Transposon Tn916 mutagenesis in Clostridium botulinum.

The study of toxinogenesis and other properties in Clostridium botulinum is limited by the absence of genetic methods that enable construction of defined mutants. In this study, tetracycline-resistant transposon Tn916 in Enterococcus faecalis was conjugatively transferred in filter matings to group I Clostridium botulinum strains Hall A and 113B. The Tn916 transfer frequencies to C. botulinum ranged from 10(-8) to 10(-5) Tcr transconjugant per recipient depending on the donor strain. Southern blot analyses of EcoRI or HindIII chromosomal digests extracted from randomly selected Tcr transconjugants showed that the transposon inserted at different sites in the recipient chromosome, and the copy number of Tn916 varied from one to three. Tn916 insertion gave several different auxotrophic mutants. This approach should be useful for the study of genes important in growth, survival, and toxinogenesis in C. botulinum.     

Transposon Tn916 mutagenesis in Clostridium botulinum.

The study of toxinogenesis and other properties in Clostridium botulinum is limited by the absence of genetic methods that enable construction of defined mutants. In this study, tetracycline-resistant transposon Tn916 in Enterococcus faecalis was conjugatively transferred in filter matings to group I Clostridium botulinum strains Hall A and 113B. The Tn916 transfer frequencies to C. botulinum ranged from 10(-8) to 10(-5) Tcr transconjugant per recipient depending on the donor strain. Southern blot analyses of EcoRI or HindIII chromosomal digests extracted from randomly selected Tcr transconjugants showed that the transposon inserted at different sites in the recipient chromosome, and the copy number of Tn916 varied from one to three. Tn916 insertion gave several different auxotrophic mutants. This approach should be useful for the study of genes important in growth, survival, and toxinogenesis in C. botulinum.     

Transposon mutagenesis in Actinobacillus pleuropneumoniae with a Tn10 derivative.

A transposon mutagenesis procedure functional in the gram-negative swine pathogen Actinobacillus pleuropneumoniae was developed for the first time. The technique involved the use of a suicide conjugative plasmid, pLOF/Km, carrying a mini-Tn10 with an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible transposase located outside the mobile element (M. Herrero, V. de Lorenzo, and K. N. Timmis, J. Bacteriol. 172:6557-6567, 1990). The plasmid was mobilized from Escherichia coli to A. pleuropneumoniae through the RP4-mediated broad-host-range conjugal transfer functions provided by the chromosome of the donor strain. When IPTG was present in the mating medium, A. pleuropneumoniae CM5 transposon mutants were obtained at a frequency of 10(-5), while no mutants were detected in the absence of IPTG. Since the frequency of conjugal transfer of the RP4 plasmid from E. coli to A. pleuropneumoniae CM5 was found to be as low as 10(-4), the above result indicated that the expression level of the transposase was a critical factor for obtaining a workable efficiency of transposon mutagenesis. The transposon insertions occurred at random, as determined by Southern blotting of chromosomal DNA of randomly selected mutants and by the ability to generate mutants defective for the selected phenotypes. Almost all the mutants analyzed resulted from a single insertion of the Tn10 element. About 1.2% of the mutants resulted from the cointegration of pLOF/Km into the A. pleuropneumoniae chromosome. The applicability of this transposon mutagenesis system was verified on other A. pleuropneumoniae strains of different serotypes. The usefulness of this transposon mutagenesis system in genetic studies of A. pleuropneumoniae is discussed.     

Transposon Tn5 mutagenesis in Rhodopseudomonas palustris

Abstract The potential of the antibiotic resistance transposon Tn5 for random insertion mutagenesis in Rhodopseudomonas palustris was assessed. The Tn5 containing suicide vector plasmid pSUP2021, was transferred from Escherichia coli to Rhodopseudomonas palustris and kanamycin-resistant transconjugants arose at a frequency of 2.7×10⁻⁷ per recipient. In the majority of transconjugants tested, Tn5 was found to have successfully transposed to yield a single chromosomal insertion, with the concomitant loss of the vector plasmid through segregation. Two Tn5 mutants, one defective in carotenoid synthesis, and one exhibiting a reduced anaerobic growth rate on aromatic acids, were partially characterised. This is the first study to show that Tn5 mutagenesis can be applied successfully to isolate mutants of Rhodopseudomonas palustris .     

Transposon mutagenesis by Tn4560 and applications with avermectin-producing Streptomyces avermitilis.

