Transfer of Tn1545 and Tn916 to Clostridium acetobutylicum

Tn1545, a conjugative transposon originally discovered in Streptococcus pneumoniae, has been transferred from Enterococcus faecalis and Bacillus subtilis to Clostridium acetobutylicum NCIB 8052. Transfer between different strains of C. acetobutylicum has also been observed. Insertion of Tn1545 into the C. acetobutylicum chromosome occurred at multiple sites, as shown by Southern hybridization. Although ermAM (erythromycin-resistance) was the most satisfactory marker for primary selection of transconjugants, all three Tn1545-encoded antibiotic resistance genes (aphA-3, ermAM, and tetM) were apparently expressed in C. acetobutylicum. Our results indicate that Tn1545 is potentially useful for undertaking mutagenesis and mutational cloning in this industrially important organism. Transfer of another conjugative transposon, Tn916, from E. faecalis to C. acetobutylicum NCIB 8052 was also apparently detected. Circumstantial evidence suggests that there may be a hot spot for Tn916 insertion in the C. acetobutylicum NCIB 8052 chromosome.     

Transposition of Tn1545-delta 3 in the pathogenic Neisseriae: a genetic tool for mutagenesis.

The ability to study the virulence of pathogenic Neisseria spp. has been greatly limited by the absence of genetic tools which allow the construction of defined mutants. We have engineered a transposon system which allows random mutagenesis of the Neisseria genome at relatively high frequency. Tn1545-delta 3 is a 3.4-kb derivative of the gram-positive transposon Tn1545 encoding resistance to kanamycin. Tn1545-delta 3 was subcloned into an erythromycin-resistant derivative of the mobilizable shuttle vector pLES2 to yield the plasmid pMGC20. This latter plasmid was introduced by conjugation from Escherichia coli S17-1 into Neisseria meningitidis 8013N and Neisseria gonorrhoeae 15063G. Kanamycin-resistant 8013N and 15063G transconjugants appeared at frequencies of 10(-5) and 10(-6), respectively. Restriction enzyme analysis and Southern blot hybridization of these transconjugants showed that, in Neisseria spp., the transposon excised spontaneously from pMGC20 and integrated into chromosomal DNA. Our studies revealed that (i) transposition of Tn1545-delta 3 was in numerous, apparently distinct sites, (ii) in most cases, for each transconjugant a single copy of Tn1545-delta 3 was integrated into the chromosome, and (iii) several passages on selective media did not induce secondary transposition. The kanamycin resistance marker expressed by the transconjugants was subsequently transformed into a parental background without change in the chromosomal location of the transposon. To assess the role of the general recombination system in the transposition of Tn1545-delta 3, the recA gene of N. meningitidis has been cloned and a rec derivative of 8013N has been engineered. Similar results were obtained when this latter strain was used as recipient, suggesting that recA function were not required for Tn1545-delta 3 transposition in N. meningitidis. Transposition with Tn1545-delta 3 may be an important technique for mutagenesis of the pathogenic neisseriae.     

A new insertion sequence, ISCb1, from Clostridium beijernickii NCIMB 8052

The NCIMB 8052 strain of Clostridium beijerinckii contains nine copies of a novel insertion sequence, ISCb1, belonging to the IS4 family. The 1764 bp element has 18 bp inverted repeats at its extremities, and generates 11 bp target repeats upon insertion. It contains a 1365 bp ORF whose predicted product (455 amino acids) resembles bacterial transposases. The highly conserved DD(35)E motif is present, as are signatures characteristic of the N3 and C1 domains of bacterial transposases. Codon usage of the ORF is somewhat different from that of other C. beijerinckii genes, suggesting that ISCb1 may have been acquired from another organism by horizontal gene transfer in the evolutionary past. One ISCb1 copy lies close to the site of insertion of Tn 1545 in a mutant strain, C10, which shows a reduced tendency to degenerate (i.e. loss of the potential to form solvents) compared with the wild type. In the C10 strain, the characteristic pattern of DNA fragments detected by an IS-specific probe was altered, but this was due to the Tn1545 insertion itself, rather than an ISCb1-mediated genome re-arrangement. There is currently no evidence that the element is involved in strain degeneration, since 12 independently isolated spontaneous mutants that had lost the ability to form solvents had the same ISCb1 profile as that of the wild type strain. The element is apparently restricted to a series of closely related solvent-forming clostridia.     

