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PubMed1981

Site-specific properties of Tn7 transposition into the E. coli chromosome.

A study has been made of the insertional properties of transposon Tn7, a 14 kilobase transposable element encoding resistances to trimethoprim, streptomycin and spectinomycin. It has previously been shown that Tn7 transposes at a low frequency and with low specificity into multiple sites in large transmissible plasmids. However, Tn7 transposes with extreme specificity and at high efficiency into the E. coli chromosome. In all cases we have studied, insertion of Tn7 into the chromosome has occurred at a unique site and with a unique orientation. A combination of genetic and biochemical techniques have been used to precisely locate this site on the E. coli chromosome to minute 82 on the linkage map between markers glmS and uncA. To investigate the nature of this highly specific transpositional event, a small region of the E. coli chromosome that includes the unique site, was cloned into the plasmid vector pBR322. Subsequently a lkb restriction fragment, including the Tn7 insertion site, was sub-cloned from this plasmid into the plasmid pACYC184. We show that Tn7 transposes into both these plasmid recombinants with the frequency and specificity characteristic of the E. coli chromosome.

Chromosome Mapping↗
PubMed1992

Design of a novel system for the construction of vectors for Agrobacterium-mediated plant transformation.

The loxP-Cre site-specific recombination system of phage P1 was used to develop a novel strategy to construct cointegrate vectors for Agrobacterium-mediated plant transformation. A pTi disarmed helper plasmid (pAL1166) was constructed by replacing the oncogenic T-DNA by a loxP sequence and a spectinomycin resistance marker in the octopine-type pTiB6 plasmid. The cre gene was cloned into an unstable incP plasmid. A third plasmid, which did not replicate in Agrobacterium and contained another loxP sequence together with a kanamycin resistance marker, was used to test the system. Electroporation of this third plasmid into an Agrobacterium strain harbouring both pAL1166 and the Cre-encoding plasmid resulted in kanamycin-resistant cells containing a cointegrate between pAL1166 and the incoming plasmid. Cointegration occurred by Cre-mediated recombination at the loxP sites, and the cointegrate was stabilized in the Agrobacterium cells by the loss of the Cre-encoding plasmid shortly after the recombination event had taken place.

Bacteriophage P1↗
PubMed1995

Marker rescue from the Nicotiana tabacum plastid genome using a plastid/Escherichia coli shuttle vector.

We recently reported an 868-bp plastid DNA minicircle, NICE1, that formed during transformation in a transplastomic Nicotiana tabacum line. Shuttle plasmids containing NICE1 sequences were maintained extrachromosomally in plastids and shown to undergo recombination with NICE1 sequences on the plastid genome. To prove the general utility of the shuttle plasmids, we tested whether plastid genes outside the NICE1 region could be rescued in Escherichia coli. The NICE1-based rescue plasmid, pNICER1, carries NICE1 sequences for maintenance in plastids, the ColE1 ori for maintenance in E. coli and a spectinomycin resistance gene (aadA) for selection in both systems. In addition, pNICER1 carries a defective kanamycin resistance gene, kan*, to target the rescue of a functional kanamycin resistance gene, kan, from the recipient plastid genome. pNICER1 was introduced into plastids where recombination could occur between the homologous kan/kan* sequences, and subsequently rescued in E. coli to recover the products of recombination. Based on the expression of kanamycin resistance in E. coli and the analysis of three restriction fragment polymorphisms, recombinant kan genes were recovered at a high frequency. Efficient rescue of kan from the plastid genome in E. coli indicates that NICE1-based plasmids are suitable for rescuing mutations from any part of the plastid genome, expanding the repertoire of genetic tools available for plastid biology.

Cloning, Molecular↗
PubMed1981

Translocation of sequences encoding antibiotic resistance from the chromosome to a receptor plasmid in Salmonella ordonez.

