Primary structure of an aminoglycoside 6''-N-acetyltransferase AAC(6'')-4, fused in vivo with the signal peptide of the Tn3-encoded beta-lactamase.

The gene aacA4 encoding an aminoglycoside 6'-N-acetyltransferase, AAC(6')-4, was cloned from a natural multiresistance plasmid, and its nucleotide sequence was determined. The gene was 600 base pairs (bp) long, and the AAC(6')-4 had a calculated molecular size of 22.4 kilodaltons and an isoelectric point of 5.35. The sequence of the 17 N-terminal amino acids was determined from the purified enzyme. The AAC(6')-4 gene was part of a resistance gene cluster, and its expression was under the control of the regulatory sequences of the beta-lactamase encoded by Tn3. The five N-terminal amino acids were identical to those of the signal peptide of the Tn3-encoded beta-lactamase, and the entire 5' region of aacA4, as far as it was sequenced (354 bp, including the promoter and the ribosome-binding site sequences), was identical to that of the beta-lactamase gene. This led us to presume an in vivo fusion between the beta-lactamase and the acetyltransferase genes. The latter was followed, in a polycistronic arrangement, by an aminoglycoside 3",9-adenylyltransferase gene, aadA, with an intergenic region of 68 bp. At a distance of ca. 1.3 kilobases in the 3' direction, we found remnants of a second Tn3-like element specifying an active beta-lactamase. At their 5' extremities, the two incomplete copies of Tn3, which were in tandem orientation, were interrupted within the resolvase gene. We speculate that Tn3-related sequences have played a role in the process of selection and dissemination of the AAC(6')-4 gene, which specifies resistance to amikacin and related aminoglycosides.     

The chloramphenicol acetyltransferase gene of Tn2424: a new breed of cat.

We have sequenced the gene coding for the chloramphenicol acetyltransferase of Tn2424 of plasmid NR79. This gene codes for a protein of 23,500 Da, and the derived protein sequence is similar to those of the chromosomal chloramphenicol acetyltransferases of Agrobacterium tumefaciens and Pseudomonas aeruginosa and of unidentified open reading frames, which may encode chloramphenicol acetyltransferases, adjacent to the ermG macrolide-lincosamide-streptogramin resistance gene of Bacillus sphaericus and the vgb virginiamycin resistance gene of Staphylococcus aureus. Weaker similarity to the LacA (thiogalactoside acetyltransferase) and CysE (serine acetyltransferase) proteins of Escherichia coli and the NodL protein of Rhizobium leguminosarum is also observed. There is no significant similarity to any other chloramphenicol acetyltransferase genes, such as that of Tn9. The Tn2424 cat gene is part of a 4.5-kb region which also contains the aacA1a aminoglycoside-6'-N-acetyltransferase gene; Tn2424 is similar to Tn21 except for the presence of this region. Sequences flanking the cat gene are typical of those flanking other genes inserted into pVS1-derived "integrons" by a site-specific recombinational mechanism.     

Evolution of complex resistance transposons from an ancestral mercury transposon.

The molecular interrelationship of a transposon family which confers multiple antibiotic resistance and is assumed to have been generated from an ancestral mercury transposon was analyzed. Initially, the transposons Tn2613 (7.2 kilobases), encoding mercury resistance, and Tn2608 (13.5 kilobases), encoding mercury, streptomycin, and sulfonamide resistances, were isolated and their structures were analyzed. Next, the following transposons were compared with respect to their genetic and physical maps: Tn2613 and Tn501, encoding mercury resistance; Tn2608 and Tn21, encoding mercury, streptomycin, and sulfonamide resistance; Tn2607 and Tn4, encoding streptomycin, sulfonamide, and ampicillin resistance; and Tn2603, encoding mercury, streptomycin, sulfonamide, and ampicillin resistance. The results suggest that the transposons encoding multiple resistance were evolved from an ancestral mercury transposon.     

Characterization of Tn2411 and Tn2410, two transposons derived from R-plasmid R1767 and related to Tn2603 and Tn21

Two transposable elements, Tn2410 and Tn2411, were isolated from Salmonella typhimurium R-factor R1767. They have sizes of 18.5 and 18.0 kilobases, respectively. Tn2411 mediates resistance to streptomycin, sulfonamides, and mercury. In Tn2410, the streptomycin resistance gene was replaced by a gene coding for the production of the beta-lactamase OXA-2, which is responsible for ampicillin resistance. Physical and functional maps of both transposons were compared with those of Tn21, Tn4, and Tn2603. From these data it appeared that Tn21 could be an ancestral transposon from which Tn2411, Tn2410, Tn2603, and Tn4 were evolved by the addition or deletion of small DNA segments.     