Clostridium beijerinckii and Clostridium difficile detoxify methylglyoxal by a novel mechanism involving glycerol dehydrogenase

In contrast to gram-negative bacteria, little is known about the mechanisms by which gram-positive bacteria degrade the toxic metabolic intermediate methylglyoxal (MG). Clostridium beijerinckii BR54, a Tn1545 insertion mutant of the NCIMB 8052 strain, formed cultures that contained significantly more (free) MG than wild-type cultures. Moreover, BR54 was more sensitive to growth inhibition by added MG than the wild type, suggesting that it has a reduced ability to degrade MG. The single copy of Tn1545 in this strain lies just downstream from gldA, encoding glycerol dehydrogenase. As a result of antisense RNA production, cell extracts of BR54 possess significantly less glycerol dehydrogenase activity than wild-type cell extracts (H. Liyanage, M. Young, and E. R. Kashket, J. Mol. Microbiol. Biotechnol. 2:87-93, 2000). Inactivation of gldA in both C. beijerinckii and Clostridium difficile gave rise to pinpoint colonies that could not be subcultured, indicating that glycerol dehydrogenase performs an essential function in both organisms. We propose that this role is detoxification of MG. To our knowledge, this is the first report of targeted gene disruption in the C. difficile chromosome.     

Transfer of conjugative elements from rumen and human Firmicutes bacteria to Roseburia inulinivorans

Studies on Firmicutes bacteria from the gut are hampered by a lack of gene transfer systems. Here the human colonic anaerobe Roseburia inulinivorans A2-194 was shown to be a transfer recipient for two conjugative transposons, Tn1545 from Eubacterium cellulosolvens and TnK10 from Clostridium saccharolyticum K10.     

An integrative vector exploiting the transposition properties of Tn1545 for insertional mutagenesis and cloning of genes from gram-positive bacteria.

We have constructed and used an integrative vector, pAT112, that takes advantage of the transposition properties (integration and excision) of transposon Tn1545. This 4.9-kb plasmid is composed of: (i) the replication origin of pACYC184; (ii) the attachment site (att) of Tn1545; (iii) erythromycin-and kanamycin-resistance-encoding genes for selection in Gram- and Gram+ bacteria; and (iv) the transfer origin of IncP plasmid RK2, which allows mobilization of the vector from Escherichia coli to various Gram+ recipients. Integration of pAT112 requires the presence of the transposon-encoded integrase, Int-Tn, in the new host. This vector retains the insertion specificity of the parental element Tn1545 and utilises it to carry out insertional mutagenesis, as evaluated in Enterococcus faecalis. Since pAT112 contains the pACYC184 replicon and lacks most of the restriction sites that are commonly used for molecular cloning, a gene from a Gram+ bacterium disrupted with this vector can be recovered in E. coli by cleavage of genomic DNA, intramolecular ligation and transformation. Regeneration of the gene, by excision of pAT112, can be obtained in an E. coli strain expressing the excisionase and integrase of Tn1545. The functionality of this system was illustrated by characterization of an IS30-like structure in the chromosome of En. faecalis. Derivatives pAT113 and pAT114 contain ten unique cloning sites that allow screening of recombinants having DNA inserts by alpha-complementation in E. coli carrying the delta M15 deletion of lacZ alpha. These vectors are useful to clone and introduce foreign genes into the genomes of Gram+ bacteria.     

Transposon mutagenesis of Mycoplasma gallisepticum by conjugation with enterococcus faecalis and determination of insertion site by direct genomic sequencing

Few genetic systems for studying mycoplasmas exist, but transposon Tn916 has been shown to transpose into the genomes of some species and can be used as an insertional mutagen. In the current study, the ability of Enterococcus faecalis to serve as a donor for the conjugative transfer of transposon Tn916 into the genome of the avian pathogen Mycoplasma gallisepticum strain PG31 was examined. Transconjugants were obtained at a frequency of > or =6 x 10(-8) per recipient CFU. To determine the transposon insertion site, an oligonucleotide primer corresponding to the 3' end of Tn916 was designed for the purpose of directly sequencing genomic DNA without PCR amplification. Using the direct sequencing approach, Tn916 was shown to insert into any of numerous sites in the M. gallisepticum genome. This is the first report of conjugal transposition of Tn916 into the M. gallisepticum genome. The ability to determine transposon insertion sites in mycoplasmas by genomic sequencing has not been previously described and allows rapid sequence analysis of transposon-generated mutants.     

