Amikacin resistance mediated by multiresistance transposon Tn2424

Tn2424, a multiresistance transposon 25 kilobases long, was isolated from IncFII plasmid NR79. Tn2424 transposed resistance to sulfonamides, streptomycin and spectinomycin, mercuric chloride, chloramphenicol, and amikacin with a frequency of 6 X 10(-5). Resistance to amikacin was mediated by a 6'-N-acetyltransferase, which conferred higher levels of resistance in Pseudomonas aeruginosa than in Escherichia coli. A restriction analysis and cloning experiments resulted in a physical and functional map of Tn2424. Comparison by a heteroduplex technique revealed that Tn2424 includes the total sequence of Tn21 and two additional DNA fragments that are 1.8 and 4 kilobases long.     

Mechanism-based inactivation of VanX, a D-alanyl-D-alanine dipeptidase necessary for vancomycin resistance

VanX is a zinc-dependent D-Ala-D-Ala amino dipeptidase required for high-level resistance to vancomycin. The enzyme is also able to process dipeptides with bulky C-terminal amino acids [Wu, Z., Wright, G. D., and Walsh, C. T. (1995) Biochemistry 34, 2455-2463]. We took advantage of this observation to design and synthesize the dipeptide-like D-Ala-D-Gly(SPhip-CHF(2))-OH (7) as a potential mechanism-based inhibitor. VanX-mediated peptide cleavage generates a highly reactive 4-thioquinone fluoromethide which is able to covalently react with enzyme nucleophilic residues, resulting in irreversible inhibition. Inhibition of VanX by 7 was time-dependent (K(irr) = 30+/-1 microM; k(inact) = 7.3+/- 0.3 min(-1)) and active site-directed, as deduced from substrate protection experiments. Nucleophilic compounds such as sodium azide, potassium cyanide, and glutathione did not protect the enzyme from inhibition, indicating that the generated nucleophile inactivates VanX before leaving the active site. The failure to reactivate the dead enzyme by gel filtration or pH modification confirmed the covalent nature of the reaction that leads to inactivation. Inactivation was associated with the elimination of fluoride ion as deduced from (19)F NMR spectroscopy analysis and with the production of fluorinated thiophenol dimer 12. These data are consistent with suicide inactivation of VanX by dipeptide 7. The small size of the VanX active site and the presence of a number of nucleophilic side chains at the opening of the active site gorge [Bussiere, D. E., et al. (1998) Mol. Cell 2, 75-84] associated with the high observed partition ratio of 7500+/-500 suggest that the inhibitor is likely to react at the entrance of the active site cavity.     

Continuous assay for VanX, the D-alanyl-D-alanine dipeptidase required for high-level vancomycin resistance.

The reaction of L-alanine-p-nitroanilide with VanX was studied in an effort to develop a continuous assay for VanX activity for future kinetic and inhibition studies. VanX, containing Zn(II), Co(II), Fe(II), or Ni(II), catalyzes the hydrolysis of L-alanine-p-nitroanilide producing L-alanine and p-nitroaniline as products; the formation of the latter product (epsilon(404nm) = 10, 700 M(-1) cm(-1)) can be continuously monitored using UV-VIS spectrophotometry. Zn(II)-, Co(II)-, Fe(II)-, and Ni(II)-containing VanX exhibit saturation kinetics when L-alanine-p-nitroanilide is used as the substrate with K(m) and k(cat) values ranging from 300 to 700 microM and 0.028 to 0.080 s(-1), respectively. Inhibition studies using O-[(1S)-aminoethylhydroxyphosphinyl]-D-lactic acid as the inhibitor and L-alanine-p-nitroanilide as the substrate yielded a K(i) of 400 +/- 8 microM at pH 7.0. These studies reveal a continuous assay of VanX activity which could be used to further study the kinetic mechanism of VanX and to allow for the development of high-throughput screening for inhibitors of VanX.     

利用Tn5转座子介导突变提高大肠杆菌丁醇生产水平

目前,对于构建高产丁醇大肠杆菌工程菌株的工作,主要是对丁醇通路和相关途径的基因进行理性改造。为进一步提升菌株的丁醇生产能力,需要发掘基因组上可影响丁醇生产能力的基因,但这很难通过已有认识或计算机模型进行预测。本工作以一株实验室前期构建的产丁醇大肠杆菌工程菌株为研究对象,利用Tn5转座子构建了一个含有1 196个菌株的突变文库。丙酮酸是丁醇的前体,并且在发酵终产物中,副产物丙酮酸的含量与丁醇的含量呈反相关,因此,可以利用丙酮酸的含量来间接反映丁醇的含量,而丙酮酸可用二硝基苯肼显色法进行快速测定,基于此,建立了96孔板——酶标仪快速筛选方法。利用该方法成功筛选到了比对照菌株丁醇产量提高了29%、49%、56%的3个突变体菌株。利用反向PCR及测序的方法,确定了其转座子插入位置分别为:pyk A、tdk、cad C基因。这些基因可以作为进一步提高菌株丁醇产量的靶点,同时这种利用Tn5转座子筛选基因靶标的策略也为构建其他微生物细胞工厂提供了新思路。     

Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147.

