The Construction and Monitoring of Genetically Tagged,Plasmid-Containing,Naphthalene-Degrading Strains in Soil

A genetically tagged, plasmid-containing, naphthalene-degrading strain, Pseudomonas putida KT2442(pNF142:: TnMod-OTc), has been constructed. The presence of the gfp gene (which codes for green fluorescent protein) and the kanamycin and rifampicin resistance genes in the chromosome of this strain allows the strain’s fate in model soil systems to be monitored, whereas a minitransposon, inserted into naphthalene biodegradation plasmid pNF142 and containing the tetracycline resistance gene, makes it possible to follow the horizontal transfer of this plasmid between various bacteria. Plasmid pNF142::TnMod-OTc is stable in strain P. putida KT2442 under nonselective conditions. The maximal specific growth rate of this strain on naphthalene is found to be higher than that of the natural host of plasmid pNF142. When introduced into a model soil system, the genetically tagged strain is stable and competitive for 40 days. The transfer of labeled plasmid pNF142::TnMod-OTc to natural soil bacteria, predominantly fluorescent pseudomonads, has been detected.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 526–532.Original Russian Text Copyright © 2005 by Filonov, Akhmetov, Puntus, Esikova, Gafarov, Izmalkova, Sokolov, Kosheleva, Boronin.     

Mobilization of vancomycin resistance by transposon-mediated fusion of a VanA plasmid with an Enterococcus faecium sex pheromone-response plasmid

A striking feature of recent outbreaks of vancomycin-resistant (Vm^R) enterococci is the apparent horizontal dissemination of resistance determinants. The plasmids pHKK702 and pHKK703 from Enterococcus faecium clinical isolate R7 have been implicated in the conjugal transfer of Vm^R. pHKK702 is a 41-kb plasmid that contains an element indistinguishable from the glycopeptide-resistance transposon Tn1546. pHKK703 is an approx. 55-kb putative sex pheromone-response plasmid that is required for conjugative mobilization of pHKK702. During experiments in which strain R7 was used as a donor, a highly conjugative Vm^R transconjugant was isolated that formed constitutive cellular aggregates. Restriction analyses and DNA hybridizations revealed that the transconjugant harbored a single plasmid of approx. 92 kb and this plasmid (pHKK701) was composed of DNA from both pHKK702 and pHKK703. Results from DNA sequence analyses showed that a 39-kb composite transposon (Tn5506) from pHKK702 had inserted into pHKK703. The left end of Tn5506 contained a single insertion sequence (IS) element, IS1216V2, whereas the right end was composed of a tandem IS structure consisting of the novel 1065-bp IS1252 nested within an IS1216V1 element. Transposition of Tn5506 from pHKK702 to pHKK703 created an 8-bp target sequence duplication at the site of insertion and interrupted an ORF (ORFX) that was 91% identical to that of prgX, a gene proposed to negatively regulate sex pheromone response of the E. faecalis plasmid, pCF10. We propose that the interruption of ORFX by Tn5506 led to the constitutive cellular aggregation phenotype and thereby enhanced the efficiency with which Vm^R was transferred. Similar IS1216V-mediated transposition events may contribute to the horizontal spread of glycopeptide resistance among enterococci in nature.     

Construction of a thermo-sensitive pRI857 vector for efficient DNA capturing in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To establish a positive cloning system with a zero background for high-throughput DNA cloning purpose.

Results

The cloning vector, pRI857, and the genomic-library construction vector, pRI857-BAC, were constructed based on the mechanism of expression of the thermo-sensitive cI857 repressor gene that can stringently repress the P_R promoter and kanamycin resistance gene (P_R-kan ^R ) at 30 °C, but have no effect on P_R-kan ^R gene at 37 °C or at higher temperatures. When the pRI857 vectors were transformed into E. coli with or without a target foreign DNA fragment inserted at the BfrBI site of the cI857 gene, only colonies with the foreign DNA fragment survive. We extended this method to construct a pRI857-BAC vector for genomic library cloning which displays an efficiency of ~10⁷ cfu per µg of genomic DNA, with no empty vectors detected.

