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PubMed2006

Plasmid pBP136 from Bordetella pertussis represents an ancestral form of IncP-1beta plasmids without accessory mobile elements.

The complete 41,268 bp nucleotide sequence of the IncP-1beta plasmid pBP136 from the human pathogen Bordetella pertussis, the primary aetiological agent of whooping cough, was determined and analysed. This plasmid carried a total of 46 ORFs: 44 ORFs corresponding to the genes in the conserved IncP-1beta backbone, and 2 ORFs similar to the XF1596 and XF1597 genes with unknown function of the plant pathogen Xylella fastidiosa. Interestingly, pBP136 had no accessory genes carrying genetic traits such as antibiotic or mercury resistance and/or xenobiotic degradation. Moreover, pBP136 had only two of the kle genes (kleAE) that have been reported to be important for the stability of IncP-1 plasmid in Pseudomonas aeruginosa. Phylogenetic analysis of the Kle proteins revealed that the KleA and KleE of pBP136 were phylogenetically distant from those of the present IncP-1 plasmids. In contrast, IncC1 and KorC, encoded upstream and downstream of the kle genes respectively, and the replication-initiation protein, TrfA, were closely related to those of the IncP-1beta 'R751 group'. These results suggest that (i) pBP136 without any apparent accessory genes diverged early from an ancestor of the present IncP-1beta plasmids, especially those of the R751 group, and (ii) the kle genes might be incorporated independently into the backbone region of the IncP-1 plasmids for their stable maintenance in various host cells.

Bacterial Proteins↗
PubMed2005

Constitutively high expression of the histidine biosynthetic pathway contributes to nickel tolerance in hyperaccumulator plants.

Plants that hyperaccumulate Ni exhibit an exceptional degree of Ni tolerance and the ability to translocate Ni in large amounts from root to shoot. In hyperaccumulator plants in the genus Alyssum, free His is an important Ni binding ligand that increases in the xylem proportionately to root Ni uptake. To determine the molecular basis of the His response and its contribution to Ni tolerance, transcripts representing seven of the eight enzymes involved in His biosynthesis were investigated in the hyperaccumulator species Alyssum lesbiacum by RNA gel blot analysis. None of the transcripts changed in abundance in either root or shoot tissue when plants were exposed to Ni, but transcript levels were constitutively higher in A. lesbiacum than in the congeneric nonaccumulator A. montanum, especially for the first enzyme in the biosynthetic pathway, ATP-phosphoribosyltransferase (ATP-PRT). Comparison with the weak hyperaccumulator A. serpyllifolium revealed a close correlation between Ni tolerance, root His concentration, and ATP-PRT transcript abundance. Overexpression of an A. lesbiacum ATP-PRT cDNA in transgenic Arabidopsis thaliana increased the pool of free His up to 15-fold in shoot tissue, without affecting the concentration of any other amino acid. His-overproducing lines also displayed elevated tolerance to Ni but did not exhibit increased Ni concentrations in either xylem sap or shoot tissue, suggesting that additional factors are necessary to recapitulate the complete hyperaccumulator phenotype. These results suggest that ATP-PRT expression plays a major role in regulating the pool of free His and contributes to the exceptional Ni tolerance of hyperaccumulator Alyssum species.

ATP Phosphoribosyltransferase↗
PubMed1999

Cloning and functional characterization of an Arabidopsis nitrate transporter gene that encodes a constitutive component of low-affinity uptake.

The Arabidopsis CHL1 (AtNRT1) gene encodes an inducible component of low-affinity nitrate uptake, which necessitates a "two-component" model to account for the constitutive low-affinity uptake observed in physiological studies. Here, we report the cloning and characterization of a CHL1 homolog, AtNRT1:2 (originally named NTL1), with data to indicate that this gene encodes a constitutive component of low-affinity nitrate uptake. Transgenic plants expressing antisense AtNRT1:2 exhibited reduced nitrate-induced membrane depolarization and nitrate uptake activities in assays with 10 mM nitrate. Furthermore, transgenic plants expressing antisense AtNRT1:2 in the chl1-5 background exhibited an enhanced resistance to chlorate (7 mM as opposed to 2 mM for the chl1-5 mutant). Kinetic analysis of AtNRT1:2-injected Xenopus oocytes yielded a K(m) for nitrate of approximately 5.9 mM. In contrast to CHL1, AtNRT1:2 was constitutively expressed before and after nitrate exposure (it was repressed transiently only when the level of CHL1 mRNA started to increase significantly), and its mRNA was found primarily in root hairs and the epidermis in both young (root tips) and mature regions of roots. We conclude that low-affinity systems of nitrate uptake, like high-affinity systems, are composed of inducible and constitutive components and that with their distinct functions, they are part of an elaborate nitrate uptake network in Arabidopsis.