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.     

Evolution of Tn21-related transposons: isolation of Tn2425, which harbours IS161

The isolation of two multi-resistance transposons, Tn2425 and Tn1831, and their relation to Tn21 and Tn2424, is described. A 1.7 kb segment present in Tn2424 and Tn2425 was identified as an IS element by rec-independent transposition, resulting in a cointegrate structure that carries two direct repeated copies of the IS element. By the isolation of this IS element we demonstrated that transposition is one mechanism leading to sequence variations in Tn21-like structures, especially in the region between the mer operon and the sul gene.     

A chromosomally located transposon in Pseudomonas aeruginosa

A new transposon, Tn2521, coding for carbenicillin, streptomycin, spectinomycin, and sulfanilamide resistance, has been identified in Pseudomonas aeruginosa. The transposon occurs naturally in the chromosome of clinical strains of P. aeruginosa isolated in geographically separated hospitals. This has been demonstrated by its transductional linkage to the pur-136 marker and also by Southern hybridization. Tn2521 is 6.8 kilobases, can transpose from the chromosome to both IncP-1 and IncP-2 plasmid genomes, and has a pattern of restriction endonuclease sites unlike that of any previously described transposon. The carbenicillin resistance carried by Tn2521 is due to the PSE-4 type of beta-lactamase.     

Structure and function of hot spots providing signals for site-directed specific recombination and gene expression in Tn21 transposons

Tn21- and Tn3-related transposons are widespread and carry various resistance determinants. The insertion points of different resistance genes were precisely defined in Tn2424, Tn1696, Tn2410, Tn4000 and its derivatives and compared to the corresponding sites in Tn7, pSA, R388, R46, Tn2603, Tn1331 and in Tn3-related elements. Insertional 'hot spots' located at the 3' end of different genes comprised 55 nucleotides and yielded more than 90% homology to the corresponding consensus sequence, termed hs1. Elements of this class were found to direct recA-independent generation of deletions. Flanking the 5' ends, hs2 (CTAAAACAAAGTTA) comprised the terminal nucleotides of hs1. Functional properties of hot spots as recognition sites for site-specific recombination and regulation of gene expression indicate that they might be involved in transfer, stable inheritance and expression of prokaryotic genes.     

Identification of citrate utilization transposon Tn3411 from a naturally occurring citrate utilization plasmid.

We have isolated a new transposon, Tn3411, encoding citrate-utilizing ability, from a naturally occurring citrate utilization (Cit) plasmid, pOH3001. Citrate transposon Tn3411 was transposed from pOH3001 to lambda b519 b515 cI857 S7 (abbreviated lambda bb) phage, and further from the resulting lambda bb:Tn3411 to a vector plasmid, pBR322, in recA-deficient strains. The Cit+ plasmids (pOH2 and pOH3) constructed by the integration of Tn3411 into pBR322 were examined by restriction endonuclease and heteroduplex analysis. The results obtained were as follows: (i) Tn3411 was 7.4 kilobases long and flanked by small inverted repeats, and it contained one more pair of inverted repeats at the opposite orientation in the internal region, thus making alternate repeats; and (ii) the Cit+ structure gene was located on the fragment (5.5 kilobases) between two SalI cleavage sites on Tn3411.     

Tn4351 transposes in Bacteroides spp. and mediates the integration of plasmid R751 into the Bacteroides chromosome.

The gene for resistance to erythromycin and clindamycin, which is carried on the conjugative Bacteroides plasmid, pBF4, has been shown previously to be part of an element (Tn4351) that transposes in Escherichia coli. We have now introduced Tn4351 into Bacteroides uniformis 0061 on the following two suicide vectors: (i) the broad-host-range IncP plasmid R751 (R751::Tn4351) and (ii) pSS-2, a chimeric plasmid which contains 33 kilobases of pBF4 (including Tn4351) cloned into the IncQ plasmid RSF1010 and which is mobilized by R751. When E. coli HB101, carrying either R751::Tn4351 or R751 and pSS-2, was mated with B. uniformis under aerobic conditions, Emr transconjugants were detected at a frequency of 10(-6) to 10(-5) (R751::Tn4351) or 10(-8) to 10(-6) (R751 and pSS-2). In matings involving pSS-2, all Emr transconjugants contained simple insertions of Tn4351 in the chromosome, whereas in matings involving R751::Tn4351, about half of the Emr transconjugants had R751 cointegrated with Tn4351 in the chromosome. Of the Emr transconjugants, 13% were auxotrophs. Bacteroides spp. which had R751 cointegrated with Tn4351 in the chromosome did not transfer R751 or Tn4351 to E. coli HB101 or to isogenic B. uniformis, nor did the intergrated R751 mobilize pE5-2, an E. coli-Bacteroides shuttle vector that contains a transfer origin that is recognized by R751.     

