was inserted into the BamHI site of transposon Tn5. This transposon was called Tn5-rpsL. Tn5-rpsL may be useful in microbiological studies when one wants to cure various bacterial genera of certain plasmid(s). A streptomycin-resistant (SmR) derivative of the host bacterial strain is first isolated. The plasmid(s) later to be cured are then labelled with Tn5-rpsL, which makes the cells Sm-sensitive. These cells can regain their resistance to Sm if they lose the Tn5-rpsL-tagged plasmid. Thus, plasmid-free bacteria are easily selected among SmR survivors. The frequency of occurrence of the plasmid-less variants of plasmid-containing wild-type Salmonella typhimurium measured by this method is given as an example.     

Pseudo-transposition of a Tn5 derivative in Neisseria gonorrhoeae

Abstract We constructed a Tn5 derivative for potential use in transposon mutagenesis of Neisseria gonorrhoeae . It was incorporated into the chromosome apparently at random following transformation, but the insertion events were dependent on a functional RecA and independent of a functional transposase. Furthermore, in most cases there was an incomplete transposon inserted with little or no IS50 insertion sequence. These observations suggest that TnJ transposition may not be possible in N. gonorrhoeae and that this organism may have an unexplored illegitimate recombination system.     

Cloning and characterization of the dxs gene, encoding 1-deoxy-d-xylulose 5-phosphate synthase from Agrobacterium tumefaciens, and its overexpression in Agrobacterium tumefaciens

A newly isolated gene dxs11 from Agrobacterium tumefaciens (KCCM 10413), an organism with potential for the industrial production of ubiquinone-10 (UbiQ(10)), encoding a 1-deoxy-d-xylulose 5-phosphate synthase (Dxs), was cloned in Escherichia coli and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1920bp, capable of encoding a polypeptide of 640 amino acids residues with a calculated isoelectric point of pH 5.63 and a molecular mass of 68,054Da. The homodimeric enzyme was overexpressed in E. coli and purified as an active soluble form. The enzyme required thiamine diphosphate and a divalent metal ion, either Mg(2+) or Mn(2+), for enzymatic activity. The enzyme had an optimal pH and temperature of 8.0 and 37 degrees C, respectively, with a k(cat) of 26.8s(-1) and a k(cat)/K(m) of 0.67 and 1.17s(-1)M(-1) for pyruvate and d-glyceraldehyde 3-phosphate, respectively. A. tumefaciens Dxs showed a comparable catalytic efficiency to other Dxs proteins. The dxs11 gene was transformed into A. tumefaciens KCCM 10413, and the resulting recombinant, A. tumefaciens pGX11, showed higher UbiQ(10) production (502.4mg/l) and content (8.3mg/gDCW) than A. tumefaciens KCCM 10413, by 21.9 and 23.9%, respectively. This work describes Dxs from A. tumefaciens, an organism with the potential for industrial UbiQ(10) production, and the first metabolic engineering study with the non-mevalonate pathway enzyme in A. tumefaciens.     

Construction of a Tn5 derivative determining resistance to gentamicin and spectinomycin using a fragment cloned from R1033

A physical and genetic map of the IncP plasmid R1033 was constructed: restriction fragments were subcloned and antibiotic resistance genes were located. The map is consistent with previous reports that R1033 is a derivative of RP4 carrying a 16-kb transposon Tn1696 which contains the antibiotic-resistance determinants present on R1033 but not on RP4. A BamHI fragment from R1033, determining resistance to gentamicin, spectinomycin and streptomycin, was cloned into Tn5, replacing the central Bg/II fragment that determined kanamycin resistance, producing a recombinant transposon Tn5-GmSpSm. This was shown to transpose in Rhizobium leguminosarum at a frequency similar to that of the parental Tn5.     

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.     

Nucleotide sequence of a gene from the Pseudomonas transposon Tn501 encoding mercuric reductase

We have determined the nucleotide sequence of the merA gene from the mercury-resistance transposon Tn501 and have predicted the structure of the gene product, mercuric reductase. The DNA sequence predicts a polypeptide of Mr 58 660, the primary structure of which shows strong homologies to glutathione reductase and lipoamide dehydrogenase, but mercuric reductase contains as additional N-terminal region that may form a separate domain. The implications of these comparisons for the tertiary structure and mechanism of mercuric reductase are discussed. The DNA sequence presented here has an overall G+C content of 65.1 mol%, typical of the bulk DNA of Pseudomonas aeruginosa from which Tn501 was originally isolated. Analysis of the codon usage in the merA gene shows that codons with C or G at the third position are preferentially utilized.     

Alteration of surface properties in a Tn5 mutant strain of Rhizobium trifolii 0403.

