DFR1 Gene Research Articles

Characterization of multiple antibiotic resistance of culturable microorganisms and metagenomic analysis of total microbial diversity of marine fish sold in retail shops in Mumbai, India.

Marine fish species were analyzed for culturable and total metagenomic microbial diversity, antibiotic resistance (AR) pattern, and horizontal gene transfer in culturable microorganisms. We observed a high AR microbial load of 3 to 4 log CFUg-1. Many fish pathogens like Providencia, Staphylococcus, Klebsiella pneumoniae, Enterobacter, Vagococcus, and Aeromonas veronii were isolated. Photobacterium and Vibrio were two major fish and human pathogens which were identified in the fish metagenome. Other pathogens that were identified were Shewanella, Acinetobacter, Psychrobacter, and Flavobacterium. Most of these pathogens were resistant to multiple antibiotics such as erythromycin, kanamycin, neomycin, streptomycin, penicillin, cefotaxime, bacitracin, rifampicin, trimethoprim, ciprofloxacin, and doxycycline with a high multiple antibiotic resistance index of 0.54-0.77. The fish microflora showed high prevalence of AR genes like bla TEM, Class I integron, tetA, aph(3')-IIIa, ermB, aadA, and sul1. Nineteen of 26 AR isolates harbored Class I integrons showing high co-resistance to trimethoprim, kanamycin, doxycycline, and cefotaxime. Mobile R-plasmids from 6 of the 12 AR pathogens were transferred to recipient E. coli after conjugation. The transconjugants harbored the same R-plasmid carrying bla CTX-M, dfr1, tetA, bla TEM, and cat genes. This study confirms that fish is a potential carrier of AR pathogens which can enter the human gut via food chain. To the best of our knowledge, this is the first study in the Indian subcontinent reporting a direct evidence of spread of AR pathogens to humans from specific marine fish consumption.

The sequence diversity and expression among genes of the folic acid biosynthesis pathway in industrial Saccharomyces strains.

Folic acid is an important vitamin in human nutrition and its deficiency in pregnant women's diets results in neural tube defects and other neurological damage to the fetus. Additionally, DNA synthesis, cell division and intestinal absorption are inhibited in case of adults. Since this discovery, governments and health organizations worldwide have made recommendations concerning folic acid supplementation of food for women planning to become pregnant. In many countries this has led to the introduction of fortifications, where synthetic folic acid is added to flour. It is known that Saccharomyces strains (brewing and bakers' yeast) are one of the main producers of folic acid and they can be used as a natural source of this vitamin. Proper selection of the most efficient strains may enhance the folate content in bread, fermented vegetables, dairy products and beer by 100% and may be used in the food industry. The objective of this study was to select the optimal producing yeast strain by determining the differences in nucleotide sequences in the FOL2, FOL3 and DFR1 genes of folic acid biosynthesis pathway. The Multitemperature Single Strand Conformation Polymorphism (MSSCP) method and further nucleotide sequencing for selected strains were applied to indicate SNPs in selected gene fragments. The RT qPCR technique was also applied to examine relative expression of the FOL3 gene. Furthermore, this is the first time ever that industrial yeast strains were analysed regarding genes of the folic acid biosynthesis pathway. It was observed that a correlation exists between the folic acid amount produced by industrial yeast strains and changes in the nucleotide sequence of adequate genes. The most significant changes occur in the DFR1 gene, mostly in the first part, which causes major protein structure modifications in KKP 232, KKP 222 and KKP 277 strains. Our study shows that the large amount of SNP contributes to impairment of the selected enzymes and S. cerevisiae and S. pastorianus produce reduced amounts of the investigated metabolite. The results obtained here yield a list of genetically stable yeast strains which can be implemented as a starter culture in the food industry.

Dianthus pseudocrinitus (Caryophyllaceae), a new species from Northeast of Iran identified by morphological and molecular data

A new species from southwest of Bojnord, NE of Iran is described and illustrated here as Dianthus pseudocrinitus (Caryophyllaceae). This species is morphologically similar to D. crinitus subsp. turcomanicus, but can be distinguished from the latter by bract number, width of lower and upper leaves, margin of thickness of outermost bracts, length of calyx, length and width of anther, branches of stem, type of sheath of lower leaf, and tip shape of petal fimbria. The new species is also similar to D. orientalis subsp. stenocalyx in terms of floral characters, but they differ by several non- overlapping morphological characters. Results obtained from the morphological data are consistent with those obtained from the molecular phylogenetic trees based on sequences of the two copies of DFR1 gene, confirming phylogenetic affinity of the new species to D. crinitus subsp. turcomanicus.

