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43700.9508Cloning of genes responsible for acetic acid resistance in Acetobacter aceti. Five acetic acid-sensitive mutants of Acetobacter aceti subsp. aceti no. 1023 were isolated by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Three recombinant plasmids that complemented the mutations were isolated from a gene bank of the chromosome DNA of the parental strain constructed in Escherichia coli by using cosmid vector pMVC1. One of these plasmids (pAR1611), carrying about a 30-kilobase-pair (kb) fragment that conferred acetic acid resistance to all five mutants, was further analyzed. Subcloning experiments indicated that a 8.3-kb fragment was sufficient to complement all five mutations. To identify the mutation loci and genes involved in acetic acid resistance, insertional inactivation was performed by insertion of the kanamycin resistance gene derived from E. coli plasmid pACYC177 into the cloned 8.3-kb fragment and successive integration into the chromosome of the parental strain. The results suggested that three genes, designated aarA, aarB, and aarC, were responsible for expression of acetic acid resistance. Gene products of these genes were detected by means of overproduction in E. coli by use of the lac promoter. The amino acid sequence of the aarA gene product deduced from the nucleotide sequence was significantly similar to those of the citrate synthases (CSs) of E. coli and other bacteria. The A. aceti mutants defective in the aarA gene were found to lack CS activity, which was restored by introduction of a plasmid containing the aarA gene. A mutation in the CS gene of E. coli was also complemented by the aarA gene. These results indicate that aarA is the CS gene.19902156811
19610.9497A specialized citric acid cycle requiring succinyl-coenzyme A (CoA):acetate CoA-transferase (AarC) confers acetic acid resistance on the acidophile Acetobacter aceti. Microbes tailor macromolecules and metabolism to overcome specific environmental challenges. Acetic acid bacteria perform the aerobic oxidation of ethanol to acetic acid and are generally resistant to high levels of these two membrane-permeable poisons. The citric acid cycle (CAC) is linked to acetic acid resistance in Acetobacter aceti by several observations, among them the oxidation of acetate to CO2 by highly resistant acetic acid bacteria and the previously unexplained role of A. aceti citrate synthase (AarA) in acetic acid resistance at a low pH. Here we assign specific biochemical roles to the other components of the A. aceti strain 1023 aarABC region. AarC is succinyl-coenzyme A (CoA):acetate CoA-transferase, which replaces succinyl-CoA synthetase in a variant CAC. This new bypass appears to reduce metabolic demand for free CoA, reliance upon nucleotide pools, and the likely effect of variable cytoplasmic pH upon CAC flux. The putative aarB gene is reassigned to SixA, a known activator of CAC flux. Carbon overflow pathways are triggered in many bacteria during metabolic limitation, which typically leads to the production and diffusive loss of acetate. Since acetate overflow is not feasible for A. aceti, a CO(2) loss strategy that allows acetic acid removal without substrate-level (de)phosphorylation may instead be employed. All three aar genes, therefore, support flux through a complete but unorthodox CAC that is needed to lower cytoplasmic acetate levels.200818502856
81520.9489The sequence of the mer operon of pMER327/419 and transposon ends of pMER327/419, 330 and 05. Three different, independently isolated mercury-resistance-conferring plasmids, pMER327/419, pMER330 and pMER05, from cultures originating from the river Mersey (UK), contain identical regulatory merR genes and transposon ends. The mer determinant from pMER327/419 contains an additional potential ORF (ORF F) located between merP and merA when compared with the archetypal Tn501. Although these plasmids confer narrow-spectrum resistance (resistance to Hg2+, but not organomercurials) their merR genes encode a potential organomercurial-sensing protein. Transposition of the mer of pMER05 into plasmid RP4 was demonstrated and, as with Tn502 and Tn5053, insertion occurred at a specific region. The sequence of pMER05 is identical at the 'left' and 'right' termini and across merR to Tn5053, which was independently isolated from the chromosome of a Xanthomonas sp. bacteria from the Khaidarkan mercury mine in Kirgizia, former Soviet Union [Kholodii et al., J. Mol. Biol. 230 (1993a) 1103-1107]. The transpositional unit of pMER05 is, like that of Tn5053, bounded by DNA homologous to the imperfect 25-bp inverted repeats (IR) of the In2 integron, which brackets antibiotic-resistance cassettes in Tn21 subgroup transposons. At one end of the transposable element, and internal to the In2-like IR, is a 38-bp IR which closely resembles the IR that bounds Tn21.19948063107