Salmonella ordonez strain BM2000 carries kanamycin (Km), ampicillin (Ap), spectinomycin (Sp), chloramphenicol (Cm), tetracycline (Tc), and sulfonamide (Su) resistance and production of colicin Ib (Cib). The Km and Cib characters were carried by a 97 kb IncI1 plasmid (pIP565). In addition to the Km and Cib traits, all or part of the other antibiotic resistance (R) determinants could be transferred by conjugation from S. ordonez to Escherichia coli where all the acquired characters are borne by an IncI1 plasmid, designated complete or partial composite plasmid respectively. DNA from pIP565 and composite plasmids and total DNA from strain BM2000 were studied by agarose and polyacrylamide gel electrophoresis following digestion with restriction endonucleases, and by Southern hybridization. These comparative analyses enabled us a) to show that acquisition by pIP565 of resistance to all or some of the antibiotics was due to the insertion of a single DNA fragment into the receptor plasmid; b) to detect two types of composite plasmids with regard to the specificity of insertion into pIP565 and the mapping of the inserts; c) to demonstrate that the ApCmSpSuTc resistance determinants were integrated into S. ordonez BM2000 chromosomal DNA; d) to map the restriction fragments of the translocatable sequence integrated into strain BM2000 chromosome or into pIP565. The results obtained suggest that two distinct mechanisms for the translocation of the R determinants coexist in S. ordonez BM2000. Recombination between two of the four directly repeated copies of the IS-like sequence (IS1522) present in S. ordonez chromosome leads to the circularisation of all or part of the ApCmSpSuTc R determinants and is followed by either 1) a second recombination with the copy of IS1522 in pIP565 (Type I composite plasmids), or 2) transposition of precise groups of characters in various sites of pIP565 (Type II composite plasmids).

Chromosomes, Bacterial↗
PubMed1995

Influence of salt concentration on the susceptibility of moderately halophilic bacteria to antimicrobials and its potential use for genetic transfer studies.

The influence of salinity on the susceptibility of 13 moderately halophilic collection strains belonging to the genera Chromohalobacter, Deleya, Halomonas, Vibrio, and Volcaniella to 10 common antimicrobials has been studied. Three different patterns of tolerance were found when salinity was varied from 10 to 1% (wt/vol) total salts in the testing media. The first one included the responses to ampicillin and rifampicin, where only minimal effects on the susceptibility were found. All moderate halophiles showed a high sensitivity to rifampicin regardless of the salt concentration. In the second group, including the responses to the aminoglycosides gentamycin, kanamycin, neomycin, and streptomycin, a remarkable and gradual increase of the toxicity was detected at lower salinities. Thirdly, the highest heterogeneity was found for the rest of antimicrobials assayed (trimethoprim, nalidixic acid, spectinomycin, and tetracycline), where the effect of salinity was moderate and dependent on both the individual strain and the antimicrobial tested. The data presented here should facilitate genetic studies on moderate halophiles. Thus, they simplify the design of selection media for genetic exchange experiments. Besides, by using low-salinity media, genes encoding resistance to a number of antimicrobials, especially to aminoglycosides, can be used as genetic markers for plasmids or transposons to be transferred to these extremophiles.

Anti-Bacterial Agents↗
PubMed1986

Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae.

A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tylr) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tylr phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyls) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyls mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyls mutant obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage lambda Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tylr gene is not closely linked to tylosin biosynthetic genes.

Cloning, Molecular↗
PubMed1983

An amber replication mutant of F plasmid mapped in the minimal replication region.

We have constructed a mini-F derivative (pKP1013) consisting of a 5.4 kilobase pairs (kb) segment (44.0 to 49.4 kb) of mini-F and fragments carrying the chloramphenicol and spectinomycin resistance genes that originated from the R plasmid NR1. The plasmid pKP1013 replicates autonomously in a manner indistinguishable from that of the parental mini-F. An amber mutant defective in replication has been isolated from pKP1013 by localized mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine. The virtual absence of incorporation of [3H]-thymidine into the plasmid DNA as well as the kinetics of appearance of plasmid-free segregants suggest that plasmid DNA synthesis is primarily affected under nonpermissive conditions. The amber mutation has been mapped within the 530 base pairs (bp) region that extends from 45.25 (XmaI) to 45.78 Kb (PstI) by extensive analysis of in vitro recombinants constructed from rep+ and rep- plasmids.

Chromosome Mapping↗
PubMed1988

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

A 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 containing 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 30,126 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.