Transposon Tn5 mutagenesis of Pseudomonas fluorescens to isolate mutants deficient in antibacterial activity

Abstract Pseudomonas fluorescens was subjected to insertion mutagenesis studies using the transposon Tn5-GM to generate mutants deficient in antibacterial activity minus mutants. The transposon located on the temperature-sensitive plasmid pCHR84 was conjugally transferred into the non-pathogenic pseudomonad using the triparental mating procedure. Random integration of Tn 5 -GM into the chromosome of P. fluorescens was achieved by heat ttreatment of the transformed cells at 42°C. Approximately 2% of transconjugants revealed an auxotrophic phenotype indicating efficient integration of the employed transposon into the chromosome of P. fluorescens . One transposon insertion mutant was obtained showing an antibacterial activity minus phenotype. This mutant (MM-7) was found to be defective in the production of an unidentified antibacterial compound against B. subtilis . These results introduce Tn 5 transposon mutagenesis as a new useful tool for the molecular analysis of P. fluorescens .     

A pathway for the evolution of the plasmid NTP16 involving the novel kanamycin resistance transposon Tn4352

The kanamycin resistance determinant of the drug resistance plasmid NTP16 has been characterized by DNA sequencing and has been shown to possess all of the structural features of a transposable element. It is made up of a 1040-bp central region encoding a protein identical to the aminoglycoside 3'-phosphotransferase of Tn903, flanked by direct repeats of an element identical to IS26. This novel transposon has been designated Tn4352. Analysis of the host sequences flanking the transposon reveal that they are derived from a Tn3-like element, and contain no 8 base pair target size duplications which are normally created by the insertion of IS26-like elements. Comparison to the Tn3 sequence shows that the flanking sequences are noncontiguous within Tn3, with the clear implication that NTP16 has evolved from a similar plasmid encoding only ampicillin resistance (presumably NTP1) by the insertion of Tn4352 into the Tn3-like element, followed by a substantial deletion. The sequence analysis suggests that the initial insertion was into the tnpR gene of the ampicillin transposon, followed by a deletion extending to a specific site within tnpA.     

Excision of a conjugative transposon in vitro by the Int and Xis proteins of Tn916.

The roles of purified Int and Xis proteins of the conjugative transposon Tn 916 in excision of a deletion derivative of the closely related element Tn 1545 were investigated. At a low salt concentration (37.5 mM NaCl), Int alone was able to promote limited excision to produce a covalently closed circular form of the transposon, showing that Tn 916 Int can catalyze both DNA cleavage and strand exchange. This reaction was stimulated by Xis. At higher salt concentrations (150 mM NaCl), excision by Int alone was reduced to barely detectable levels and Xis was required for excision. The low salt, Xis-stimulated reaction was approximately 8-fold more efficient than the high salt, Xis-dependent reaction. These results reflect in vivo requirements for Int and Xis in excision.     

A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction.

We have developed a strategy to rapidly construct DNA hybridization probes for the isolation of genes disrupted by transposon Tn5 insertions. A single oligonucleotide complementary to and extending outward from the ends of the inverted repeat of Tn5 was used to prime DNA synthesis in the polymerase chain reaction. The amplified product consisted of DNA sequences adjacent to both ends of the transposon insertion. The general feasibility of the approach was tested by amplifying pBR322 sequences from a derivative of pBR322 containing a Tn5 insertion. To amplify genomic DNA sequences flanking a Tn5 insertion in the chromosome of a Pseudomonas syringae strain, circular substrates were generated by ligating EcoRI-digested genomic DNA. Tn5 was contained intact within one such circular molecule, as the transposon does not contain sites for cleavage by EcoRI. The amplified product (approximately 2.5 kb) was used as a DNA hybridization probe to isolate the homologous fragment from a cosmid library of wild-type Pseudomonas syringae genomic DNA. This approach may be applied to the efficient isolation of sequences flanking any Tn5 insertion.     