Conclusions

Cloning by indirect activation of resistance marker gene represents a novel DNA-capturing system, which can be widely applied for high-throughput DNA cloning.

     

Distribution of Tetracycline Resistance Genes and Transposons among Phylloplane Bacteria in Michigan Apple Orchards

The extent and nature of tetracycline resistance in bacterial populations of two apple orchards with no or a limited history of oxytetracycline usage were assessed. Tetracycline-resistant (Tc^r) bacteria were mostly gram negative and represented from 0 to 47% of the total bacterial population on blossoms and leaves (versus 26 to 84% for streptomycin-resistant bacteria). A total of 87 isolates were screened for the presence of specific Tc^r determinants. Tc^r was determined to be due to the presence of Tet B in Pantoea agglomerans and other members of the family Enterobacteriacae and Tet A, Tet C, or Tet G in most Pseudomonas isolates. The cause of Tc^r was not identified in 16% of the isolates studied. The Tc^r genes were almost always found on large plasmids which also carried the streptomycin resistance transposon Tn5393. Transposable elements with Tc^r determinants were detected by entrapment following introduction into Escherichia coli. Tet B was found within Tn10. Two of eighteen Tet B-containing isolates had an insertion sequence within Tn10; one had IS911 located within IS10-R and one had Tn1000 located upstream of Tet B. Tet A was found within a novel variant of Tn1721, named Tn1720, which lacks the left-end orfI of Tn1721. Tet C was located within a 19-kb transposon, Tn1404, with transposition genes similar to those of Tn501, streptomycin (aadA2) and sulfonamide (sulI) resistance genes within an integron, Tet C flanked by direct repeats of IS26, and four open reading frames, one of which may encode a sulfate permease. Two variants of Tet G with 92% sequence identity were detected.     

A pBRINT family of plasmids for integration of cloned DNA into the Escherichia coli chromosome

Plasmid pBRINT is an efficient vector for chromosomal integration of cloned DNA into the lacZ gene of Escherichia coli [Balbás et al., Gene 136(1993) 211–213]. A family of related plasmids containing different antibiotic-resistance markers (Cm^R or Gm^R or Km^R and a larger multiple cloning site (MCS) has been constructed. This set of plasmids, whose integration efficiencies are as good as those obtained with the prototype plasmid pBRINT, constitutes a collection of tools that allow rapid and easy integration of cloned DNA, at the chromosomal level. Their functionality as integration vectors has been ascertained by integrating the Vitreoscilla sp. hemoglobin-encoding gene and the Photobacterium leiognathi lux genes. To evaluate the level of expression obtained after chromosomal integration, we constructed strains carrying one or two copies of the cat gene integrated in the chromosome, and compared their enzymatic activities with those obtained from a strain carrying cat on a multicopy plasmid.     

The isolation and characterisation of a plaque-forming derivative of bacteriophage Mu carrying a fragment of Tn3 conferring ampicillin resistance.

Summary We have isolated a plaque-forming derivative of phage Mu which carries a determinant for Ap^R. The biological properties of this MuAp phage are similar to those of normal Mu. Its genome contains a 1.1 kb substitution where Mu DNA from the right end of the G region has been replaced by a similar length of DNA from the transposon Tn3. This fragment of Tn3 DNA carries the Ap^R gene, but is no longer capable of independent transposition.     

Isolation of P22 Specialized Transducing Phage following F''-Episome Fusion

A P22 specialized transducing phage has been constructed which carries the structural gene for aspartate transcarbamylase (ATCase). This gene (pyrB) was first brought close to the P22 attachment site by fusing an F' pyrB⁺ episome to an F' prolac episome which carries a P22 prophage attachment site. A prophage was added to these fused F' episomes and the lysogen was UV-induced. The specialized transducing phage was isolated from the resulting lysate. The phage also carries argI, the structure gene for ornithine transcarbamylase.     