Sequence determination of the flanking regions of the vancomycin resistance van gene cluster carried by pIP816 in Enterococcus faecium BM4147 revealed similarity to transposons of the Tn3 family. Imperfect inverted repeats (36 of 38 bp) delineated a 10,851-bp element designated Tn1546. The 4-kb region located upstream from the vanR gene contained two open reading frames (ORF) transcribed in opposite directions. The deduced amino acid sequence of ORF1 (988 residues) displayed, respectively, 56 and 42% identity to those of the transposases of Tn4430 from Bacillus thuringiensis and of Tn917 from Enterococcus faecalis. The product of ORF2 (191 residues) was related to the resolvase of Tn917 (33% amino acid identity) and to the Res protein (48%) of plasmid pIP404 from Clostridium perfringens. Tn1546 transposed consecutively in Escherichia coli from plasmid pUC18 into pOX38 and from pOX38 into various sites of pBR329. Transposition was replicative, led to the formation of cointegrates, and produced a 5-bp duplication at the target site. Southern hybridization and DNA amplification revealed the presence of Tn1546-related elements in enterococci highly resistant to glycopeptides. Analysis of sequences surrounding these elements indicated that transposition plays a role in dissemination of the van gene cluster among replicons of human clinical isolates of E. faecium.     

途径工程及Tn5转座子介导突变提高大肠杆菌丙酮酸生产

为了开发丙酮酸高产菌株,以大肠杆菌MG1655为出发菌株,通过基因敲除阻断副产物途径构建了产丙酮酸大肠杆菌工程菌KLPP。进一步利用p UT Mini-Tn5载体进行转座子随机突变,构建了含有7 197个单克隆的突变体文库。使用基于丙酮酸的二硝基苯肼显色法,建立了96孔板-酶标仪快速筛选方法,经过两轮的筛选,成功筛选到了6个突变体菌株,比KLPP丙酮酸产量提高了38%、31%、19%、28%、44%和14%。利用全基因组重测序确定了其转座子插入的位置,进而确定了可能影响丙酮酸产量的基因位点,为后续菌株改造工作奠定了基础。     

New chromogenic dipeptide substrate for continuous assay of the D-alanyl-D-alanine dipeptidase VanX required for high-level vancomycin resistance.

A direct continuous UV-Vis spectrophotometric assay has been developed for VanX, a D-alanyl-D-alanine aminodipeptidase necessary for vancomycin resistance. This method is based on the hydrolysis of the alternative substrate D-alanyl-alpha-(R)-phenylthio-glycine D-Ala-D-Gly(S-Ph)-OH (H-DAla-DPsg-OH, 5a). Spontaneous decomposition of the released phenylthioglycine generates thiophenol, which is quantified using Ellman's reagent. The dipeptide behaved as an excellent substrate of VanX, exhibiting Michaelis-Menten kinetics with a kcat of 76 +/- 5/s and a KM of 0.83 +/- 0.08 mm (kcat = 46 +/- 3/s and KM = 0.11 +/- 0.01 mm for D-Ala-D-Ala). Determination of the kinetic parameters of the previously reported mechanism-based inhibitor D-Ala-D-Gly(SPhip-CHF2)-OH (H-D-Ala-DPfg-OH, 5c) [Araoz, R., Anhalt, E., René, L., Badet-Denisot, M.-A., Courvalin, P. & Badet, B. (2000) Biochemistry 39, 15971-15979] using the substrate reported in the present study yielded values of Kirr of 22 +/- 1 microM and kinact of 9.3 +/- 0.4/min in good agreement with values previously obtained in our laboratory (Kirr = 30 +/- 1 mm; kinact = 7.3 +/- 0.3/min). In addition, inhibition by the competing substrate D-Ala-D-Ala resulted in determination of a Ki = 70 +/- 6 microM close to the previously reported KM value. These results demonstrate that the present assay is a convenient, rapid and sensitive tool in the search for VanX inhibitors.     