53030.9480Location of the genes for anthranilate synthase in Streptomyces venezuelae ISP5230: genetic mapping after integration of the cloned genes. The anthranilate synthase (trpEG) genes in Streptomyces venezuelae ISP5230 were located by allowing a segregationally unstable plasmid carrying cloned S. venezuelae trpEG DNA and a thiostrepton resistance (tsr) marker to integrate into the chromosome. The integrated tsr was mapped by conjugation and transduction to a location close to tyr-2, between arg-6 and trpA13. A genomic DNA fragment containing trpC from S. venezuelae ISP5230 was cloned by complementation of a trpC mutation in Streptomyces lividans. Evidence from restriction enzyme analysis of the cloned DNA fragments, from Southern hybridization using the cloned trp DNA as probes, and from cotransduction frequencies, placed trpEG at a distance of 12-45 kb from the trpCBA cluster. The overall arrangement of tryptophan biosynthesis genes in the S. venezuelae chromosome differs from that in other bacteria examined so far.19938515229
80340.9479Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii. Azotobacter vinelandii contains a heterodimeric, membrane-bound [NiFe]hydrogenase capable of catalyzing the reversible oxidation of H2. The beta and alpha subunits of the enzyme are encoded by the structural genes hoxK and hoxG, respectively, which appear to form part of an operon that contains at least one further potential gene (open reading frame 3 [ORF3]). In this study, determination of the nucleotide sequence of a region of 2,344 bp downstream of ORF3 revealed four additional closely spaced or overlapping ORFs. These ORFs, ORF4 through ORF7, potentially encode polypeptides with predicted masses of 22.8, 11.4, 16.3, and 31 kDa, respectively. Mutagenesis of the chromosome of A. vinelandii in the area sequenced was carried out by introduction of antibiotic resistance gene cassettes. Disruption of hoxK and hoxG by a kanamycin resistance gene abolished whole-cell hydrogenase activity coupled to O2 and led to loss of the hydrogenase alpha subunit. Insertional mutagenesis of ORF3 through ORF7 with a promoterless lacZ-Kmr cassette established that the region is transcriptionally active and involved in H2 oxidation. We propose to call ORF3 through ORF7 hoxZ, hoxM, hoxL, hoxO, and hoxQ, respectively. The predicted hox gene products resemble those encoded by genes from hydrogenase-related operons in other bacteria, including Escherichia coli and Alcaligenes eutrophus.19921624446
81950.9470Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257. Trimethoprim resistance mediated by the Staphylococcus aureus multi-resistance plasmid pSK1 is encoded by a structure with characteristics of a composite transposon which we have designated Tn4003. Nucleotide sequence analysis of Tn4003 revealed it to be 4717 bp in length and to contain three copies of the insertion element IS257 (789-790 bp), the outside two of which are flanked by directly repeated 8-bp target sequences. IS257 has imperfect terminal inverted repeats of 27-28 bp and encodes for a putative transposase with two potential alpha-helix-turn-alpha-helix DNA recognition motifs. IS257 shares sequence similarities with members of the IS15 family of insertion sequences from Gram-negative bacteria and with ISS1 from Streptococcus lactis. The central region of the transposon contains the dfrA gene that specifies the S1 dihydrofolate reductase (DHFR) responsible for trimethoprim resistance. The S1 enzyme shows sequence homology with type I and V trimethoprim-resistant DHFRs from Gram-negative bacteria and with chromosomally encoded DHFRs from Gram-positive and Gram-negative bacteria. 5' to dfrA is a thymidylate synthetase gene, designated thyE.19892548057