Transposition of Tn4551 in Bacteroides fragilis: identification and properties of a new transposon from Bacteroides spp.

Tn4551, a clindamycin resistance (Ccr) transposon from the R plasmid pBI136, was cloned onto an Escherichia coli-Bacteroides shuttle vector which could replicate normally in E. coli but was maintained unstably in Bacteroides fragilis. To aid in cloning and to ensure maintenance of Tn4551 in E. coli, a kanamycin resistance determinant (Kmr) was inserted in the transposon. The transposon-bearing shuttle vector pFD197 was transformed into B. fragilis 638, and putative insertions of Tn4551::Kmr were identified by screening for resistance to clindamycin and plasmid content. Southern hybridization analyses were used to verify integration of the transposon in the B. fragilis chromosome, and the frequency of insertion was estimated at 7.8 X 10(-5) events per generation. In 57% of the isolates tested a second integration event also occurred. This second insertion apparently involved just a single copy of the 1.2-kilobase repeat sequence which flanks the transposon. In addition, Tn4551::Kmr appeared to function as a transposon in E. coli. Evidence for this was obtained by the isolation of transposon insertions into the bacteriophage P1 genome. Finally, the transposon vector, pFD197, could be mobilized to other B. fragilis strains in which transposition was detected. Mobilization from the strain 638 background was via a conjugation like process, but occurred in the absence of known conjugative elements or other detectable plasmids. This result suggested the presence of a host-encoded transfer system in this B. fragilis strain.     

Molecular cloning into Tn5 and integration in the Pseudomonas aeruginosa chromosome: a tool for heterologous gene expression

The DNA primase gene of the promiscuous IncP-1 conjugative plasmid RP1, encoding two polypeptides of 118 and 80 kDa, was inserted into the transposon Tn5 in Escherichia coli. The derivative transposon, Tn2523, was then transposed to a temperature-sensitive replication mutant of the promiscuous IncP-1 conjugative plasmid R68 at permissive temperature and the plasmid transferred to Pseudomonas aeruginosa strain PAO. The latter strain was then grown at non-permissive temperature to identify transposition of Tn2523 into the P. aeruginosa chromosome. Immunological and enzymic analysis showed the expression of functional primase polypeptides in the constructed P. aeruginosa strain. This strain also restored wild-type conjugational transfer proficiency, by complementation, to mutants of the IncP-1 plasmid R18 affected in transfer from P. aeruginosa to P. stutzeri or to Acinetobacter calcoaceticus due to transposon Tn7 insertion mutations in the primase gene. This strategy of cloning into a transposon and integration into the bacterial chromosome should facilitate genetic manipulation and studies of gene expression in a range of Gram-negative bacteria.     

Behavior and target site selection of conjugative transposon Tn916 in two different strains of toxigenic Clostridium difficile

The insertion sites of the conjugative transposon Tn916 in the anaerobic pathogen Clostridium difficile were determined using Illumina Solexa high-throughput DNA sequencing of Tn916 insertion libraries in two different clinical isolates: 630ΔE, an erythromycin-sensitive derivative of 630 (ribotype 012), and the ribotype 027 isolate R20291, which was responsible for a severe outbreak of C. difficile disease. A consensus 15-bp Tn916 insertion sequence was identified which was similar in both strains, although an extended consensus sequence was observed in R20291. A search of the C. difficile 630 genome showed that the Tn916 insertion motif was present 100,987 times, with approximately 63,000 of these motifs located in genes and 35,000 in intergenic regions. To test the usefulness of Tn916 as a mutagen, a functional screen allowed the isolation of a mutant. This mutant contained Tn916 inserted into a gene involved in flagellar biosynthesis.     

High incidence of transposon Tn3 insertions into a replication control gene of the chimeric R/Ent plasmid pCG86 of Escherichia coli.

Insertion of transposon Tn3 into the chimeric R/Ent plasmid pCG86 occurred preferentially into a replication control gene, generating mutants with increased plasmid copy number. In two mutants, the nucleotide sequence of the region of the gene containing the Tn3 insertion was determined.