Efficient insertional mutagenesis in group A streptococci mediated by Tn917 transposon

The replication-conditional thermosensitive vector pTV1-OK (repATs, Kan^r) harbouring the transposon Tn917 (Em^r) was successfully used to mutagenise a clinical Streptococcus pyogenes isolate (CS101). In the initial studies, conditions were established for electrotransformation of the pTV1-OK vector into CS101. Transformants selected on media containing Kan at 29°C were shown to become Kan^s at 39°C and to carry the transposon-linked Em^r marker. One such transformant was chosen for transposition studies. Upon temperature shift, transposition was achieved with a frequency of approximately 0.01% with a plasmid curing efficiency of close to 100%. Southern blot analysis demonstrated that the majority of mutants contained a single copy of Tn917 and showed no evidence for preferential sites of integration (“hot spots”). Screening of Tn917 libraries of S. pyogenes has led to the identification of mutants lacking either haemolysing or plasminogen activating activity. Mutants defective in each of these activities were identified at a frequency of approximately one in 1000 to 4000 colonies. These findings suggest that the pTV1-OK vector can be used for transposon mutagenesis of S. pyogenes and that this strategy will be valuable for identifying virulence factors and regulatory mechanisms in these bacteria.     

Specific DNA cleavage mediated by the integrase of conjugative transposon Tn916.

The conjugative transposon Tn916 encodes a protein called INT(Tn916) which, based on DNA sequence comparisons, is a member of the integrase family of site-specific recombinases. Integrase proteins such as INT(lambda), FLP, and XERC/D that promote site-specific recombination use characteristic, conserved amino acid residues to catalyze the cleavage and ligation of DNA substrates during recombination. The reaction proceeds by a two-step transesterification reaction requiring the formation of a covalent protein-DNA intermediate. Different requirements for homology between recombining DNA sites during integrase-mediated site-specific recombination and Tn916 transposition suggest that INT(Tn916) may use a reaction mechanism different from that used by other integrase recombinases. We show that purified INT(Tn916) mediates specific cleavage of duplex DNA substrates containing the Tn916 transposon ends and adjacent bacterial sequences. Staggered cleavages occur at both ends of the transposon, resulting in 5' hydroxyl protruding ends containing coupling sequences. These are sequences that are transferred with the transposon from donor to recipient during conjugative transposition. The nature of the cleavage products suggests that a covalent protein-DNA linkage occurs via a residue of INT(Tn916) and the 3'-phosphate group of the DNA. INT(Tn916) alone is capable of executing the strand cleavage step required for recombination during Tn916 transposition, and this reaction probably occurs by a mechanism similar to that of other integrase family site-specific recombinases.     

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.     

Some properties of the chloramphenicol resistance transposon Tn9

Summary We have isolated variants of the plasmid RTF which have received the transposon Tn9 from bacteriophage P1Cm. We have shown by the formation of heteroduplex molecules between one RTF: Tn9 derivative and R100.1 that Tn9 is homologous to the r-determinant region of R100.1 which carries the determinants for chloramphenicol resistance. This suggests that Tn9 was derived from an r-det like structure by deletion, possibly mediated by one of the flanking IS1 elements. In spite of the similarity in structure between Tn9 and r-det however, we have demonstrated two distinct differences in the behavior of these two elements: 1) Tn9 but nor r-det, is able to amplify, by a recA dependent mechanism, when cells harboring RTF::Tn9 are grown in the presence of chloramphenicol, and 2) Tn9, unlike r-det, does not form extrachromosomal circular molecules when RTF::Tn9 is tegrated into the bacterial chromosome.     

Stoichiometry of metal-tetracycline/H+ antiport mediated by transposon Tn10-encoded tetracycline resistance protein in Escherichia coli.

The tetracycline resistance protein (TetA) endoded by transposon Tn10 mediates the efflux of divalent cation-tetracycline chelating complexes [Yamaguchi, A., Udagawa, T. and Sawai, T. (1990) J. Biol. Chem. 265, 4809-4813]. It was confirmed that protons were antiported with the complexes through an electrically-neutral process because the antiport consumed delta pH but not delta psi. The quantitative relationship between delta pH and delta pTC determined by a flow-dialysis method clearly indicated a 1:1 stoichiometry of the monocationic metal-tetracycline/H+ exchange.     

Structure of fosfomycin resistance protein FosA from transposon Tn2921

The crystal structure of fosfomycin resistance protein FosA from transposon Tn2921 has been established at a resolution of 2.5 A. The protein crystallized without bound Mn(II) and K+, ions crucial for efficient catalysis, providing a structure of the apo enzyme. The protein maintains the three-dimensional domain-swapped arrangement of the paired betaalphabetabetabeta-motifs observed in the genomically encoded homologous enzyme from Pseudomonas aeruginosa (PA1129). The basic architecture of the active site is also maintained, despite the absence of the catalytically essential Mn(II). However, the absence of K+, which has been shown to enhance enzymatic activity, appears to contribute to conformational heterogeneity in the K(+)-binding loops.     

