Evolved neomycin phosphotransferase from an isolate of Klebsiella pneumoniae

A new aminoglycoside resistance gene (aphA1-IAB) confers high-level resistance to neomycin. The sequence of aphA1-IAB is closely related to aphA1 found in the transposons Tn4352, Tn903 and Tn602. For example, aphA1-IAB differs from aphA1-903 at five nucleotides that result in four amino acid replacements. The enzyme encoded by aphA1-IAB has a significantly higher turnover number with neomycin, kanamycin and G418 as substrates than does the aphA1-903 enzyme. A parsimonious phylogenetic tree suggests that aphA1-IAB evolved from an ancestral form that is closely related or identical to the aphA1 found in Tn903. The excess of replacement substitutions over silent substitutions in aphA1-IAB, as well as its convergence toward aphA3 from Staphylococcus aureus, is indicative of selective evolution. Our hypothesis to explain these results is that aphA1-IAB evolved under the selective pressure of neomycin use in relatively recent times.     

Tn602: A naturally occurring relative of Tn903 with direct repeats

We report the characterization of Tn602, a transposon encoding resistance to kanamycin and related aminoglycosides present on the R-plasmid pGD10. Tn602 is highly homologous to the previously characterized Tn903, present on the R-plasmid R6, in that it consists of a gene for aminoglycoside-phosphotransferase-3'-I (homologous to that of Tn903) flanked by copies of an IS-element homologous to IS903. Tn602 differs from Tn903 in the following respects: the flanking IS-elements (IS602) are in direct rather than inverted orientation as in Tn903; the fusion points between the IS-elements and the central region are different from those in Tn903; and several sequence changes, detected by the loss and acquisition of restriction sites, show the two repeats of IS602 to be nonidentical and different from IS903, IS102, and IS903.B. These structural details suggest that Tn602 and Tn903 evolved separately from related modules.     

Evolutionary changes in the structural and regulatory regions of aminoglycoside-3'-phosphotransferase type I gene of E. coli

To investigate the evolutionary relationships between the aph(3') genes from different plasmids, the nucleotide sequence of the aph(3') gene from the E. coli R plasmid was determined and compared with the known aph(3') genes of Tn903 and Tn4352. Three point mutations in the structural part of the cloned aph(3') gene caused amino acid changes in the enzyme molecule at positions 19, 27 and 48 beginning from the start codon. The structural part of the gene was followed by two stop codons and a long DNA region containing no nucleotide sequences homologous to the sequences of Tn903 or Tn4352. Both the cloned aph(3') gene and Tn4352 were limited on the left by the spacer sequence and the insertion sequence IS176. Twenty one base pairs deletion abolished the -35 sequence of the promoter suggested for the aph(3') gene of Tn4352 and resulted in formation of a fusion promoter utilizing the -35 box of IS176 and the -10 box of the aph(3') gene. The distance between the -35 and -10 sequences changed from 18 to 17 bp. Changes in the cloned aph(3') gene and the flanking DNA regions resulted in formation of a new promoter and loss of the right IS176 element.     

Nucleotide sequence of the kanamycin resistance determinant of plasmid RP4: Homology to other aminoglycoside 3′-phosphotransferases

The kanamycin resistance determinant of the broad-host-range plasmid RP4 encodes an aminoglycoside 3'-phosphotransferase of type I. The nucleotide sequence of the kanamycin resistance gene (Kmr) and the right end of the insertion element IS8 of plasmid RP4 has been determined. The gene (816 bp) is located between IS8 and the region (Tra 1) encoding plasmid factors mediating bacterial conjugation. Kmr and Tra 1 are transcribed toward each other. The nucleotide sequence has been compared to five related aphA genes originating from gram-negative and gram-positive organisms and from antibiotic producers. Among these that of Tn903 shares the highest degree of similarity (60%) with the RP4 gene. Significant similarities were also detected between the amino acid sequences of the six enzymes. The C-terminal domains of six different aminoglycoside 3'-phosphotransferases (APH(3'] are highly conserved. They are substantially similar to segments of a variety of enzymes using ATP as cofactor. The role of the C-terminal sequences of APH(3') as potential domains for ATP recognition and binding is discussed.     