Amino Acid Sequence↗
PubMed1975

Expression of ribosomal protein genes in Escherichia coli.

Streptomycin or spectinomycin treatment of an E. coli strain, carrying the strR and spcR alleles on the chromosome and the wild-type (sensitive) alleles on the episome, selects for inactivation of the relevant sensitive allele. After Mu induced mutagenesis, in the absence of selection against extended deletions upon the episome, a large proportion of stro mutants are also spco, and vice versa. However, when markers flanking the strA and spcA gene cluster are simultaneously selected, effectively eliminating long deletions, the majority of stro mutants continue to express the spcs allele, and vice versa. Insofar as inactivation after Mu treatment is due to prophage insertion within or proximal to the genes in question, this result indicates that the genes strA and spcA are not parts of a single operon. In virtue of the high frequency of extended deletions observed in the absence of suitable counter-selection, we must place a word of caution upon the use of phage Mu-1 as a means of isolating polar mutations and defining transcriptional units.

Bacterial Proteins↗
PubMed1982

Mutagenesis of Alcaligenes eutrophus by insertion of the drug-resistance transposon Tn5.

Drug-resistance element Tn5 coding for kanamycin resistance was used for mutagenesis of Alcaligenes eutrophus strain H16. The vehicle for introducing Tn5 into A. eutrophus was plasmid pJB4JI harboured by Escherichia coli. Kanamycin-resistant transconjugants occurred at a frequency of approximately 5 x 10(-8). One third of the transconjugants exhibited other plasmid-coded resistances such as gentamycin and spectinomycin. However, the latter markers were not stably maintained in the new host. Among the kanamycin-resistant transconjugants three classes of mutants were found: (i) Auxotrophic mutants occurred at a frequency of 0.8% and showed requirements for histidine, methionine, aspartate or isoleucine. Out of eleven auxotrophic mutants examined eight reverted to prototrophy. However, none of the revertants was kanamycin-sensitive. (ii) Mutants unable to grow with fructose as the carbon source occurred at a frequency of almost 10%. (iii) Mutants which had lost the ability to grow autotrophically with hydrogen and carbon dioxide were found at a frequency of 1%. Further analyses revealed that this class of mutants was either defective in carbon dioxide fixation or impaired in hydrogen metabolism.

Alcaligenes↗
PubMed1982

Genetic map of the crown gall suppressive IncW plasmid pSa.

A genetic map of the W incompatibility group plasmid pSa has been prepared through the construction of deletion derivatives of pSa and the cloning of various fragments of pSa in pBR322. Phenotypic analysis of these derivatives has identified the location of genes encoding resistance to chloramphenicol, sulfonamides, spectinomycin, streptomycin, kanamycin, gentamycin, and tobramycin. Information sufficient for the replication of the plasmid in both Escherichia coli and Agrobacterium tumefaciens is contained within a 4 kilobase pair region. Two regions have been identified as involved in the transfer of the plasmid; one of these regions is also involved in the inhibition of oncogenesis by pSa when it is present in an oncogenic strain of A. tumefaciens. Certain of the deletion derivatives of pSa are potential vectors for the cloning and analysis of A. tumefaciens Ti plasmid DNA.

Anti-Bacterial Agents↗
PubMed1997

Surveillance of sexually transmitted diseases in Switzerland, 1973-1994: evidence of declining trends in gonorrhoea and syphilis.

The HIV/AIDS epidemic has led to growing interest in the epidemiology of sexually transmitted diseases (STDs) in Switzerland. STD surveillance data from three sources are presented: reports from six policlinics of dermatovenereology since 1973, laboratory reports of Neisseria gonorrhoeae, Treponema pallidum and Chlamydia trachomatis since 1988, and reports by the Swiss Sentinel Network between June 1991 and December 1993. The data indicate that there has been a decline in the number of cases of syphilis and, in particular, gonorrhoea since the early 1980s in Switzerland. Since many factors could explain the declines (e.g. more effective treatments, more widespread use of antibiotics, marked changes in behaviour such as increased levels of condom use) it is difficult to identify their exact causes. Evidence for two of the before-mentioned factors exists and these probably played important roles in the declines. Firstly, as a result of the emergence of penicillinase producing strains of N. gonorrhoeae, the introduction of new treatments for gonorrhoea in the early 1980s which were simpler to administer and more effective (in particular the use of spectinomycin). Secondly, the national AIDS prevention campaign which began in 1987 and has been associated with major increases in condom use in the Swiss population.