Action Potentials↗
PubMed1985

Evolution of plasmid-coded resistance to broad-spectrum cephalosporins.

A clinical isolate of Klebsiella ozaenae with transferable resistance to broad-spectrum cephalosporins produces a beta-lactamase determined by plasmid pBP60. The beta-lactamase had the same isoelectric point as SHV-1 (7.6). From heteroduplex analysis, an extensive homology between the two bla genes could be deduced; therefore, the new beta-lactamase was designated SHV-2. Enzymatic studies revealed that SHV-2 was able to hydrolyze broad-spectrum cephalosporins due to an increased affinity of these compounds for the enzyme. The assumption that SHV-2 is a natural mutant of SHV-1 was strongly supported by the isolation of a laboratory mutant of SHV-1 that showed activities similar to those of SHV-2.

Cephalosporins↗
PubMed2005

Six groups of the OXY beta-Lactamase evolved over millions of years in Klebsiella oxytoca.

The diversity and evolution of the class A OXY beta-lactamase from Klebsiella oxytoca were investigated and compared to housekeeping gene diversity. The entire bla(OXY) coding region was sequenced in 18 clinical isolates representative of the four K. oxytoca beta-lactamase gene groups bla(OXY-1) to bla(OXY-4) and of two new groups identified here, bla(OXY-5) (with four isolates with pI 7.2 and one with pI 7.7) and bla(OXY-6) (with four isolates with pI 7.75 and three with pI 8.1). Genes bla(OXY-5) and bla(OXY-6) showed 99.8% within-group nucleotide similarity but differed from each other by 4.2% and from bla(OXY-1), their closest relative, by 2.5% and 2.9%, respectively. Antimicrobial susceptibility to beta-lactams was similar among OXY groups. Nucleotide sequence diversity of the 16S rRNA (1,454 bp), rpoB (940 bp), gyrA (383 bp), and gapDH (573 bp) genes was in agreement with the beta-lactamase gene phylogeny. Strains with bla(OXY-1), bla(OXY-2), bla(OXY-3), bla(OXY-4), and bla(OXY-6) genes formed five phylogenetic groups, named KoI, KoII, KoIII, KoIV, and KoVI, respectively. Isolates harboring bla(OXY-5) appeared to represent an emerging lineage within KoI. We estimated that the bla(OXY) gene has been evolving within K. oxytoca for approximately 100 million years, using as calibration the 140-million-year estimation of the Escherichia coli-Salmonella enterica split. These results show that the bla(OXY) gene has diversified along K. oxytoca phylogenetic lines over long periods of time without concomitant evolution of the antimicrobial resistance phenotype.

Amino Acid Sequence↗
PubMed2004

Interaction of Bacillus thuringiensis toxins with larval midgut binding sites of Helicoverpa armigera (Lepidoptera: Noctuidae).