Inheritance of evolved glyphosate resistance in <Emphasis Type="Italic">Conyza canadensis</Emphasis> (L.) Cronq.

N-(phosphonomethyl)glycine (glyphosate) resistance was previously reported in a horseweed [Conyza (=Erigeron) canadensis (L.) Cronq.] population from Houston, DE (P ₀ ^R ). Recurrent selection was performed on P ₀ ^R , since the population was composed of susceptible (5%) and resistant (95%) phenotypes. After two cycles of selection at 2.0 kg ae glyphosate ha^–1, similar glyphosate rates that reduced plant growth by 50%, glyphosate rates that inflicted 50% mortality in the population, and accumulations of half of the maximum detectable shikimic acid concentration were observed between the parental P ₀ ^R and the first (RS₁) and second (RS₂) recurrent generations. In addition, RS₁ and RS₂ did not segregate for resistance to glyphosate. This suggested that the RS₂ population comprised a near-homozygous, glyphosate-resistant line. Whole-plant rate responses estimated a fourfold resistance increase to glyphosate between RS₂ and either a pristine Ames, IA (P ₀ ^P ) or a susceptible C. canadensis population from Georgetown, DE (P ₀ ^S ). The genetics of glyphosate resistance in C. canadensis was investigated by performing reciprocal crosses between RS₂ and either the P ₀ ^P or P ₀ ^S populations. Evaluations of the first (F₁) and second (F₂) filial generations suggested that glyphosate resistance was governed by an incompletely dominant, single-locus gene (R allele) located in the nuclear genome. The proposed genetic model was confirmed by back-crosses of the F₁ to plants that arose from achenes of the original RS₂, P ₀ ^P , or P ₀ ^S parents. The autogamous nature of C. canadensis, the simple inheritance model of glyphosate resistance, and the fact that heterozygous genotypes (F₁) survived glyphosate rates well above those recommended by the manufacturer, predicted a rapid increase in frequency of the R allele under continuous glyphosate selection. The impact of genetics on C. canadensis resistance management is discussed.     

Genomic islands 1 and 2 play key roles in the evolution of extensively drug-resistant ST235 isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa are noscomially acquired, opportunistic pathogens that pose a major threat to the health of burns patients and the immunocompromised. We sequenced the genomes of P. aeruginosa isolates RNS_PA1, RNS_PA46 and RNS_PAE05, which displayed resistance to almost all frontline antibiotics, including gentamicin, piperacillin, timentin, meropenem, ceftazidime and colistin. We provide evidence that the isolates are representatives of P. aeruginosa sequence type (ST) 235 and carry Tn6162 and Tn6163 in genomic islands 1 (GI1) and 2 (GI2), respectively. GI1 disrupts the endA gene at precisely the same chromosomal location as in P. aeruginosa strain VR-143/97, of unknown ST, creating an identical CA direct repeat. The class 1 integron associated with Tn6163 in GI2 carries a bla_GES-5–aacA4–gcuE15–aphA15 cassette array conferring resistance to carbapenems and aminoglycosides. GI2 is flanked by a 12 nt direct repeat motif, abuts a tRNA-gly gene, and encodes proteins with putative roles in integration, conjugative transfer as well as integrative conjugative element-specific proteins. This suggests that GI2 may have evolved from a novel integrative conjugative element. Our data provide further support to the hypothesis that genomic islands play an important role in de novo evolution of multiple antibiotic resistance phenotypes in P. aeruginosa.     