Tn903 induces inverted duplications in the chromosome of bacteriophage lambda

Specialized transducing strains of bacteriophage lambda have been isolated that carry the transposable kanamycin resistance element, Tn903. Tn903 carries an inverted duplication of 1130 base-pairs flanking the kanamycin resistance gene. Often, when λ::Tn903 particles are infected into bacterial cells, the lambda chromosome is rearranged into a defective lambda plasmid which replicates with the bacterial cell. The formation of the defective plasmids (called Tn903λdv) is most likely induced by the Tn903 insertion itself. This follows from the fact that the novel DNA sequence found in these plasmids, with respect to the ancestral λTn903 chromosome, is always adjacent to the Tn903 element. Physical chromosomal mapping of these plasmids shows that they contain large inverted duplications of lambda sequences situated about the Tn903 insertion. The formation of the Tn903λdv plasmids from the ancestral λTn903 is not dependent on the recombination functions provided through the phage red gene or the host recA gene.     

Completion of the nucleotide sequence of the central region of Tn5 confirms the presence of three resistance genes.

The DNA sequence of the region located downstream from the kanamycin resistance gene of Tn5 up to the right inverted repeat IS50R has been determined. This completes the determination of the sequence of Tn5 which is 5818 bp long. The 2.7 Kb central region contains three resistance genes: the kanamycin-neomycin resistance gene, a gene coding for resistance to CL990 an antimitotic-antibiotic compound of the bleomycin family and a third gene that confers streptomycin resistance in some bacterial species but is cryptic in E. coli. A Tn5* mutant able to express streptomycin resistance in E. coli was isolated. With this mutant, it was demonstrated that in E. coli the expression of the three resistance genes is coordinated in a single operon.     

Characterisation of plasmids purified from Acetobacter pasteurianus 2374

Four cryptic plasmids pAP1, pAP2, pAP3, and pAP4 with their replication regions AP were isolated from Gram-negative bacteria Acetobacter pasteurianus 2374 and characterised by sequence analyses. All plasmids were carrying the kanamycin resistance gene. Three of four plasmids pAP2, pAP3, and pAP4 encode an enzyme that confers ampicillin resistance to host cells. Moreover, the tetracycline resistance gene was identified only in pAP2 plasmid. All plasmids are capable to coexist with each other in Acetobacter cells. On the other hand, the coexistence of more than one plasmid is excluded in Escherichia coli. The nucleotide sequence of replication regions showed significant homology. The nucleotide and protein sequence analyses of resistance genes of all plasmids were compared with transposons Tn3, Tn10, and Tn903 which revealed significant differences in the primary structure, however no functional changes of gene were obtained.     

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.     

Sequence homology between the tetracycline-resistance determinants of Tn10 and pBR322

The Tn10 tetracycline resistance gene, tetA, encodes a tetracycline-inducible protein with an apparent Mr of 36 X 10(3). We have determined the nucleotide sequence of the Tn10 tetA gene. The extent of the tetA gene was determined by analysis of amino-terminal and carboxy-terminal deletion mutants. We conclude that a single Tn10 gene, the tetA gene, is sufficient to confer tetracycline resistance. The predicted Mr of the tetA protein is 43.2 X 10(3). The sequence homology between the Tn10 tetA gene and the pBR322 tetracycline resistance determinant (49% nucleotide homology, 44% amino acid homology) indicates that these phenotypically distinct tetracycline-resistance determinants must have evolved from a common ancestral sequence. The markedly hydrophobic character of the predicted amino acid sequences of the Tn10 tetA and pBR322 tet-coded proteins suggests that a substantial portion of these proteins may be embedded within the cytoplasmic membrane.     