Instability of Tn5 inserts in cyanobacterial cloning vectors.

Transposon Tn5 was used to produce random insertions in two hybrid cloning vectors for the unicellular cyanobacterium Anacystis nidulans. The transposon-containing plasmids were used to localize essential replication functions and to characterize the stability of large inserts in these vectors. The effect of the insertions on plasmid function was tested by transformation into a derivative of A. nidulans that had been cured of the endogenous plasmid used to construct the vectors. A region of approximately 4 kilobases was essential for successful plasmid transformation and replication. This region has also been shown to be involved in plasmid replication by deletion analysis. High rates of excision of Tn5 inserts within this region and restoration of normal replication function were observed when transformants were selected by using a resistance marker outside the replication region in the absence of selection for the transposon-coded kanamycin resistance. Transposon inserts outside this region were not deleted.     

Cointegrate formation between homologous plasmids in Escherichia coli.

Conjugation experiments were performed in which the donor was Escherichia coli K-12 strain KP245 containing either R plasmid NR1 plus an ampicillin-resistant derivative of ColE1 (*ColE1::Tn3, called RSF2124) or NR1 plus RSF2124 carrying a cloned EcoRI fragment of NR1. The recipient was the polA amber mutant JG112, in which RSF2124 cannot replicate. Ampicillin-resistant transconjugants can arise only when the genes for ampicillin resistance are linked to NR1 or are transposed to the host chromosome. When EcoRI fragment A of NR1 (20.5 kilobases) was cloned to RSF2124, the frequency of cotransfer of ampicillin resistance with tetracycline resistance was 25 to 60%. Plasmid DNA from these ampicillin-resistant transconjugant cells was analyzed by gel electrophoresis and was shown to be a cointegrate of NR1 and the RSF2124 derivative. Analysis of plasmid DNA isolated from donor cultures showed that the cointegrates were present before conjugation, which indicates that the mating does not stimulate cointegrate formation. When the cloned fragment was EcoRI fragment H of NR1 (4.8 kilobases), the frequency of cotransfer of ampicillin resistance with tetracycline resistance was about 4%, and the majority of the ampicillin-resistant transconjugants were found to contain cointegrate plasmids. When the donor contained NR1 and RSF2124, the frequency of cotransfer of ampicillin resistance was less than 0.1%, and analysis of plasmid DNA from the ampicillin-resistant transconjugants showed that Tn3 had been transposed onto NR1. These data suggest that plasmids which share homology may exist in cointegrate form to a high degree within a host cell.     

Detection and characterization of Tn2501, a transposon included within the lactose transposon Tn951

The DNA sequence spanning coordinates 9.9 to 16.4 kilobases of the lactose transposon Tn951 ( Cornelis et al., Mol. Gen. Genet. 160:215-224, 1978) constitutes a transposable element by itself. Unlike Tn951 ( Cornelis et al., Mol. Gen. Genet. 184:241-248, 1981), this element, called Tn2501 , transposes in the absence of any other transposon. Transposition of Tn2501 proceeds through transient cointegration and duplicates 5 base pairs of host DNA. Tn2501 is flanked by nearly perfect inverted repeats (44 of 48), related to the inverted repeats of Tn21 ( Zheng et al., Nucleic Acids Res. 9:6265-6278, 1982). Unlike Tn21 , Tn2501 does not confer mercury resistance.     

Molecular genetics of mutants of Rhizobium leguminosarum which fail to fix nitrogen

Summary Mutants of Rhizobium leguminosarum which failed to fix nitrogen within nodules on peas were isolated following the insertion of the transposon Tn5 into pRL1JI, a Rhizobium plasmid known to carry the genes for nitrogenase. The sites of the Tn5 insertions were identified by restriction endonuclease mapping of cloned fragments of DNA from the mutant strains. One group of mutants was located within 4 kilobases of the structural genes for nitrogenase and another was located about 30 kilobases from this region. Two mutants from the first group, one of which appeared to be affected in a nitrogenase gene, induced nodules that contained bacterioids, but the number of plant cells containing bacteroids was less than in a normal nodule. Another group of mutants, which was located about 30 kilobases from the nitrogenase genes failed to form bacterioids. Electron microscopy of the nodules induced by these mutants indicated that there was a defect in their release from infection threads.     