A symbiotically defective mutant strain of Rhizobium trifolii, UR251, was obtained by transposon Tn5 mutagenesis of R. trifolii 0403 rif and recognized by its partially ineffective (Fix +/-) phenotype on white clover plants. UR251 had a single Tn5 insertion in plasmid DNA, a wild-type plasmid pattern, and no detectable Mu DNA sequences originally present in the vector used for Tn5 mutagenesis. Agglutination by the clover lectin trifoliin A and attachment to clover root hairs was higher with UR251 than with the wild-type strain. The capsular polysaccharide (CPS) of UR251 was altered, as shown by a slower rate of CPS depolymerization with a CPS beta-lyase, PD-I; more pyruvate and less acetate and 3-hydroxybutanoate noncarbohydrate substitutions as quantitated by 1H nuclear magnetic resonance; and a higher pyruvyl transferase activity (enzymatic pyruvylation of lipid-bound saccharides). The site of increased pyruvylation in the CPS of UR251 was on the terminal galactose of the branch of the repeating oligosaccharide unit. These results show that the level of noncarbohydrate substitutions of the CPS as well as pyruvyl transferase activity are altered in R. trifolii UR251 and that trifoliin A-binding ability and clover root hair attachment are improved in this mutant strain of R. trifolii 0403 rif.     

Transposon Tn5-induced mutagenesis of Rhizobium japonicum yielding a wide variety of mutants

When the "suicide" vector pSUP1011, which carries transposon Tn5 (Kmr), was introduced into Rhizobium japonicum USDA 110, kanamycin-resistant (Kmr) colonies were detected at a frequency (4.2 X 10-6) ca. 30 times greater than the spontaneous kanamycin resistance frequency (1.4 X 10-7). Ten thousand Kmr mutants were isolated and tested for nutritional auxotrophy. Auxotrophs were detected at a frequency of 0.5%. The following classes of auxotrophs were identified: adenine- (three), histidine- (three), glutamate- (five), adenine plus thiamine- (nine), uracil- (three), pantothenic acid- (one), tryptophan- (three), and methionine- (three). Mutants blocked in symbiotic nitrogen fixation (Fix-) were also identified at a frequency of 3%. The glutamate auxotrophs were studied in more detail, and all five showed an altered expression of nitrogenase activity in free-living cultures.     

高温胁迫对根瘤菌Tn5在土壤中的存活及其表型表达的影响

研究了３株弗氏中华根瘤菌（Ｒｈｉｚｏｂｉｕｍｆｒｅｄｉｉ）Ｔｎｓ突变株于适宜温度和高温胁迫两种条件下在土壤中的存活和Ｔｎｓ表型的表达．在适宜温度（２８℃）条件下的灭菌和未灭菌土壤中的存活研究表明生物因素抑制了突变株和野生型的生长．但野生型和突变株的存活种群密度之间无显著差异（Ｐ＝０．０１）．在高温胁迫（４０℃）条件下,土壤中野生型和突变株的种群密度迅速下降,其中部分ＯＮ－２和ＯＮ－３细胞丢失了Ｔｎｓ表型,说明部分细菌的Ｔｎ５表型在高温胁迫条件下不能表达．     

Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster

For insertional mutagenesis of Pseudomonas aeruginosa, a derivative of the kanamycin-resistance (KmR) transposon Tn5 was constructed (Tn5-751) that carried the trimethoprim-resistance (TpR) determinant from plasmid R751 as an additional marker. Double selection for KmR and TpR avoided the isolation of spontaneous aminoglycoside-resistant mutants which occur at high frequencies in P. aeruginosa. As a delivery system for the recombinant transposon, plasmid pME305, a derivative of the broad-host-range plasma RP1, proved effective; pME305 is temperature-sensitive at 43 degrees C for maintenance in Escherichia coli and P. aeruginosa and deleted for IS21 and the KmR and primase genes. In matings with an E. coli donor carrying pME9(= pME305::Tn5-751), transposon insertion mutants of P. aeruginosa PAO were recovered at approx. 5 X 10(-7)/donor at 43 degrees C. Among Tn5-751 insertional mutants 0.9% were auxotrophs. A thr::Tn5-751 mutation near the recA-like locus rec-102 is useful for the construction of recombination-deficient strains. Several arc::Tn5-751 mutants could be isolated that were defective in anaerobic utilization of arginine as an energy source. From three of these mutants the arc gene region was cloned into an E. coli vector plasmid. Since Tn5-751 has a single EcoRI site between the TpR and KmR genes, EcoRI-generated fragments carrying either resistance determinant plus adjacent chromosomal DNA could be selected separately in E. coli. Thus, a restriction map of the arc region was constructed and verified by hybridization experiments. The arc genes were tightly clustered, confirming earlier genetic evidence.     