53160.9470p-Aminobenzoic acid and chloramphenicol biosynthesis in Streptomyces venezuelae: gene sets for a key enzyme, 4-amino-4-deoxychorismate synthase. Amplification of sequences from Streptomyces venezuelae ISP5230 genomic DNA using PCR with primers based on conserved prokaryotic pabB sequences gave two main products. One matched pabAB, a locus previously identified in S. venezuelae. The second closely resembled the conserved pabB sequence consensus and hybridized with a 3.8 kb NcoI fragment of S. venezuelae ISP5230 genomic DNA. Cloning and sequence analysis of the 3.8 kb fragment detected three ORFs, and their deduced amino acid sequences were used in BLAST searches of the GenBank database. The ORF1 product was similar to PabB in other bacteria and to the PabB domain encoded by S. venezuelae pabAB. The ORF2 product resembled PabA of other bacteria. ORF3 was incomplete; its deduced partial amino acid sequence placed it in the MocR group of GntR-type transcriptional regulators. Introducing vectors containing the 3.8 kb NcoI fragment of S. venezuelae DNA into pabA and pabB mutants of Escherichia coli, or into the Streptomyces lividans pab mutant JG10, enhanced sulfanilamide resistance in the host strains. The increased resistance was attributed to expression of the pair of discrete translationally coupled p-aminobenzoic acid biosynthesis genes (designated pabB/pabA) cloned in the 3.8 kb fragment. These represent a second set of genes encoding 4-amino-4-deoxychorismate synthase in S. venezuelae ISP5230. In contrast to the fused pabAB set previously isolated from this species, they do not participate in chloramphenicol biosynthesis, but like pabAB they can be disrupted without affecting growth on minimal medium. The gene disruption results suggest that S. venezuelae may have a third set of genes encoding PABA synthase.200111495989
300070.9464A large conjugative Acinetobacter baumannii plasmid carrying the sul2 sulphonamide and strAB streptomycin resistance genes. Acinetobacter baumannii is an important nosocomial pathogen that often complicates treatment because of its high level of resistance to antibiotics. Though plasmids can potentially introduce various genes into bacterial strains, compared to other Gram-negative bacteria, information about the unique A. baumannii plasmid repertoire is limited. Here, whole genome sequence data was used to determine the plasmid content of strain A297 (RUH875), the reference strain for the globally disseminated multiply resistant A. baumannii clone, global clone 1(GC1). A297 contains three plasmids. Two known plasmids were present; one, pA297-1 (pRAY*), carries the aadB gentamicin, kanamycin and tobramycin resistance gene and another is an 8.7kb cryptic plasmid often found in GC1 isolates. The third plasmid, pA297-3, is 200kb and carries the sul2 sulphonamide resistance gene and strAB streptomycin resistance gene within Tn6172 and a mer mercuric ion resistance module elsewhere. pA297-3 transferred sulphonamide, streptomycin and mercuric ion resistance at high frequency to a susceptible A. baumannii recipient, and contains several genes potentially involved in conjugative transfer. However, a relaxase gene was not found. It also includes several genes encoding proteins involved in DNA metabolism such as partitioning. However, a gene encoding a replication initiation protein could not be found. pA297-3 includes two copies of a Miniature Inverted-Repeat Transposable Element (MITE), named MITE-297, bracketing a 77.5kb fragment, which contains several IS and the mer module. Several plasmids related to but smaller than pA297-3 were found in the GenBank nucleotide database. They were found in different A. baumannii clones and are wide spread. They all contain either Tn6172 or a variant in the same position in the backbone as Tn6172 in pA297-3. Some related plasmids have lost the segment between the MITE-297 copies and retain only one MITE-297. Others have segments of various lengths between two MITE-297 copies, and these can be derived from the region in pA297-3 via a deletion adjacent to IS related to IS26 such as IS1007 or IS1007-like. pA297-3 and its relatives represent a third type of conjugative Acinetobacter plasmid that contributes to the dissemination of antibiotic resistance in this species.201627601280