In 1996, Bt-cotton (cotton expressing a Bacillus thuringiensis toxin gene) expressing the Cry1Ac protein was commercially introduced to control cotton pests. A threat to this first generation of transgenic cotton is the evolution of resistance by the insects. Second-generation Bt-cotton has been developed with either new B. thuringiensis genes or with a combination of cry genes. However, one requirement for the "stacked" gene strategy to work is that the stacked toxins bind to different binding sites. In the present study, the binding of (125)I-labeled Cry1Ab protein ((125)I-Cry1Ab) and (125)I-Cry1Ac to brush border membrane vesicles (BBMV) of Helicoverpa armigera was analyzed in competition experiments with 11 nonlabeled Cry proteins. The results indicate that Cry1Aa, Cry1Ab, and Cry1Ac competed for common binding sites. No other Cry proteins tested competed for either (125)I-Cry1Ab or (125)I-Cry1Ac binding, except Cry1Ja, which competed only at the highest concentrations used. Furthermore, BBMV from four H. armigera populations were also tested with (125)I-Cry1Ac and Cry1Ab to check the influence of the insect population on the binding results. Finally, the inhibitory effect of selected sugars and lectins was also determined. (125)I-Cry1Ac binding was strongly inhibited by N-acetylgalactosamine, sialic acid, and concanavalin A and moderately inhibited by soybean agglutinin. In contrast, (125)I-Cry1Ab binding was only significantly inhibited by concanavalin A. These results show that Cry1Ac and Cry1Ab use different epitopes for binding to BBMV.

Animals↗
PubMed1998

Bacillus thuringiensis and its pesticidal crystal proteins.

During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism's pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.

Amino Acid Sequence↗
PubMed1998

Spatial heterogeneity and insect adaptation to toxins.

Behavioral responses of insect herbivores to toxins are examined in managed and natural systems with reference to two important but largely ignored factors: heterogeneity in toxin distributions and the nature of the relationship between behavioral responses and physiological adaptation to the same toxins. Heterogeneous toxin distributions, which provide the opportunity for behavioral responses, are ubiquitous in managed and natural systems. Insect herbivores have evolved a wide variety of behavioral responses to such toxins. The nature of behavioral responses reflects toxin apparency, mode of action, and the extent to which sublethal effects influence behavior. The interaction between these behavioral responses to heterogeneously distributed toxins and physiological mechanisms of tolerance has influenced the evolution of insecticide resistance in managed systems and the evolution of plant defensive strategies in natural systems. An understanding of this interaction could lead to more evolutionarily stable methods of crop protection.

Journal Article↗
PubMed1999

Genotypic evolution in a quinolone-resistant Neisseria gonorrhoeae isolate from a patient with clinical failure of levofloxacin treatment.

Recently, a reduction in the antimicrobial susceptibility of clinical isolates of Neisseria gonorrhoeae to newer fluoroquinolones including levofloxacin in vitro has been recognized in Japan. We examined the quinolone resistance mechanisms in N. gonorrhoeae isolates from a patient with clinical failure of levofloxacin treatment. Man with gonococcal urethritis was treated with oral 100 mg levofloxacin 3 times daily for 7 days. However, clinical failure of the treatment was observed. The minimum inhibitory concentration of levofloxacin for the posttreatment isolate (4.0 microg/ml) was 4-fold higher than that for the pretreatment isolate (1.0 microg/ml). To analyze quinolone resistance mechanisms in the set of isolates, we performed DNA sequencing of the quinolone resistance-determining regions within the gyrA and parC genes. Moreover, we assayed the intracellular levofloxacin and norfloxacin accumulation level in these isolates. The pretreatment isolate contained three substitutions compared to susceptible wild-type isolate, including serine to phenylalanine at position 91 and aspartic acid to asparagine at position 95 in the GyrA protein, and serine to proline at position 88 in the ParC protein. The posttreatment isolate had four substitutions, including the same three substitutions and an additional glutamic acid to glutamine substitution at position 91 in ParC. There was no significant difference in the level of accumulation of levofloxacin and norfloxacin between the pretreatment and posttreatment isolates. Our results indicate that levofloxacin selects a mutant having an additional alteration within the gene cording for the ParC protein during treatment, which may have enhanced quinolone resistance in the organism.

Adult↗
PubMedDate not supplied

An introduction to ALS-inhibiting herbicides.