Mutagenesis by insertion of drug resistance transposon Tn7 into a vibrio species

A halotolerant, collagenolytic strain of Vibrio sp. was conjugated with an Escherichia coli strain carrying plasmid RP4. The plasmid was transferred to and maintained in the Vibrio and could be subsequently transferred in matings to suitably marked stains of the same species. After conjugation with an E. coli carrying the cointegrate plasmid RP4::Mu cts61::Tn7, Vibrio transconjugants were selected that carried Tn7 inserted into the bacterial chromosome. A large proportion of these transconjugants were auxotrophic, showing that plasmid suicide by Mu can be used to isolate Tn7-derived mutants in Vibrio. Approximately half of the auxotrophs isolate Tn7-derived mutants in Vibrio. Approximately half of the auxotrophs isolated were ilv mutants, all of which exhibited the same phenotype. Thus, although Tn7 insertion can induce auxotrophy, including trp, thy, his and ura, in Vibrio, there does appear to be a hot spot for integration in the ilv operon.     

Sequence requirements for target activity in site-specific recombination mediated by the Int protein of transposon Tn 1545

Excision and integration of Tn 1545 occur by reciprocal site-specific recombination between 6 (or 7)bp variable sequences present in the recombining attachment (atf) sites and designated overlap regions. We devised an assay for Tn1545 transposition in which derivatives containing the cis-acting transposition sequences (atfTn 1545) integrate into a target replicon when complemented in trans by the transposon-encoded integrase Int-Tn. This assay was used to determine the characteristics of the DNA sequence that influence target site selection. Characterization of several integration sites indicated that a 20 bp segment, designated attB, contains the sequences required for target activity. It also appeared that (i) the target activity depends upon the extent of homology between the 7bp segments flanking the overlap regions in attB and attTn1545, and (ii) the degree of homology between the two recombining overlap regions does not affect the level of target activity and has no influence on integration orientation.     

In vivo isolation of a pPJ3a-lambda cosmid mediated by the Tn2301 transposon

From an E. coli cell harboring plasmid pPJ3b (= pPJ3a::Tn2301) and infected with phage λ, we have isolated two defective phages having inserted pPJ3a DNA and Tn2301 in their genomes. One of them has been extensively characterized: it behaves like a cosmid, i.e., upon injection into the cell, its DNA circularizes and replicates as a plasmid (pPJ10); it can be packaged again in λ heads, provided the presence of a phage helper. Furthermore, heteroduplex analysis has shown that in pPJ10, the transposon Tn2301 is inverted compared to its direction in pPJ3b. We give evidence suggesting that this type of inversion is in part mediated by Tn2301.     

Tetracycline resistance transposon Tn1721: recA-dependent gene amplification and expression of tetracycline resistance

The 7.1-megadalton transposon Tn1721 codes for inducible tetracycline resistance (Tcr). The transposable element consists of a "minor transposon" (3.6 megadaltons) encoding functions required for transposition and a "tet region" (3.5 megadaltons) encoding resistance. Multiple tandem repeats of the tet region can be generated by recA-dependent gene amplification. This feature of Tn1721 has been used to analyze the relationship between gene dosage and Tcr. Derivatives of plasmid R388:Tn1721 containing from one to nine copies of the tet region were isolated and separately transformed into recA host cells, where they are stably maintained. The results of the study of Tcr in these strains were as follows: (i) the uninduced, "basal" level of Tcr was linearly related to gene dosage between 4 and 36 copies of tet per chromosome equivalent; (ii) the underlying mechanism could not be attributed to reduced accumulation of the drug; and (iii) induction with tetracycline elicited a four- to fivefold reduction in drug accumulation, independent of the gene dosage.     

A gene and its product required for transposition of resistance transposon Tn2603.

Tn2603 is a multiple-resistance transposon encoding resistance to ampicillin, streptomycin, sulfonamide, and mercury and having a molecular size of 20 kilobase pairs, with 200-base-pair inverted repeats at both ends. The essential sites and functions of Tn2603 which are required for its transposition were determined through construction and characterization of various deletion mutants affecting the efficiency of transposition. Deletions were introduced in plasmid pMK1::Tn2603 by partial digestion with restriction endonuclease EcoRI in vitro. Analysis of deletion mutants showed that the inverted repeat segments at both ends of the trans-acting diffusible product(s) encoded in the right-hand side of the central portion were required for the transposition of Tn2603. An essential gene product was revealed as a protein having a molecular weight of 110,000 by analysis of polypeptides synthesized in Escherichia coli minicells. This protein was assumed to be the so-called transposase.