Adolescent↗
PubMed1992

Diversity of aminoglycoside resistance in Enterobacter cloacae in Greece.

Ninety Enterobacter cloacae strains isolated from 12 Greek hospitals were examined in terms of epidemiological types and resistance mechanisms. Using O serotyping 69% of the strains were assigned to a specific serotype and overall 16 different serotypes were identified. The combination of serotyping, phagetyping and biotyping efficiently discriminated most of the strains, indicating that single epidemic strains were not prevalent, although serotypes 3, 7, and group II predominated. Eight representative strains, all resistant to gentamicin, tobramycin, amikacin and netilmicin, were further examined for transferability and mechanisms of resistance. Aminoglycoside resistance was found to be transferable in most strains, and 13 R plasmids of 40-120 MDa molecular weight were detected. The enzymes detected consisted of three enzymes active against gentamicin [ANT(2h'), AAC(3)-I and AAC(3)-V]; three active against tobramycin [ANT(2"), AAC(3)-V and AAC(6')-I]; two active against netilmicin [AAC(3)-V and AAC(6')-I]; and one active against amikacin [AAC(6')-I]. APH(3') and ANT (3"), which modify neomycin and streptomycin plus spectinomycin respectively, were also found. Overall up to five aminoglycoside modifying enzymes were detected on the same R plasmid, AAC(6')-I plus ANT(2") being the most prevalent. The high incidence of multiresistance in Enterobacter cloacae and the fact that resistance is due to enzymatic inactivation of the antibiotics, indicate that in Greece this species might act as a gene pool for the spread of resistance to other bacteria of clinical relevance.

Aminoglycosides↗
PubMed1996

Transgenic white clover. Studies with the auxin-responsive promoter, GH3, in root gravitropism and lateral root development.

We report improved method for white clover (Trifolium repens) transformation using Agrobacterium tumefaciens. High efficiencies of transgenic plant production were achieved using cotyledons of imbibed mature seed. Transgenic plants were recovered routinely from over 50% of treated cotyledons. The bar gene and phosphinothricin selection was shown to be a more effective selection system than nptII (kanamycin selection) or aadA (spectinomycin selection). White clover was transformed with the soybean auxin responsive promoter, GH3, fused to the GUS gene (beta-glucuronidase) to study the involvement of auxin in root development. Analysis of 12 independent transgenic plants showed that the location and pattern of GUS expression was consistent but the levels of expression varied. The level of GH3:GUS expression in untreated plants was enhanced specifically by auxin-treatment but the pattern of expression was not altered. Expression of the GH3:GUS fusion was not enhanced by other phytohormones. A consistent GUS expression pattern was evident in untreated plants presumably in response to endogenous auxin or to differences in auxin sensitivity in various clover tissues. In untreated plants, the pattern of GH3:GUS expression was consistent with physiological responses which are regarded as being auxin-mediated. For the first time it is shown that localised spots of GH3:GUS activity occurred in root cortical tissue opposite the sites where lateral roots subsequently were initiated. Newly formed lateral roots grew towards and through these islands of GH3:GUS expression, implying the importance of auxin in controlling lateral root development. Similarly, it is demonstrated for the first time that gravistimulated roots developed a rapid (within 1 h) induction of GH3:GUS activity in tissues on the non-elongating side of the responding root and this induction occurred concurrently with root curvature. These transgenic plants could be useful tools in determining the physiological and biochemical changes that occur during auxin-mediated responses.

Agrobacterium tumefaciens↗
PubMed1992

Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants.