82080.9457Nucleotide sequence analysis of a transposon (Tn5393) carrying streptomycin resistance genes in Erwinia amylovora and other gram-negative bacteria. A class II Tn3-type transposable element, designated Tn5393 and located on plasmid pEa34 from streptomycin-resistant strain CA11 of Erwinia amylovora, was identified by its ability to move from pEa34 to different sites in plasmids pGEM3Zf(+) and pUCD800. Nucleotide sequence analysis reveals that Tn5393 consists of 6,705 bp with 81-bp terminal inverted repeats and generates 5-bp duplications of the target DNA following insertion. Tn5393 contains open reading frames that encode a putative transposase (tnpA) and resolvase (tnpR) of 961 and 181 amino acids, respectively. The two open reading frames are separated by a putative recombination site (res) consisting of 194 bp. Two streptomycin resistance genes, strA and strB, were identified on the basis of their DNA sequence homology to streptomycin resistance genes in plasmid RSF1010. StrA is separated from tnpR by a 1.2-kb insertion element designated IS1133. The tnpA-res-tnpR region of Tn5393 was detected in Pseudomonas syringae pv. papulans Psp36 and in many other gram-negative bacteria harboring strA and strB. Except for some strains of Erwinia herbicola, these other gram-negative bacteria lacked insertion sequence IS1133. The prevalence of strA and strB could be accounted for by transposition of Tn5393 to conjugative plasmids that are then disseminated widely among gram-negative bacteria.19938380801
39790.9455PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and lambda-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriT(RK2) for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriT(RK2) to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.200312563033
3018100.9455The large Bacillus plasmid pTB19 contains two integrated rolling-circle plasmids carrying mobilization functions. Plasmid pTB19 is a 27-kb plasmid originating from a thermophilic Bacillus species. It was shown previously that pTB19 contains an integrated copy of the rolling-circle type plasmid pTB913. Here we describe the analysis of a 4324-bp region of pTB19 conferring resistance to tetracycline. The nucleotide sequence of this region revealed all the characteristics of a second plasmid replicating via the rolling-circle mechanism. This sequence contained (i) the tetracycline resistance marker of pTB19, which is highly similar to other tetL-genes of gram-positive bacteria; (ii) a hybrid mob gene, which bears relatedness to both the mob-genes of pUB110 and pTB913; (iii) a palU type minus origin identical to those of pUB110 and pTB913; and (iv) a plus origin of replication similar to that of pTB913. A repB-type replication initiation gene sequence identical to that of pTB913 was present, which lacked the middle part (492 bp), thus preventing autonomous replication of this region. The hybrid mob gene was functional in conjugative mobilization of plasmids between strains of Bacillus subtilis.19911946749
335110.9452Construction and characterization of a replication-competent retroviral shuttle vector plasmid. We constructed two versions of an RCASBP-based retroviral shuttle vector, RSVP (RCASBP shuttle vector plasmid), containing either the zeocin or blasticidin resistance gene. In this vector, the drug resistance gene is expressed in avian cells from the long terminal repeat (LTR) promoter, whereas in bacteria the resistance gene is expressed from a bacterial promoter. The vector contains a bacterial origin of replication (ColE1) to allow circular viral DNA to replicate as a plasmid in bacteria. The vector also contains the lac operator sequence, which binds to the lac repressor protein, providing a simple and rapid way to purify the vector DNA. The RSVP plasmid contains the following sequence starting with the 5" end: LTR, gag, pol, env, drug resistance gene, lac operator, ColE1, LTR. After this plasmid was transfected into DF-1 cells, we were able to rescue the circularized unintegrated viral DNA from RSVP simply by transforming the Hirt DNA into Escherichia coli. Furthermore, we were able to rescue the integrated provirus. DNA from infected cells was digested with an appropriate restriction enzyme (ClaI) and the vector-containing segments were enriched using lac repressor protein and then self-ligated. These enriched fractions were used to transform E. coli. The transformation was successful and we did recover integration sites, but higher-efficiency rescue was obtained with electroporation. The vector is relatively stable upon passage in avian cells. Southern blot analyses of genomic DNAs derived from successive viral passages under nonselective conditions showed that the cassette (drug resistance gene-lac operator-ColE1) insert was present in the vector up to the third viral passage for both resistance genes, which suggests that the RSVP vectors are stable for approximately three viral passages. Together, these results showed that RSVP vectors are useful tools for cloning unintegrated or integrated viral DNAs.200211799171