Genetic analysis of the pAD1 pheromone response in Streptococcus faecalis, using transposon Tn917 as an insertional mutagen

The conjugative plasmid pAD1 (56.7 kilobases) in Streptococcus faecalis has been shown to confer a mating response to the sex pheromone cAD1 excreted by recipient strains. The response is characterized by the synthesis of a proteinaceous adhesin which coats the surface of the pAD1 -containing donor cell and facilitates the formation of mating aggregates. Donors exposed to cAD1 -containing filtrates of recipients undergo self-aggregation (clumping), an event believed to be associated with an interaction between the adhesin and a binding substance always present on the surface of both recipients and donors. To analyze the molecular processes involved in the mating response, mutants were generated by the erythromycin resistance transposon Tn917 . Transpositions to pAD1 in S. faecalis DS16 gave rise to a number of derivatives that exhibited "constitutive clumping" and the ability to transfer at high frequencies in short (10-min) matings. These mutants fell into two subclasses, which exhibited colony morphologies that were "dry" or "normal". The Tn917 insertions were mapped by restriction enzyme analysis to two separate clusters, designated traA and traB. The dry colony subclass corresponded to traA and represented a span of 1.5 kilobases, whereas the normal subclass corresponded to traB and spanned 1.3 kilobases. The two clusters were separated by 1.7 kilobases in which insertions of Tn917 did not affect the ability to respond normally to cAD1 . Neither type of constitutive clumper produced cAD1 . Another series of insertions exhibited reduced donor potential. In two cases, the reduction in transfer was three to four orders of magnitude; these mapped in traA . In two other cases, the reduction was one to two orders of magnitude. These mapped outside of traA and traB, and one was associated with an increase in plasmid copy number.     

High-frequency transformation of Brevibacterium lactofermentum protoplasts by plasmid DNA.

An efficient polyethylene glycol-assisted method for transformation of Brevibacterium lactofermentum protoplasts that uses plasmid vectors has been developed. Two small plasmids, pUL330 (5.2 kilobases) and pUL340 (5.8 kilobases), both containing the kanamycin resistance gene from transposon Tn5 and the replication origin of the natural plasmid pBL1 of B. lactofermentum, were selected as vectors. Supercoiled forms of the plasmids yielded a 100-fold higher transformation frequency than did linear forms. The optimal transformation frequency was achieved with 10 ng of DNA in 1 ml of transformation buffer. Higher concentrations of plasmid DNA resulted in a decrease in transformation frequency per microgram of DNA. Optimal transformation was obtained with 25 to 35% polyethylene glycol 6000. Under optimal conditions, 10(6) transformants per microgram of DNA were obtained.     

Physical mapping of transposon Tn5 insertions defines a gene cluster functional in nitrous oxide respiration by Pseudomonas stutzeri.

By transposon Tn5 mutagenesis, 19 strains of Pseudomonas stutzeri were acquired that had defects in nitrous oxide respiration (Nos- phenotype). A physical map of the mutants showed nearly random Tn5 insertions into genomic DNA within a single region ca. 8 kilobases long. Mutants were characterized immunochemically, enzymatically, and chemically. Several functions related to the synthesis and regulation of nitrous oxide reductase were associated with this DNA region, indicating that in P. stutzeri part of the genetic information necessary to respire nitrous oxide is clustered.     

Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.

The construction and purification of recombinant baculovirus vectors for the expression of foreign genes in insect cells by standard transfection and plaque assay methods can take as long as 4 to 6 weeks. This period can be reduced to several days by using a novel baculovirus shuttle vector (bacmid) that can replicate in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. The bacmid is a recombinant virus that contains a mini-F replicon, a kanamycin resistance marker, and attTn7, the target site for the bacterial transposon Tn7. Expression cassettes comprising a baculovirus promoter driving expression of a foreign gene that is flanked by the left and right ends of Tn7 can transpose to the target bacmid in E. coli when Tn7 transposition functions are provided in trans by a helper plasmid. The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells.     

Isolation and characterization of Tn5 insertion mutants of Pseudomonas syringae pv. syringae altered in the production of the peptide phytotoxin syringotoxin.

A syringotoxin-producing strain of Pseudomonas syringae pv. syringae (B457) was subjected to Tn5 mutagenesis by the transposon vector pSUP1011. Analyses of auxotrophs obtained suggested simple random insertions of Tn5. Syringotoxin-negative mutants arose at a frequency of about 0.28%. In a Southern blot analysis, the loss of toxin production was associated with Tn5 insertions into chromosomal EcoRI fragments of about 10.5, 17.8, and 19.3 kilobases. Data from a Southern blot analysis of SstI-digested DNA from these mutants suggest that the 10.5- and 17.8-kilobase EcoRI fragments may be adjacent to or near each other. Mutants that produced only 3 to 4% wild-type toxin levels also were identified.