Genetic manipulations in Rhizobium meliloti utilizing two new transposon Tn5 derivatives

Summary Two derivatives of the prokaryotic transposon Tn5 were constructed in vitro. In Tn5-233, the central area of Tn5, which carries resistance to kanamycin/neomycin, bleomycin and streptomycin, is replaced by a fragment carrying resistance to the aminocyclitol antibiotics gentamycin/kanamycin and streptomycin/spectinomycin. In Tn5-235, the Escherichia coli -galactosidase gene is inserted within the streptomycin resistance gene of Tn5, and constitutively expressed from a Tn5 promoter. Both constructs transpose with about the same frequency as Tn5 in Escherichia coli and Rhizobium meliloti. When a Tn5-derivative is introduced into an R. meliloti strain which already contains a different Tn5-derivative, in situ transposon replacement is obtained at high frequency, presumably by a pair of crossovers between the IS50 sequences at the ends of the incoming and resident transposons. In this way we converted a previously isolated recA::Tn5 mutant into the corresponding recA::Tn5-233 strain, which can now be used as a genetic background in the study of complementation of other Tn5-induced mutations. We also replaced the drug markers of several Tn5-induced exo mutants, which we were then able to map relative to each other by transduction with phage M12. In a strain carrying Tn5-235 located near Tn5-233, we were able to isolate deletions of the intervening markers, presumably resulting from general recombination between the two transposons, by screening for loss of the Lac⁺ phenotype. Unlike Tn5 itself, resident Tn5-233 does not appear to suppress transposition of another incoming Tn5-derivative.Abbreviations bp base pairs - Nm neomycin - Km kanamycin - Sm streptomycin - Sp spectinomycin - Gm gentamycin - Tc tetracycline - Tp trimethoprim - Ot oxytetracycline - Rf rifampicin - Xgal 5-bromo-4-chloro-3-indolyl--d-galactoside     

Isolation and characterization of transposon Tn5-induced symbiotic mutants of Rhizobium loti

Rhizobium loti NZP2037 and NZP2213, each cured of its single large indigenous plasmid, formed effective nodules on Lotus spp., suggesting that the symbiotic genes are carried on the chromosome of these strains. By using pSUP1011 as a vector for introducing transposon Tn5 into R. loti NZP2037, symbiotic mutants blocked in hair curling (Hac), nodule initiation (Noi), bacterial release (Bar), and nitrogen fixation (Nif/Cof) on Lotus pedunculatus were isolated. Cosmids complementing the Hac, Noi, and Bar mutants were isolated from a pLAFR1 gene library of NZP2037 DNA by in planta complementation and found to contain EcoRI fragments of identical sizes to those into which Tn5 had inserted in the mutants. The cosmids that complemented the mutants of these phenotypic classes did not share common fragments, nor did cosmids that complemented four mutants within the Noi class, suggesting that these symbiotically important regions are not tightly linked on the R. loti chromosome.     

Tn5563, a transposon encoding putative mercuric ion transport proteins located on plasmid pRA2 of Pseudomonas alcaligenes

Sequence analysis of pRA2, an endogenous 33-kb plasmid from Pseudomonas alcaligenes NCIB 9867 (strain P25X), revealed the presence of a 6256-bp transposon of the Tn3 family, designated Tn5563. Tn5563, which is flanked by two 39-bp inverted repeats, encodes a transposase, a resolvase, and two open reading frames which share amino acid sequence similarities with the mercuric ion transport proteins MerT and MerP encoded by several mer operons. However, no other mer operon genes were found on Tn5563. Sequencing of a RP4::Xln hybrid plasmid indicates possible interactions between pRA2 and the P25X chromosome mediated by Tn5563.     

Expression of Rhizobium meliloti nod genes in Rhizobium and Agrobacterium backgrounds.

Rhizobium meliloti nod genes are required for the infection of alfalfa. Induction of the nodC gene depends on a chemical signal from alfalfa and on nodD gene expression. By using a nodC-lacZ fusion, we have shown that the induction of the R. meliloti nodC gene and the expression of nodD occur at almost normal levels in other Rhizobium backgrounds and in Agrobacterium tumefaciens, but not in Escherichia coli. Xanthomonas campestris, or Pseudomonas savastanoi. Our results suggest that bacterial genes in addition to nodDABC are required for nod gene response to plant cells. We have found that inducing activity is present in other plant species besides alfalfa. Acetosyringone, the A. tumefaciens vir gene inducer, does not induce nodC.     

Construction of a physical map of a kanamycin (Km) transposon, Tn5, and a comparison to another km transposon, Tn903

Summary A cleavage map of Tn5, a kanamycin (Km) transposon from plasmid JR67, was constructed from pMKI, a composite plasmid of ColE1 and Tn5, and compared to that of Tn903, a Km transposon from plasmid R6-5. The two transposons showed marked heterogeneity in both the structural gene for Km resistance and the inverted repeat regions as evidenced by their distinctly different restriction maps. This result suggests separate paths of evolution for the two Km transposons.