Herbicides that inhibit acetolactate synthase (ALS), the enzyme common to the biosynthesis of the branch-chain amino acids (valine, leucine, and isoleucine), affect many species of higher plants as well as bacteria, fungi, yeasts, and algae. The novel mechanism of action attributed to ALS inhibitors, their effect on the reproduction of some plant species, their potency at extremely low concentrations, and the rapid evolution of resistance to these herbicides in some plants and microorganisms are characteristics that set ALS inhibitors apart from their predecessors. This class of chemicals affects seedling growth. Older plants exhibit varied signs of malformation, stunting, and reduced seed production. These herbicides are so potent that they can affect plants at levels that are undetectable by any standard chemical protocol. Weeds quickly become resistant to ALS inhibitors, presumably because these herbicides have a single mode of action and because many have long residual activity. Concern now is directed towards developing the technology to detect very low concentrations of ALS inhibitors in the environment and their indirect effects on plant and animal health.

Acetolactate Synthase↗
PubMed2001

Cytomegalovirus treatment options in immunocompromised patients.

Cytomegalovirus (CMV) infection was recognised in congenitally infected infants in the first half of the 20th century. Following the increased use of immunosuppressive regimens for bone marrow and solid organ transplantation, various manifestations of CMV disease were recognised. Milder symptoms included fever, anorexia and malaise but severe symptoms included pneumonitis, hepatitis, gastrointestinal ulceration, choreoretinitis and encephalopathy, all with a high morbidity or mortality. With the onset of the AIDS epidemic, manifestations of CMV became evident, predominantly retinitis. Ganciclovir used intravenously has been the principal anti-CMV agent investigated. However, ganciclovir has problems with suboptimal efficacy, toxicity, poor oral bioavailability and evolution of resistant strains. Additional studies have been performed on foscarnet and cidofovir, although the use of both have been limited by their nephrotoxicity. Combination therapy with ganciclovir and foscarnet for resistant strains has been used. There are promising newer drugs like the methylenecyclopropane nucleoside analogues and benzimidazole. The most novel compound is the antisense oligonucleotide fomivirsen that has been evaluated principally in CMV retinitis. The role of immunotherapy with either immunoglobulin prophylaxis or the novel adoptive immunotherapy needs further evaluation.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Genetic variation at the tomato Cf-4/Cf-9 locus induced by EMS mutagenesis and intralocus recombination.

The interaction between tomato (Lycopersicon esculentum) and the leaf mold pathogen Cladosporium fulvum is an excellent model for investigating disease resistance gene evolution. The interaction is controlled in a gene-for-gene manner by Cf genes that encode type I transmembrane extracellular leucine-rich repeat glycoproteins that recognize their cognate fungal avirulence (Avr) proteins. Cf-4 from L. hirsutum and Cf-9 from L. pimpinellifolium are located at the same locus on the short arm of tomato chromosome 1 in an array of five paralogs. Molecular analysis has shown that one mechanism for generating sequence variation in Cf genes is intragenic sequence exchange through unequal crossing over or gene conversion. To investigate this we used a facile genetic selection to identify novel haplotypes in the progeny of Cf-4/Cf-9 trans-heterozygotes that lacked Cf-4 and Cf-9. This selection is based on the ability of Avr4 and Avr9 to induce Cf-4- or Cf-9-dependent seedling death. The crossovers were localized to the same intergenic region defining a recombination hotspot in this cross. As part of a structure-function analysis of Cf-9 and Cf-4, nine EMS-induced mutant alleles have been characterized. Most mutations result in single-amino-acid substitutions in their C terminus at residues that are conserved in other Cf proteins.

Alleles↗
PubMedDate not supplied

[The evolution of in-vitro resistance in Plasmodium falciparum to antimalarials in a gold-prospecting area in the state of Amapá between 1983 and 199)].

This study evaluates the sensitivity of P. falciparum in vitro to antimalarial drugs in an area of gold mining exploration in Amapá State during the period of 1983 to 1990. The following tests were done, 75 in vitro studies with chloroquine and quinine, 74 with amodiaquine and 76 with mefloquine. The results showed 81% of resistance to chloroquine and 27% to amodiaquine while resistant strains to quinine and mefloquine were found. Also for these two quinolinomethanols a loss of sensitivity was noticed in the period of study. An association between resistance to chloroquine and decrease of sensitivity to quinine was also evident with the same strains.

Animals↗
PubMed1992

Molecular biological approaches to the study of vectors in relation to malaria control.