Nucleotide sequence of the kanamycin resistance determinant of the pneumococcal transposon Tn1545: evolutionary relationships and transcriptional analysis of aphA-3 genes

The nucleotide sequence of the kanamycin resistance determinant aphA-3 encoded by transposon Tn1545 from Streptococcus pneumoniae was determined and compared to those of plasmids pJH1 and pIP1433 from Streptococcus faecalis and Campylobacter coli, respectively. The three sequences were found to be identical and differed by two substitutions and the deletion of a codon from that of plasmid pSH2 from Staphylococcus aureus. Comparison of the 5' noncoding sequences indicated that the regions containing the aphA-3 gene in pJH1 and in Tn1545 evolved independently by deletion from a sequence similar to that found in pIP1433. In the latter plasmid, aphA-3 is transcribed from a promoter, P1, which is flanked by two 12-base pair direct repeats. The rearrangement observed in pJH1 removed one of these recombinogenic sites and altered the -10 and 3' flanking sequences of P1. The promoter thus generated. P1', allows expression of similar level of kanamycin resistance as P1. However, fusion experiments carried out with a promotorless chloramphenicol acetyltransferase gene indicated that the canonical promoter P1 is significantly less efficient than P1'. From analysis of the thermodynamic properties of these promoters, we conclude that this difference in strength reflects the melting properties of the -10 sequences. The transition from pIP1433 to pJH1 may correspond to the progression of a molecule structurally unstable to a more stable one combined with the need to maintain an efficient promoter upstream of the aphA-3 gene. The deletion event in Tn1545, which occurred between the two 12-base pair directly repeated sequences, removed P1 in its entirety.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Transposon-Like Structure of IS26-Tetracycllne,and Kanamycin Resistance Determinant Derived from Transferable R Plasmid of Fish Pathogen,Pasteurella piscicida

Tetracycline (pp-tet), and kanamycin (pp-kan) resistance genes were cloned from a transferable R plasmid of fish pathogen Pasteurella piscicida, and complete nucleotide sequences were determined. The pp-tet was a class D Tet determinant constructed with the tetA resistance gene of 1,182 bp encoding a protein with a deduced molecular mass of 41 kDa and the tetR repressor gene of 654 bp encoding a product of 24 kDa. The pp-tet was highly homologous to the tet(D) of plasmid RA1 isolated from Aeromonas hydrophila with two nucleotide differences in the tetR, and of plasmid pIP173 from Salmonella ordonez with two nucleotide differences in the tetA. The pp-kan contained 813 bp encoding a 31 kDa protein of 271 amino acids, and was classified into type aph-Ic. It was identical to the aphA7 in the IAB operon of pBWH77, in which was originally found an isolate of Klebsiella pneumoniae, in its nucleotide sequences and hybrid promoter construction. The genes were connected by an insertion sequence IS 26 of 820 bp, and were flanked by repeated copies in direct orientation at the 3′ flanking region of the pp-tetA and in inverted orientation at the 3′ flanking region of the pp-kan. The genetic elements are organized like a complex transposon by close linkage of the IS 26 and the pp-tet and -kan.     

The nitrogen-regulated Bacillus subtilis nrgAB operon encodes a membrane protein and a protein highly similar to the Escherichia coli glnB-encoded PII protein.

Expression of beta-galactosidase encoded by the nrg-29::Tn917-lacZ insertion increases 4,000-fold during nitrogen-limited growth (M.R. Atkinson and S. H. Fisher, J. Bacteriol. 173:23-27, 1991). The chromosomal DNA adjacent to the nrg-29::Tn917-lacZ insertion was cloned and sequenced. Analysis of the resulting nucleotide sequence revealed that the Tn917-lacZ transposon was inserted into the first gene of a dicistronic operon, nrgAB. The nrgA gene encodes a 43-kDa hydrophobic protein that is likely to be an integral membrane protein. The nrgB gene encodes a 13-kDa protein that has significant sequence similarity with the Escherichia coli glnB-encoded PII protein. Primer extension analysis revealed that the nrgAB operon is transcribed from a single promoter. The nucleotide sequence of this promoter has significant similarity with the -10 region, but not the -35 region, of the consensus sequence for Bacillus subtilis sigma A-dependent promoters.     

Polarity of Tn5 insertion mutations in Escherichia coli.

