Coregulation of dihydrofolate reductase and thymidylate synthase B in bacillus subtilis

A 2.0-kb fragment of Bacillus subtilis 168 chromosomal DNA has been shown to contain both the dihydrofolate reductase (dfrA) and thymidylate synthase B (thyB) genes. In addition to the close proximity of dfrA and thyB, the expression of these genes seems to be regulated coordinately. Mutations that map near or within the dfrA gene resulted in coordinate increases in both dihydrofolate reductase and thymidylate synthase B activities. Also, when trimethoprim, a specific inhibitor of dihydrofolate reductase and thymidylate synthase B activities. Also, when trimethoprim, a specific inhibitor of dihydrofolate reductase, was added to growing cells, both dihydrofolate reductase and thymidylate synthase B activities increased coordinately.     

Cloning and mapping of the dihydrofolate reductase gene of Bacillus subtilis

The structural gene for dihydrofolate reductase (dfrA) from the Bacillus subtilis 168 chromosome has been cloned, along with the thyB gene, on a 4.5-kb insert contained on chimeric plasmid pER1. The presence of the dfrA gene on pER1 was demonstrated by showing that: (i) transformation of Escherichia coli strains RUE10(Thy-) and RUE11(Thy+) with pER1 resulted in a 60 to 130-fold increase in dihydrofolate reductase (DFRase) activity with a turnover number characteristic of that of B. subtilis and (ii) pER1-mediated transformation of trimethoprim-resistant E. coli strain D05, which overproduced a DFRase with a decreased affinity for trimethoprim, resulted in a 41-fold increase in DFRase activity with an affinity for trimethoprim similar to that of the B. subtilis enzyme. The dfrA gene was mapped to the 200 degrees region of the B. subtilis chromosome, and the gene order was established as thyB dfrA ilvA. Furthermore, the dfrA gene was shown to be linked closely (95-99% cotransformation) to the thyB gene.     

Construction of a new shuttle expression vector for Bacillus subtilis and Escherichia coli by using a polycistronic system

A shuttle vector has been constructed by fusing the Bacillus subtilis trimethoprim-resistance-carrying (TpR) plasmid pNC601 with the Escherichia coli plasmid pBR322. The resultant plasmid pNBL1 can replicate in both B. subtilis and E. coli, conferring Tp resistance on both cells and ampicillin resistance (ApR) on E. coli. The B. subtilis dihydrofolate reductase operon (dfr) on pNC601 and therefore on pNBL1 consists of the thymidylate synthase B gene (thyB) and the TpR-dihydrofolate reductase gene lacking the C-terminal seven codons (designated as drfA' as compared with the complete dfrA gene). A direct-expression vector pNBL3 has been constructed by inserting synthetic oligodeoxynucleotides containing a Bacillus ribosome-binding site (RBS) and the ATG codon downstream from dfrA' on pNBL1. When the E. coli lacZ gene was placed downstream from the dfrA' gene in pNBL3, efficient synthesis of beta-galactosidase was observed in both cells, showing that the polycistronic expression system is suitable for directing expression of heterologous genes. Translational efficiency of the lacZ gene on pNBL3 was further examined in B. subtilis by changing the sequence upstream from lacZ. Unlike the results previously reported [Sprengel et al., Nucleic Acids Res. 13 (1985) 893-909], when RBS was present, the high level of lacZ expression was preserved irrespective of spacing between the stop codon of the upstream dfrA' gene and the start codon of the downstream lacZ gene. However, in the absence of RBS, the spacing between both genes affected lacZ expression. That is, translational coupling of dfrA'-lacZ was observed, although the translational efficiency was very low.     

Thymidylate synthesis and aminopterin resistance in Bacillus subtilis

Wilson, Melba Carr (Brown University, Providence, R.I.), James L. Farmer, and Frank Rothman. Thymidylate synthesis and aminopterin resistance in Bacillus subtilis. J. Bacteriol. 92:186-196. 1966.-The thymine-requirement of Bacillus subtilis 168 thy results from mutation in two unlinked genes (i.e., genetic loci) designated thyA and thyB. The thyB gene is located between the met and ile markers. Both thyA(+)thyB and thyA thyB(+) strains are phenotypically thy(+). ThyA(+)thyB strains resemble the wild type in their sensitivity to aminopterin, poor incorporation of exogenous thymine into deoxyribonucleic acid (DNA), and high level of thymidylate synthetase activity in crude extracts. ThyA thyB(+) strains are resistant to aminopterin in the presence of thymine, incorporate exogenous thymine into DNA, and have no detectable thymidylate synthetase activity. Experiments designed to elucidate the role of the thyB gene indicate that it specifies an alternate pathway of thymidylate synthesis, similar to thymidylate synthetase but requiring a cofactor other than tetrahydrofolate. The mechanism of selection of thymine-requiring mutants by aminopterin is revealed by these results.     