The Tn3-like Streptomyces transposon Tn4560 was used to mutagenize Streptomyces avermitilis, the producer of anthelmintic avermectins and the cell growth inhibitor oligomycin. Tn4560 transposed in this strain from a temperature-sensitive plasmid to the chromosome and from the chromosome to a plasmid with an apparent frequency of about 10(-4) to 10(-3) at both 30 and 39 degrees C. Auxotrophic and antibiotic nonproducing mutations were, however, obtained only with cultures that were kept at 37 or 39 degrees C. About 0.1% of the transposon inserts obtained at 39 degrees C caused auxotrophy or abolished antibiotic production. The sites of insertion into the S. avermitilis chromosome were mapped. Chromosomal DNA fragments containing Tn4560 insertions in antibiotic production genes were cloned onto a Streptomyces plasmid with temperature-sensitive replication and used to transport transposon mutations to other strains, using homologous recombination. This technique was used to construct an avermectin production strain that no longer makes the toxic oligomycin.     

Transposon Tn7. cis-Acting sequences in transposition and transposition immunity

We have identified and characterized the cis-acting sequences at the termini of the bacterial transposon Tn7 that are necessary for its transposition. Tn7 participates in two kinds of transposition event: high-frequency transposition to a specific target site (attTn7) and low-frequency transposition to apparently random target sites. Our analyses suggest that the same sequences at the Tn7 ends are required for both transposition events. These sequences differ in length and nucleotide structure: about 150 base-pairs at the left end (Tn7L) and about 70 base-pairs at the right end (Tn7R) are necessary for efficient transposition. We also show that the ends of Tn7 are functionally distinct: a miniTn7 element containing two Tn7R ends is active in transposition but an element containing two Tn7L ends is not. We also report that the presence of Tn7's cis-acting transposition sequences anywhere in a target replicon inhibits subsequent insertion of another copy of Tn7 into either an attTn7 target site or into random target sites. The inhibition to an attTn7 target site is most pronounced when the Tn7 ends are immediately adjacent to attTn7. We also show that the presence of Tn7R's cis-acting transposition sequences in a target replicon is necessary and sufficient to inhibit subsequent Tn7 insertion into the target replicon.     

Reduction of uranium by Desulfovibrio desulfuricans.

The possibility that sulfate-reducing microorganisms contribute to U(VI) reduction in sedimentary environments was investigated. U(VI) was reduced to U(IV) when washed cells of sulfate-grown Desulfovibrio desulfuricans were suspended in a bicarbonate buffer with lactate or H2 as the electron donor. There was no U(VI) reduction in the absence of an electron donor or when the cells were killed by heat prior to the incubation. The rates of U(VI) reduction were comparable to those in respiratory Fe(III)-reducing microorganisms. Azide or prior exposure of the cells to air did not affect the ability of D. desulfuricans to reduce U(VI). Attempts to grow D. desulfuricans with U(VI) as the electron acceptor were unsuccessful. U(VI) reduction resulted in the extracellular precipitation of the U(IV) mineral uraninite. The presence of sulfate had no effect on the rate of U(VI) reduction. Sulfate and U(VI) were reduced simultaneously. Enzymatic reduction of U(VI) by D. desulfuricans was much faster than nonenzymatic reduction of U(VI) by sulfide, even when cells of D. desulfuricans were added to provide a potential catalytic surface for the nonenzymatic reaction. The results indicate that enzymatic U(VI) reduction by sulfate-reducing microorganisms may be responsible for the accumulation of U(IV) in sulfidogenic environments. Furthermore, since the reduction of U(VI) to U(IV) precipitates uranium from solution, D. desulfuricans might be a useful organism for recovering uranium from contaminated waters and waste streams.     

Reduction of uranium by Desulfovibrio desulfuricans.

The possibility that sulfate-reducing microorganisms contribute to U(VI) reduction in sedimentary environments was investigated. U(VI) was reduced to U(IV) when washed cells of sulfate-grown Desulfovibrio desulfuricans were suspended in a bicarbonate buffer with lactate or H2 as the electron donor. There was no U(VI) reduction in the absence of an electron donor or when the cells were killed by heat prior to the incubation. The rates of U(VI) reduction were comparable to those in respiratory Fe(III)-reducing microorganisms. Azide or prior exposure of the cells to air did not affect the ability of D. desulfuricans to reduce U(VI). Attempts to grow D. desulfuricans with U(VI) as the electron acceptor were unsuccessful. U(VI) reduction resulted in the extracellular precipitation of the U(IV) mineral uraninite. The presence of sulfate had no effect on the rate of U(VI) reduction. Sulfate and U(VI) were reduced simultaneously. Enzymatic reduction of U(VI) by D. desulfuricans was much faster than nonenzymatic reduction of U(VI) by sulfide, even when cells of D. desulfuricans were added to provide a potential catalytic surface for the nonenzymatic reaction. The results indicate that enzymatic U(VI) reduction by sulfate-reducing microorganisms may be responsible for the accumulation of U(IV) in sulfidogenic environments. Furthermore, since the reduction of U(VI) to U(IV) precipitates uranium from solution, D. desulfuricans might be a useful organism for recovering uranium from contaminated waters and waste streams.     

