Lars Sundström

Site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. Sequence characterization of dhfrV and sulI and a recombination active locus of Tn21

SummaryA new gene for trimethoprim resistance, dhfrV, found in several plasmid isolates with different characteristics, was sequenced and found to correspond to a peptide of 157 amino acids showing 75% similarity with the previously characterized, drug resistant dihydrofolate reductase of type I. The sequenced surroundings of dhfrV in plasmid pLMO20, were found to be almost identical with genetic areas surrounding resistance genes in transposon Tn21 and in R plasmid R388. The trimethoprim resistance genes of pLMO20 and R388 and the spectinomycin resistance gene of Tn21 could be regarded as having been inserted, by recombination, into an evolutionary older structure containg the sulfonamide resistance gene, sulI. The latter gene was sequenced and found to correspond to a peptide of 279 amino acids and with a molecular weight of 30126 daltons. The inserted genes were found to be governed by a promoter situated in the highly conserved structure and also controlling expression of sulI. The insertion points of the different resistance genes were precisely defined, and at the 3′ ends of the inserted genes inverted repeats allowing the formation of stem and loop structures were found. Similar structures were found at the 3′ ends of the antibiotic resistance genes in Tn7, which could indicate similar recombination mechanisms to be effective in the evolutionary construction of all these different resistance elements.

IntI2 Integron Integrase in Tn7

Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7 and its relatives. We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon. This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies. The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7. However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E.

NON-PALINDROMIC ATTL SITES OF INTEGRONS ARE CAPABLE OF SITE-SPECIFIC RECOMBINATION WITH ONE ANOTHER AND WITH SECONDARY TARGETS

Genes borne on cassettes are mobile owing to site‐specific recombination systems called integrons, which have created various combinations of antibiotic resistance genes in R‐plasmids. In these processes, the palindromic site, attC (59‐base element), at cassette junctions has been proposed as being essential. Excised and circularized cassettes have been found to integrate with preference for an attI site at one end of the conserved sequence in integrons. In this work, we give evidence that recombination is possible in the absence of the highly organized attC sites between the more simply organized attI sites. Furthermore, at a very low frequency representing the background in our recombination assay, we observed cross‐overs between attI and secondary sites. To characterize recombination excluding the attC sites, we have used naturally occurring attI variants and constructed mutants. The cross‐over point was identified between a guanine and a thymine in attI using point mutations. Progressive deletions showed the extent of attI and identified two important regions in the conserved sequence 5′ of the cross‐over point. A region 27–36 bp 5′ of attI influenced recombination with attC sites only, whereas a sequence 9–14 bp 5′ of the cross‐over point in attI was important for recombination with both attI and attC. Recombination between attI and secondary sites could allow fusion of the conserved sequence encoding the integron site‐specific recombinase to new sequences.

Purification and partial characterization by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the recombinant transposase, TniA

A recombinant transposase, TniA, a basic DNA binding protein, was chromatographically purified and characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) methods. Escherichia coli cells, overexpressing native TniA, were ultrasonically disrupted and the clarified supernatant was used as starting material for anion-exchange chromatography on SOURCE1 15Q 4.6/100 PE (Tricorn), at pH 7.5. This initial step was proven to be a fast and simple way of removing acidic proteins like proteases. TniA was collected from the flow-through fraction and applied onto HiTrap heparin HP 5 ml in order to capture the basic TniA. This was followed by cation-exchange chromatography through Mono S 5/50 GL (Tricorn), at pH 6.5 which resulted in a purity of TniA of about 95%. The molecular mass of TniA was determined to 62 869 rel. mol. mass units with MALDI-TOF-MS and the identity of the protein was confirmed by peptide mass fingerprinting of trypsin-digested TniA. Partial amino acid sequencing was achieved after derivatization of tryptic peptides using Ettan CAF MALDI Sequencing Kit and post source decay. The fact that transposases are DNA-binding and that many of them possess basic isoelectric point values suggest that the outlined purification protocol may serve as a general method for the purification of recombinant nontagged transposases and other basic DNA-binding proteins.