Integrons and multidrug resistance among Escherichia coli causing community-acquired urinary tract infection in southern India.

Antimicrobial resistance genes are often clustered in integrons, genetic elements capable of recombination. There is a paucity of data on the prevalence and role of integrons from community-acquired infections in developing countries where resistance to co-trimoxazole is high. We determined the prevalence of integrons among Escherichia coli causing community-acquired urinary tract infection (UTI). Consecutive isolates of E. coli obtained from UTI of pregnant women at the Christian Medical College Hospital, Vellore, India, during 2002 were included. All isolates were tested for susceptibility to 16 antimicrobials using the disc diffusion method and for integrons of classes 1 and 2 by PCR. Of the 58 isolates tested, 28 (48.3%) were resistant to co-trimoxazole and trimethoprim. All these isolates carried integrons. Three additional isolates were sulfonamide resistant but integron negative. Class 1 integrons were present in 21 (36.2%) isolates. Resistance to ampicillin (p=0.000), nalidixic acid (p=0.001), chloramphenicol (p=0.02), tetracycline (p=0.004) and gentamicin (p=0.02) was significantly more common in isolates with integrons. DNA sequencing of two isolates with integrons showed the presence of aadA, dfr1 and dfr7 genes. This study demonstrated that integrons are widely prevalent in India and that they might play a role in multidrug resistance in E. coli from community-acquired UTI.

An integron cassette carrying dfr1 with 90-bp repeat sequences located on the chromosome of trimethoprim-resistant isolates of Campylobacter jejuni.

The frequent occurrence of high-level trimethoprim resistance in clinical isolates of Campylobacter jejuni was shown to be related to the acquisition of foreign resistance genes (dfrl or dfr9 or both) coding for resistant variants of the enzyme dihydrofolate reductase, the target of trimethoprim. The dfr1 gene detected on the chromosome of 40 different clinical strains of C. jejuni was studied further regarding structure and genetic organization. Most of the dfr1 genes were found as integron cassettes inserted in the chromosome. In 36% of the examined isolated, the dfr1 gene showed identity to that previously characterized in trimethoprim-resistant Escherichia coli. In 40% of the cases, however, a variant of the dfr1 gene containing a 90-bp direct repeat was detected, and in 5% of the isolates, the repeat-containing dfr1 variant was found to occur in the form of two cassettes in tandem in an integron context. The existence of the 90-bp repeat within the coding sequence of the dfr1gene was found to play a role in the adaptation of C. jejuni to ambient concentrations of trimethoprim.

High-level resistance to trimethoprim in clinical isolates of Campylobacter jejuni by acquisition of foreign genes (dfr1 and dfr9) expressing drug-insensitive dihydrofolate reductases.

The pathogenic bacterium Campylobacter jejuni has been regarded as endogenously resistant to trimethoprim. The genetic basis of this resistance was characterized in two collections of clinical isolates of C. jejuni obtained from two different parts of Sweden. The majority of these isolates were found to carry foreign dfr genes coding for resistant variants of the dihydrofolate reductase enzyme, the target of trimethoprim. The resistance genes, found on the chromosome, were dfr1 and dfr9. In about 10% of the strains, the dfr1 and dfr9 genes occurred simultaneously. About 10% of the examined isolates were found to be negative for these dfr genes and showed a markedly lower trimethoprim resistance level than the other isolates. The dfr9 and dfr1 genes were located in the context of remnants of a transposon and an integron, respectively. Two different surroundings for the dfr9 gene were characterized. One was identical to the right-hand end of the transposon Tn5393, and in the other, the dfr9 gene was flanked by only a few nucleotides of a Tn5393 sequence. The insertion of the dfr9 gene into the C. jejuni chromosome could have been mediated by Tn5393. The frequent occurrence of high-level trimethoprim resistance in clinical isolates of C. jejuni could be related to the heavy exposure of food animals to antibacterial drugs, which could lead to the acquisition of foreign resistance genes in naturally transformable strains of C. jejuni.