818120.9448Characterization of a staphylococcal plasmid related to pUB110 and carrying two novel genes, vatC and vgbB, encoding resistance to streptogramins A and B and similar antibiotics. We isolated and sequenced a plasmid, named pIP1714 (4,978 bp), which specifies resistance to streptogramins A and B and the mixture of these compounds. pIP1714 was isolated from a Staphylococcus cohnii subsp. cohnii strain found in the environment of a hospital where pristinamycin was extensively used. Resistance to both compounds and related antibiotics is encoded by two novel, probably cotranscribed genes, (i) vatC, encoding a 212-amino-acid (aa) acetyltransferase that inactivates streptogramin A and that exhibits 58.2 to 69.8% aa identity with the Vat, VatB, and SatA proteins, and (ii) vgbB, encoding a 295-aa lactonase that inactivates streptogramin B and that shows 67% aa identity with the Vgb lactonase. pIP1714 includes a 2,985-bp fragment also found in two rolling-circle replication and mobilizable plasmids, pUB110 and pBC16, from gram-positive bacteria. In all three plasmids, the common fragment was delimited by two direct repeats of four nucleotides (GGGC) and included (i) putative genes closely related to repB, which encodes a replication protein, and to pre(mob), which encodes a protein required for conjugative mobilization and site-specific recombination, and (ii) sequences very similar to the double- and single-strand origins (dso, ssoU) and the recombination site, RSA. The antibiotic resistance genes repB and pre(mob) carried by each of these plasmids were found in the same transcriptional orientation.19989661023
403130.9447Nucleotide sequence and expression of the mercurial-resistance operon from Staphylococcus aureus plasmid pI258. The mercurial-resistance determinant from Staphylococcus aureus plasmid pI258 is located on a 6.4-kilobase-pair Bgl II fragment. The determinant was cloned into both Bacillus subtilis and Escherichia coli. Mercury resistance was found only in B. subtilis. The 6404-base-pair DNA sequence of the Bgl II fragment was determined. The mer DNA sequence includes seven open reading frames, two of which have been identified by homology with the merA (mercuric reductase) and merB (organomercurial lyase) genes from the mercurial-resistance determinants of Gram-negative bacteria. Whereas 40% of the amino acid residues overall were identical between the pI258 merA polypeptide product and mercuric reductases from Gram-negative bacteria, the percentage identity in the active-site positions and those thought to be involved in NADPH and FAD contacts was above 90%. The 216 amino acid organomercurial lyase sequence was 39% identical with that from a Serratia plasmid, with higher conservation in the middle of the sequences and lower homologies at the amino and carboxyl termini. The remaining five open reading frames in the pI258 mer sequence have no significant homologies with the genes from previously sequenced Gram-negative mer operons.19873037534
344140.9446Identification of genes in Rhizobium leguminosarum bv. trifolii whose products are homologues to a family of ATP-binding proteins. The specific interaction between rhizobia and their hosts requires many genes that influence both early and late steps in symbiosis. Three new genes, designated prsD, prsE (protein secretion) and orf3, were identified adjacent to the exo133 mutation in a cosmid carrying the genomic DNA of Rhizobium leguminosarum bv. trifolii TA1. The prsDE genes share significant homology to the genes encoding ABC transporter proteins PrtDE from Erwinia chrysanthemi and AprDE from Pseudomonas aeruginosa which export the proteases in these bacteria. PrsD shows at least five potential transmembrane hydrophobic regions and a large hydrophilic domain containing an ATP/GTP binding cassette. PrsE has only one potential transmembrane hydrophobic domain in the N-terminal part and is proposed to function as an accessory factor in the transport system. ORF3, like PrtF and AprF, has a typical N-terminal signal sequence but has no homology to these proteins. The insertion of a kanamycin resistance cassette into the prsD gene of the R. leguminosarum bv. trifolii TA1 wild-type strain created a mutant which produced a normal amount of exopolysaccharide but was not effective in the nodulation of clover plants.19979141701