Progress in plant molecular biology has depended heavily on the availability of effective vectors for plant cell transformation and heterologous expression. In this paper we describe the structures of a wide array of plasmids which have proved extremely effective in (a) plant transformation, (b) expression of heterologous genes and (c) assaying the activity of transposons in transgenic plants. Constructs that confer resistance to kanamycin, hygromycin, streptomycin, spectinomycin and phosphinotricin, or that confer beta-glucuronidase (GUS) gene expression are presented. Binary vector constructs that carry polylinkers of the pUC and Bluescript types are also described. Plasmids that permit the expression of any heterologous reading frame from either nopaline synthase (nos) or octopine synthase (ocs) promoters, as well as the cauliflower mosaic virus 35S promoter, using either the nopaline synthase or octopine synthase 3' polyadenylation sequences, are presented. These constructs permit a choice of orientation of the resulting transgene of interest, relative to the orientation of the selection marker gene. Most of the plasmids described here are publicly available.

Amino Acid Oxidoreductases↗
PubMed2002

Identification of Salmonella enterica serovar Typhimurium genes associated with growth suppression in stationary-phase nutrient broth cultures and in the chicken intestine.

Over 2,800 Tn 5 insertion mutants of Salmonella enterica sv. Typhimurium were screened for the loss of ability to suppress the multiplication of a spectinomycin-resistant (Spc(r)) but otherwise isogenic S. enterica sv. Typhimurium strain, when the Spc(r) mutant was added to 24-h LB broth cultures of the mutants. Selected "growth non-suppressive" (GNS) mutants were defective in respiration (insertions in arcA and fnr), amino acid biosynthesis (aroA and aroD), nutrient uptake and its regulation (tdcC and crp), and chemotaxis (fliD). In the last GNS mutant, the transposon inactivated yhjH, an ORF with unknown function which shows sequence similarity to di-guanylate cyclase and to novel two-component signal transduction proteins. In newly hatched chickens, all of the mutants, with the exception of the fliDmutant, were also unable to suppress colonization of the alimentary tract by the parent strain inoculated 1 day later. Defined mutations in luxS or sdiA,genes which contribute to quorum sensing in S. enterica sv. Typhimurium, had no effect on the stationary-phase growth suppression. Analysis of a transcriptional fusion construct indicated that yhjH was moderately expressed in the exponential phase of growth and up-regulated upon entry into stationary phase. Expression of yhjH was also considerably suppressed by the addition of supernatant from a 24-h stationary-phase S. enterica sv. Typhimurium culture, suggesting that the gene belongs to a new sensing and signaling regulatory pathway in S. enterica sv. Typhimurium.

Amino Acid Sequence↗
PubMed1998

Characterization of a new Agrobacterium tumefaciens strain from alfalfa.

Agrobacterium tumefaciens strain 1D1609 is reported here as the first field isolate from alfalfa (Medicago sativa L.). Unlike well-characterized A. tumefaciens strains such as C58 and Ach5, strain 1D1609 is highly virulent on alfalfa and has a distinctive host range. Interestingly, strain 1D1609 is naturally resistant to kanamycin and spectinomycin. The Ti plasmid in strain 1D1609 is an octopine-type; thus, tumors formed by strain 1D1609 synthesize octopine, which is utilized by the bacterium as a sole carbon source. Reciprocal exchange of Ti plasmids between strains 1D1609 and C58 showed that both chromosomal and Ti plasmid genes in strain 1D1609 contribute specifically to tumor formation on alfalfa. In addition, the nondormant CUF101 alfalfa cultivar from which strain 1D1609 was isolated was significantly more susceptible to all Agrobacterium strains tested than was the dormant Agate cultivar.

Agrobacterium tumefaciens↗
PubMed1999

Isolation and characterization of promoter regions from Streptococcus gordonii CH1.

We aimed to identify transcription signal sequences from Streptococcus gordonii strain CH1 by random chromosomal cloning. Five genomic fragments from a Sau3A digest, which constitutively activated transcription of a promoterless spectinomycin resistance gene in this strain, were isolated and characterized. Additionally, one promoter fragment was isolated that was specifically activated under iron-limiting conditions. A sequence motif with similarity to the consensus for Fur-binding regulatory DNA sequences (Fur box) in Escherichia coli was detected within the putative promoter region. The open reading frame downstream of this region possibly encodes a transmembrane protein involved in iron uptake.

Amino Acid Sequence↗