Amplification of a Circular Episome Carrying an Inverted Repeat of the DFR1 Locus and Adjacent Autonomously Replicating Sequence Element of Saccharomyces cerevisiae

Lack of suitable amplification markers has hindered the use of the yeast system for investigating the mechanism of gene amplification in a eukaryote with a simple genome and well defined genetic system. Recently, methotrexate has been used to select for Saccharomyces cerevisiae mutants with de novo amplification of the dihydrofolate reductase gene (DFR1) (Huang, T. (1993) In Vivo Disruption and de Novo Amplification of the DFR1 Gene Encoding Dihydrofolate Reductase in Saccharomyces cerevisiae. Ph. D. thesis, University of Alberta, Edmonton, Canada). We report here the detailed structure of a DFR1 episome amplified in methotrexate-resistant strain 25-1. The extrachromosomal DNA is found predominantly as a single 11-kilobase circular molecule. It consists of a 5.5-kilobase inverted duplication that contains the DFR1 locus and adjacent ARS (autonomously replicating sequence) element. This molecular configuration mimics the inferred structure of double minute chromosomes observed in a number of mammalian amplification systems and suggests that mechanisms that generate amplified DNAs are conserved from yeast to mammals.

Effect of genomic position on amplification of the DFR1 gene in Saccharomyces cerevisiae.

Among the best studied examples of DNA amplification, as a response to drug selection, is the increased DHFR gene copy number observed in tumor cells resistant to methotrexate (MTX)1,2,. In spite of numerous studies into the mechanism of DHFR gene amplification in a variety of cell types, we do not yet have a detailed understanding of the molecular events which initiate the amplification process. Moreover, in spite of a relative wealth of information about the distribution and arrangement of amplicons observed in MTXR cells, we know very little about endogenous factors which affect the frequency of DHFR amplification-nor do we understand, at the molecular level, the process of resolution of early events into the final amplicon structures observed in drug-resistant cells.

The phenotype of a dihydrofolate reductase mutant of Saccharomyces cerevisiae

We have constructed a dihydrofolate reductase mutant ( dfr1) of Saccharomyces cerevisiae. The mutant has auxotrophic growth requirements for the C1 metabolites dTMP, adenine, histidine and methionine, similar to those of wild-type (wt) strains grown in the presence of methotrexate (MTX). However, unlike wt strains treated with MTX, the growth requirements of the dfr1 mutant are not satisfied by exogenous 5-formyltetrahydrofolic acid (FA; folinic acid) in complex (YEPD) medium. This result is surprising, as yeast cells treated with MTX are expected to be phenocopies of dfr1 mutants. The inability of the mutants to metabolize FA suggests that the DFR1 gene product may have a role in folate metabolism in addition to its well-characterized function in the reduction of dihydrofolate. From dfr1 strains, we have isolated secondary mutants whose growth can be supported by FA in YEPD medium. This FA-utilizing phenotype is attributable to recessive mutations which we have designated fou. In addition to their inability to metabolize FA, the dfr1 strains are unable to grow on medium containing the non-fermentable carbon source glycerol, suggesting that the DFR1 gene product is also required for mitochondrial function. In order to overcome this lack of respiratory activity in the dfr1 mutants, we isolated strains containing a dominant mutation, DIR, which allows growth on glycerol in the presence of antifolate drugs. When crossed into dfr1 strains, the DIR mutation conferred respiratory competence. These strains should be useful in a variety of studies on the genetics and biochemistry of folate metabolism in this simple eukaryote.

U3 small nuclear RNA (snR17A RNA) and DFR1 genes are very closely linked in Saccharomyces cerevisiae

SNR17A and DFR1 genes of Saccharomyces cerevisiae are only 313 bp apart and in the same orientation.

Mapping and sequencing of the dihydrofolate reductase gene ( DFR1) of Saccharomyces cerevisiae

The dihydrofolate reductase gene ( DFR1) from Saccharomyces cerevisiae has been mapped and sequenced. The gene was isolated on an 8.8-kb BamHI fragment from a yeast genomic library by screening of Escherichia coli transformants for resistance to trimethoprim. A 1.8-kb SalI- BamHI fragment which was able to confer methotrexate resistance in yeast also complemented an E. coli DHFR-deficient ( folA) mutant. Nucleotide sequence analysis revealed that the yeast DFR1 gene encoded a polypeptide with a predicted M r of 24230. The deduced sequence of 211 amino acid residues showed considerable homology with DHFRs from both bacterial and animal sources. The codon bias index of the DFR1 coding region is 0.0083, which indicates a random pattern of codon usage. The upstream region contains two consensus sequences required for binding of the yeast's positive regulatory factor, GCN4, suggesting that the DFR1 gene might be subject to the amino acid general control. Several potential ‘TATA’ boxes are located in the sequence 5' to the gene. Located in the 3' flanking region are homologies with several canonical sequences thought to be required for efficient transcription termination in yeast. We also mapped the DFR1 gene to a position 1.4 cM proximal to the MET7 locus on chromosome XV.