Transpositional Mutagenesis of Thiobacillus novellus and Thiobacillus versutus

Transpositional mutagenesis of Thiobacillus novellus by Tn501 was achieved by means of the incompatibility of IncP plasmids. Tn501 insertion caused three types of mutant phenotypes: isoleucine auxotrophy, lysine auxotrophy, and a reduced ability to oxidize reduced sulfur compounds and to fix CO₂. Oxidation rates for elemental sulfur (S⁰), thiosulfate (S₂O₃²⁻), and tetrathionate (S₄O₆²⁻) in mutants of the latter type were reduced relative to those of the nonmutant control strain. Incorporation of labeled bicarbonate (H¹⁴CO₃⁻) was also significantly impaired. Although suicide vehicles were not useful for the introduction of transposons into T. novellus, this method was effective for the Tn1721-induced mutagenesis of Thiobacillus versutus. Tn1721 insertions resulted in the loss of the natural resistance of T. versutus to arsenate and gentamicin and in auxotrophies for isoleucine-valine, arginine, phenylalanine, valine, and panthothenate. Transpositional mutagenesis by either method should prove to be a useful tool for further study of these and other members of the genus Thiobacillus.     

Reconstitution of the R compound allele in maize

The R^r:standard allele in maize, which conditions anthocyanin pigmentation in plant and seed tissues in the presence of appropriate complementary factors, is associated with a tandem duplication. The proximal member of the duplication carries P, the plant pigmenting determiner and the distal member member carries S, the seed pigmenting determiner. Derivatives from R^r that have lost S function are designated r^r. They represent either losses of the distal member of the duplication (P derivatives) or mutations of S to s (P s). Derivatives that have lost P function are designated R^g, and represent either losses of the proximal member of the duplication (S derivatives) or mutations of P to p (p S).—All four possible types of r^r/R^g heterozygotes were tested for their capacity to yield R^r reconstitution by crossing over. No R^r derivatives were obtained from P/S heterozygotes, a result consistent with the view that P and S occupy corresponding positions in homologous chromosome segments. R^r reconstitution was detected in both tandem duplication heterozygotes P s/S and P/p S, and was found to be about ten times more frequent in the latter. The ratio of R^r reconstitution in the two heterozygotes is a function of position of the anthocyanin marker within the duplicated segment. The data from these heterozygotes allow one to measure the distance between P and S, that is to say, the genetic length of the duplicated segment. This distance was found to be 0.16 map units. The highest frequency of R^r reconstitution was obtained from P s/p S heterozygotes, since direct pairing (see PDF) as well as the p//s type of displaced pairing have the potential to produce R^r derivatives. One of the R^g derivatives used in this study, R^g₆, was found to back-mutate in some sublines to R^r. The basis for this instability remains unknown.     

Influence of periplasmic oxidation of glucose on pyoverdine synthesis in Pseudomonas putida S11

Fluorescent pseudomonads catabolize glucose simultaneously by two different pathways, namely, the oxidative pathway in periplasm and the phosphorylative pathway in cytoplasm. This study provides evidence for the role of glucose metabolism in the regulation of pyoverdine synthesis in Pseudomonas putida S11. We have characterized the influence of direct oxidation of glucose in periplasm on pyoverdine synthesis in P. putida S11. We identified a Tn5 transposon mutant of P. putida S11 showing increased pyoverdine production in minimal glucose medium (MGM). This mutant designated as IST1 had Tn5 insertion in glucose dehydrogenase (gcd) gene. To verify the role of periplasmic oxidation of glucose on pyoverdine synthesis, we constructed mutants S11 Gcd^? and S11 PqqF^? by antibiotic cassette mutagenesis. These mutants of P. putida S11 with loss of glucose dehydrogenase gene (gcd) or cofactor pyrroloquinoline quinone biosynthesis gene (pqqF) showed increased pyoverdine synthesis and impaired acid production in MGM. In minimal gluconate medium, the pyoverdine production of wild-type strain S11 and mutants S11 Gcd^? and S11 PqqF^? was higher than in MGM indicating that gluconate did not affect pyoverdine synthesis. In MGM containing PIPES–NaOH (pH?7.5) buffer which prevent pH changes due to gluconic acid production, strain S11 produced higher amount of pyoverdine similar to mutants S11 Gcd^? and S11 PqqF^?. Therefore, it is proposed that periplasmic oxidation of glucose to gluconic acid decreases the pH of MGM and thereby influences pyoverdine synthesis of strain S11. The increased pyoverdine synthesis enhanced biotic and abiotic surface colonization of the strain S11.     