We assessed the effect of insertions of the kanamycin resistance transposon Tn5 in the lac operon of Escherichia coli on the expression of distal genes lacY and lacA (melibiose fermentation at 41 degrees C and thiogalactoside transacetylase synthesis, respectively). Every insertion mutation tested (41 in lacZ and 23 in lacY) was strongly polar. However, approximately one-third of the insertion mutants expressed distal genes at low levels due to a promoter associated with Tn5. To localize this promoter, we (i) reversed the orientation of Tn5 at several sites and (ii) replaced wild-type Tn5 with several substitution derivatives which lack Tn5's central region. Neither alteration changed the expression of distal genes. Thus, in contrast to transposons IS2 and TnA. Tn5's ability to turn on distal gene expression is not due to a promoter in its central region and therefore is not dependent on the overall orientation of Tn5 in the operon. Our results suggest that the promoter is within 186 base pairs of the ends of Tn5. It is possible that the promoter is detected in only a fraction of insertions because it overlaps Tn5-target sequence boundary.     

Nucleotide sequence of the kanamycin resistance transposon Tn903

The entire nucleotide sequence of the kanamycin resistance transposon Tn903 was determined by analyzing a mini-ColE1 derivative carrying Tn903. Tn903 was 3094 base-pairs in length and at both extremities possessed two identical inverted 1057 base-pair sequences. Furthermore, 18 bases at the ends of the 1057 base-pair sequence are themselves present in an invertedly repeated order as has been described for various insertion sequences. Analysis of initiation and termination codons in the Tn903 sequence indicated that Tn903 could possibly code for at least three high molecular weight polypeptides. One in the region between the two 1057 base-pair sequences is suggested to be the kanamycin resistance determinant (aminoglycoside 3′-phosphotransferase) from its location and size. The other polypeptides were located within the 1057 base-pair sequence and may be associated with transposition functions of Tn903.     

Insertion element IS102 resides in plasmid pSC101.

In vivo recombination was found to occur between plasmid pHS1, a temperature-sensitive replication mutant of pSC101 carrying tetracycline resistance, and plasmid ColE1 after selection for tetracycline resistance at the restrictive temperature, 42 degrees C. Extensive analysis of the physical structures of three of these recombinant plasmids, using restriction endonucleases and the electron microscope heteroduplex method, revealed that the plasmid pHS1 was integrated into different sites on ColE1. The recombinant plasmids contained a duplication of a unique 1-kilobase (kb) sequence of pHS1 in a direct orientation at the junctions between the two parental plasmid sequences. This was confirmed by comparing the nucleotide sequence of the recombinants and their parental plasmids. Nucleotide sequence analysis further revealed that nine nucleotides at the site of recombination of ColE1 were duplicated at the junction of each of the 1-kb sequences. The formation of recombinants was independent of RecA function. Based on our previous finding that a plasmid containing a deoxyribonucleic acid insertion (IS) element can recombine with a second plasmid to generate a duplication of the IS element, we conclude that the 1-kb sequence is an insertion sequence, which we named IS102. For convenience, we have also denoted the IS102 sequence as eta theta to assign the orientation of the sequence. Eighteen nucleotides at one end (eta end) were found to be repeated in an inverted orientation at the other end (theta end) of IS102. The nucleotide sequence of the eta end of the sequence was found to be identical to the sequence at the ends of the transposon Tn903, which is responsible for transposition of the kanamycin resistance gene.     

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.     

Nucleotide sequence of the AAD(2'''') aminoglycoside adenylyltransferase determinant aadB. Evolutionary relationship of this region with those surrounding aadA in R538-1 and dhfrII in R388.

The nucleotide sequence of the aadB gene which confers resistance to kanamycin, gentamicin, and tobramycin has been determined. The size of the longest reading frame is 747 bases encoding a protein of predicted size 27,992 daltons. A segment of the aadB gene sequence (including the promoter region) was found upstream of the aadA gene in R538-1 and of the dhfrII gene in R388 and the proposed promoters for these genes coincide with the aadB promoter region. The sequence homology extends upstream to the end of the sequenced regions of R388 and R538-1. Almost perfect homology was also found between the sequences 3'- to the aadB gene and 3'- to the aadA genes of R538-1 and pSa. This segment includes a 59 base element previously found flanking the Tn7 aadA gene. A model is presented for the evolution of this region of the plasmid genomes in which the 59- base element functions as an insertional "hot spot" and the possibility that this region is analogous to the aadA/aadB region of the Tn21- like transposon family is considered.     

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.