3028150.9446Novel macrolide resistance module carried by the IncP-1beta resistance plasmid pRSB111, isolated from a wastewater treatment plant. The macrolide resistance plasmid pRSB111 was isolated from bacteria residing in the final effluents of a wastewater treatment plant. The 47-kb plasmid confers resistance to azithromycin, clarithromycin, erythromycin, roxithromycin, and tylosin when it is carried by Pseudomonas sp. strain B13 and is very similar to prototype IncP-1beta plasmid pB3, which was previously isolated from an activated-sludge bacterial community of a wastewater treatment plant. The two plasmids differ in their accessory regions, located downstream of the conjugative transfer module gene traC. Nucleotide sequence analysis of the pRSB111 accessory region revealed that it contains a new macrolide resistance module composed of the genes mphR(E), mph(E), and mrx(E), which putatively encode a transcriptional regulator, a macrolide phosphotransferase, and a transmembrane transport protein, respectively. Analysis of the contributions of the individual genes of the macrolide resistance module revealed that mph(E) and mrx(E) are required for high-level macrolide resistance. The resistance genes are flanked by two insertion sequences, namely, ISPa15 and ISRSB111. Two truncated transposable elements, IS6100 and remnants of a Tn3-like transposon, were identified in the vicinity of ISRSB111. The accessory element of pRSB111 apparently replaced the Tn402-like element present on the sister plasmid, pB3, as suggested by the conservation of Tn402-specific terminal inverted repeats on pRSB111.200717101677
431160.9446Nucleotide sequence analysis of the complement resistance gene from plasmid R100. The multiple antibiotic resistance plasmid R100 renders Escherichia coli resistant to the bactericidal action of serum complement. We constructed a plasmid (pOW3) consisting of a 1,900-base-pair-long restriction fragment from R100 joined to a 2,900-base-pair-long fragment of pBR322 carrying ampicillin resistance. E. coli strains carrying pOW3 or R100 were up to 10,000-fold less sensitive to killing by serum complement than were plasmid-free bacteria or bacteria carrying pBR322. Nucleotide sequencing revealed that 875 of the 1,900 bases from R100 correspond exactly to part of the bacterial insertion sequence IS2. The remaining 1,075 bases contained only one sizeable open reading frame; it covered 729 base pairs (243 amino acids) and was preceded by nucleotide sequences characteristic of bacterial promoters and ribosome binding sites. The first 20 amino acids of the predicted protein showed features characteristic of a signal sequence. The remainder of the predicted protein showed an amino acid composition almost identical with that determined for the traT protein from the E. coli F factor. Southern blot analysis showed that the resistance gene from R100 does not hybridize to the serum resistance gene from ColV,I-K94 isolated by Binns et al.; we concluded that these genes are distinct.19826284713
3049170.9444Characterisation 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.200314511653
532180.9442Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain.199910514571
821190.9441DNA probes for studying streptothricin resistance evolution in enteric bacteria. Probes for the detection of streptothricin resistance genes have been derived from recombinant plasmids. These include the streptothricin resistance gene probe sat 1/2 derived from Tn 1826 and specific for both the sat-1 determinant of Tn 1825 and the sat-2 determinant of Tn 1826, and the probe sat D derived from and specific for the sat-1 determinant of transposon Tn 1825. A third streptothricin resistance gene probe, sat 3, represents the streptothricin resistance determinant sat-3 of the IncQ R plasmid pIE639. Hybridization studies did not reveal any sequence homology between sat-3 and the transposon-localized sat-1 and sat-2 determinants. Moreover, non of the different sat-determinants isolated from plasmids of gram negative bacteria hybridized with the analogous resistance determinant of Streptomyces noursei, which had been cloned and named nat by Krügel et al. (Gene, 1988, 62, 209-214). The sat 1/2 probe in combination with the sat D probe proved to be suitable for the identification and the differentiation of sat-1 and sat-2 determinants in different genetic environments. Streptothricin resistance genes related to those present on transposons Tn 1825 and Tn 1826 have been detected by hybridization with the probe sat 1/2 on plasmids isolated a long time ago before the application of streptothricins. The sat-3 determinant appears to be exclusively associated with the IncQ plasmid pIE639.19902166786