HrpM is involved in glucan biosynthesis,biofilm formation and pathogenicity in Xanthomonas citri ssp. citri

Xanthomonas citri ssp. citri (Xcc) is the causal agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. A biofilm‐deficient mutant was identified in a screening of a transposon mutagenesis library of the Xcc 306 strain constructed using the commercial Tn5 transposon EZ‐Tn5 <KAN‐2> Tnp Transposome (Epicentre). Sequence analysis of a mutant obtained in the screening revealed that a single copy of the EZ‐Tn5 was inserted at position 446 of hrpM, a gene encoding a putative enzyme involved in glucan synthesis. We demonstrate for the first time that the product encoded by the hrpM gene is involved in β‐1,2‐glucan synthesis in Xcc. A mutation in hrpM resulted in no disease symptoms after 4 weeks of inoculation in lemon and grapefruit plants. The mutant also showed reduced ability to swim in soft agar and decreased resistance to H ₂ O ₂ in comparison with the wild‐type strain. All defective phenotypes were restored to wild‐type levels by complementation with the plasmid pBBR1‐MCS containing an intact copy of the hrpM gene and its promoter. These results indicate that the hrpM gene contributes to Xcc growth and adaptation in its host plant.     

Stable integration and expression of heterologous genes in several lactobacilli using an integration vector constructed from the integrase and attP sequences of phage ΦAT3 isolated from Lactobacillus casei ATCC 393

An integration vector capable of stably integrating and maintaining in the chromosomes of several lactobacilli over hundreds of generations has been constructed. The major integration machinery used is based on the ΦAT3 integrase (int) and attP sequences determined previously. A novel core sequence located at the 3′ end of the tRNA^leu gene is identified in Lactobacillus fermentum ATCC 14931 as the integration target by the integration vector though most of such sequences found in other lactobacilli are similar to that determined previously. Due to the lack of an appropriate attB site in Lactococcus lactis MG1363, the integration vector is found to be unable to integrate into the chromosome of the strain. However, such integration can be successfully restored by cotransforming the integration vector with a replicative one harboring both attB and erythromycin resistance sequences into the strain. Furthermore, the integration vector constructed carries a promoter region of placT from the chromosome of Lactobacillus rhamnosus TCELL-1 which is used to express green fluorescence and luminance protein genes in the lactobacilli studied.     

Potent HIV-1 protease inhibitors incorporating squaramide-derived P2 ligands: Design,synthesis, and biological evaluation

We describe the design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors containing a squaramide-derived scaffold as the P2 ligand in combination with a (R)-hydroxyethylamine sulfonamide isostere. Inhibitor 3h with an N-methyl-3-(R)-aminotetrahydrofuranyl squaramide P2-ligand displayed an HIV-1 protease inhibitory K_i value of 0.51 nM. An energy minimized model of 3h revealed the major molecular interactions between HIV-1 protease active site and the tetrahydrofuranyl squaramide scaffold that may be responsible for its potent activity.     

An efficient chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli

Chloramphenicol (Cm) was demonstrated to be a suitable selective agent for the plasmid-mediated transformation of haploid and polyploid strains of Saccharomyces cerevisiae.A yeast/Escherichia colishuttle Cm-resistance (Cm^R)marker was constructed by inserting the CAT coding sequence from Tn9,and its associated bacterial ribosome-binding site, between a modified yeast ADC1 promoter and CYC1 terminator. When present on a 2 μm-based replicating plasmid, this marker transformed yeast as efficiently as the auxotrophic markers TRP1 and LEU2. When included in an integrating vector, single-copy transformants were formed as efficiently as with LEU2 and HIS3. Industrial yeast strains were transformed with both the replicating and integrating plasmids. The Cm^R marker could also efficiently transform E. coli. This versatile and efficient performance is currently unique for a yeast dominant marker.