Transposon mutagenesis in Staphylococcus epidermidis using the Enterococcus faecalis transposon Tn917

We transformed a clinical Staphylococcus epidermidis isolate with the Enterococcus faecalis transposon Tn917-carrying plasmid pTV1. Loss of plasmid replication was observed at 47 degrees C. Tn917 transposes efficiently and apparently randomly. The transposition frequency could be stimulated by erythromycin. Transposon mutagenesis in S. epidermidis provides a means for genetic study of the various virulence factors of this pathogen.     

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.     

Transposon mutagenesis of Xylella fastidiosa by electroporation of Tn5 synaptic complexes

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 x 10(3) and 4 x 10(3) Tn5 mutants per microg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.     

Transposon mutagenesis in halophilic eubacteria: conjugal transfer and insertion of transposon Tn5 and Tn1732 in Halomonas elongata

Abstract Molecular genetic studies of halophilic eubacteria have been limited by the lack of a suitable method for mutagenesis. To overcome this, we established a transposon mutagenesis procedure for the ectoine-producing, halophilic bacterium Halomonas elongata . We used suicide plasmids pSUP101 and pSUP102-Gm to introduce the transposons Tn5 and Tn7732 respectively into H. elongata via Escherichia coli SM10 mediated conjugation. Our finding that H. elongata is sensitive to aminoglycoside antibiotics at low salinity enabled us to apply transposons that mediate kanamycin resistance. The insertions of transposon In 1732 occurred at different sites in the chromosome of H. elongata , as proved by Southern hybridization analysis. Phenotypic analysis revealed that different auxotrophic and salt sensitive mutants were generated by mutagenesis with transposon Tn 1732 . To our knowledge this is the first report of a successful application of a transposon for direct generalized mutagenesis in a halophilic eubacterium.     

In vitro transposition of Tn552: a tool for DNA sequencing and mutagenesis.

We have explored the potential of the Tn 552 in vitro transposition reaction as a genetic tool. The reaction is simple (requiring a single protein component), robust and efficient, readily producing insertions into several percent of target DNA. Most importantly, Tn 552 insertions in vitro appear to be essentially random. Extensive analyses indicate that the transposon exhibits no significant regional or sequence specificity for target DNA and leaves no discernible 'cold' spots devoid of insertions. The utility of the in vitro reaction for DNA sequencing was demonstrated with a cosmid containing the Mycobacterium smegmatis recBCD gene cluster. The nucleotide sequence of the entire operon was determined using 71 independent Tn 552 insertions, which generated over 13.5 kb of unique sequence and simultaneously provided a comprehensive collection of insertion mutants. The relatively short ends of Tn 552 make construction of novel transposons a simple process and we describe several useful derivatives. The data presented suggest that Tn 552 transposition is a valuable addition to the arsenal of tools available for molecular biology and genomics.     

Transposon Tn5 mutagenesis in Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica

In matings between Escherichia coli 2492(pJB4JI) and Erwinia carotovora subsp. carotovora Ecc71 and E. carotovora subsp. atroseptica Eca12, Kmr Gms transconjugants were obtained at high frequencies, indicating instability of the Mu-containing plasmid pJB4JI and transposition of Tn5 into the recipient genome. This was verified by Southern blot hybridization with pRZ102 DNA containing Tn5 as the 32P-labeled probe. Examination of Kmr Gms transconjugants of Ecc71 and Eca12 disclosed that a proportion (2 to 3%) were either auxotrophic or defective in catabolism of specific carbohydrates. Spontaneous prototrophic revertants were obtained for all markers with the exception of ilv, tyr, and suc. Genetic and physical data indicate that scattered insertions of Tn5 from pJb4JI into the chromosome of Ecc71 and Eca12 produced a variety of altered phenotypes due mostly to single insertions of Tn5 not accompanied by Mu DNA.     

Transposon mutagenesis: Cloning of chromosomal DNA from the site of Tn916 insertion using polymerase chain reaction

A protocol that allows fast recovery and further analyses of chromosomal DNA adjacent to the Tn916 site of insertion is described. The procedure is based on single specific primer PCR amplification using restricted chromosomal DNA ligated into a suitable vector. Two primers, one Tn916-specific and the second vector-specific, allow amplification of the chromosomal DNA flanking the site of insertion.