To a large extent, control of malaria vectors relies on the elimination of breeding sites and the application of chemical agents. There are increasing problems associated with the use of synthetic insecticides for vector control, including the evolution of resistance, the high cost of developing and registering new insecticides and an awareness of pollution from insecticide residues. These factors have stimulated interest in the application of molecular biology to the study of mosquito vectors of malaria; focussing primarily on two aspects. First, the improvement of existing control measures through the development of simplified DNA probe systems suitable for identification of vectors of malaria. The development of synthetic, non-radioactive DNA probes suitable for the identification of species in the Anopheles gambiae complex is described with the aim of defining a simplified methodology which is suitable for entomologist in the field. The second aspect to be considered is the development of completely novel strategies through the genetic manipulation of insect vectors of malaria in order to alter their ability to transmit the disease. The major requirements for producing transgenic mosquitoes are outlined together with the progress which has been made to date and discussed in relation to the prospects which this type of approach has for the future control of malaria.

Animals↗
PubMed2001

Life history traits of Helicoverpa zea (Lepidoptera: Noctuidae) on non-Bt and Bt transgenic corn hybrids in eastern North Carolina.

Transgenic varieties of field corn that express the CrylAb B. thuringiensis (Bt) toxin in ear tissue present the potential of reducing ear feeding by the corn earworm, Helicoverpa zea (Lepidoptera: Noctuidae), and for reducing the size of populations of the insect infesting other host crops. Life history parameters of H. zea feeding on ears of conventional and Bt field corn varieties were measured in field plots in eastern North Carolina in 1997 and 1998. Transformation events investigated were Mon-810 and Bt-11. Bt corn was found to cause a steady mortality of larvae during development, but permitted approximately 15-40% survival to the prepupal stage compared with non-Bt corn. Mortality of prepupae and pupae from Bt corn was also higher than from non-Bt corn, reducing overall adult production by 65-95%. The larvae that did survive grew more slowly on Bt than on non-Bt corn, and produced pupae that weighed 33% less. Pupation and adult eclosion were delayed by 6-10 d by feeding on Bt corn ears. Corn varieties expressing Bt in ear tissue have the potential to reduce H. zea ear feeding by up to 80%, and the potential to reduce populations emerging from ear-stage corn fields to infest cotton, soybean and other crops by around 75%. To have a measurable effect on area-wide populations, Bt corn varieties would need to be planted in large proportions of corn fields. Extensive planting of varieties such as those tested here, having only moderate effects on H. zea, would raise concerns about rapid evolution of resistance.

Animals↗
PubMed2003

Effects of MON810 Bt field corn on adult emergence of Helicoverpa zea (Lepidoptera: Noctuidae).

A 3-yr study (1996-1998) was conducted to evaluate the effects of MON810 Bt corn on Helicoverpa zea (Boddie) emergence and to determine whether delayed larval development as a result of Bt intoxication results in higher levels of diapause induction and pupal mortality. In the 1997 study, there was no difference in prepupal mortality between corn types, although significantly more prepupae from Bt plots than from non-Bt plots died in emergence buckets before constructing pupal chambers in 1998. In all years, significantly fewer moths emerged from prepupae collected from Bt plots, suggesting that effects of the expressed Cry1Ab extended to the prepupal and pupal stages. Late plantings of corn showed the greatest reductions in moth emergence from Bt corn because environmental conditions were more conducive to trigger diapause at the time H. zea was developing in these plantings. This was supported by a significantly greater proportion of diapausing pupae remaining in the ground in the late plantings of both Bt and non-Bt corn. For April and early May plantings, larval feeding on Bt corn delayed the time to pupation, although there was no significant difference in moth emergence between corn types for those larvae that successfully pupated. Although Bt expression had less impact on the proportion of moths emerging, the actual number of moths emerging from Bt corn was significantly reduced because fewer larvae reached pupation. Delays in adult emergence, along with significant reductions in adult emergence from MON810 Bt corn, should reduce the rates of colonization in soybean and other late host crops but may also result in asynchrony of mating between individuals emerging from Bt and non-Bt corn. This, in turn, may contribute to the evolution of resistance to Bt corn.

Animals↗