Genetic analysis of multiple antimicrobial resistance in Salmonella isolated from diseased broilers in Egypt

To date, no information has been available on the molecular bases of antimicrobial resistance in Salmonella spp. from poultry in Egypt or even in Africa. Therefore, the objective of this study was to analyze, at the molecular level, the mechanisms of multidrug-resistance in isolates of Salmonella recovered from diseased broilers in Egypt. Twenty-one Salmonella isolates were identified; 13 of these isolates were Salmonella enterica serovar Enteritidis and eight Salmonella enterica serovar Typhimurium. 17 (81%). Salmonella isolates displayed multidrug resistance phenotypes, particularly against ampicillin, streptomycin, spectinomycin, kanamycin, tetracycline, chloramphenicol, and trimethoprim/sulfamethoxazole. PCR and DNA sequencing identified class 1 integrons in nine (42.9%) isolates and class 2 integrons in three (14.3%) isolates. The identified resistance genes within class 1 integrons were aminoglycoside adenyltransferase type A, aadA1, aadA2 and aadA5 and dihydrofolate reductase type A, dfrA1, dfrA5, dfrA12, dfrA15 and dfrA17. The β-lactamase encoding genes bla(TEM-1) and bla(CMY-2) and florfenicol resistance gene floR were also identified. Furthermore, the tetracycline resistance gene tet(A) was identified in 14 (66.7%) Salmonella isolates. To the best of our knowledge, this is the first report of the molecular basis of antimicrobial resistance in Salmonella spp. isolated from poultry in Africa.     

Transfection of DHFR- and DHFR+ mammalian cells using methotrexate-resistant mutants of mouse dihydrofolate reductase

Site-directed mutagenesis was used to generate mutants of mouse dihydrofolate reductase more resistant to methotrexate than the wild type enzyme. The mutant genes were used to transfect either DHFR- or DHFR+ cell lines. These mutants, as well as the wild type gene, were able to confer methotrexate resistance to DHFR- CHO cells. The number of selected colonies decreased with increased concentrations of methotrexate. The number of colonies observed at 10 microM methotrexate is correlated with the Ki(MTX) of the enzyme: the higher the Ki, the higher the number of colonies for the corresponding mutant. In contrast, the transfection of DHFR+ cells gave a few numbers of colonies not different for the wild type and the mutants.     

Cloning and nucleotide sequence of a negative regulator gene for Klebsiella aerogenes arylsulfatase synthesis and identification of the gene as folA.

A negative regulator gene for synthesis of arylsulfatase in Klebsiella aerogenes was cloned. Deletion analysis showed that the regulator gene was located within a 1.6-kb cloned segment. Transfer of the plasmid, which contains the cloned fragment, into constitutive atsR mutant strains of K. aerogenes resulted in complementation of atsR; the synthesis of arylsulfatase was repressed in the presence of inorganic sulfate or cysteine, and this repression was relieved, in each case, by the addition of tyramine. The nucleotide sequence of the 1.6-kb fragment was determined. From the amino acid sequence deduced from the DNA sequence, we found two open reading frames. One of them lacked the N-terminal region but was highly homologous to the gene which codes for diadenosine tetraphosphatase (apaH) in Escherichia coli. The other open reading frame was located counterclockwise to the apaH-like gene. This gene was highly homologous to the gene which codes for dihydrofolate reductase (folA) in E. coli. We detected 30 times more activity of dihydrofolate reductase in the K. aerogenes strains carrying the plasmid, which contains the arylsulfatase regulator gene, than in the strains without plasmid. Further deletion analysis showed that the K. aerogenes folA gene is consistent with the essential region required for the repression of arylsulfatase synthesis. Transfer of a plasmid containing the E. coli folA gene into atsR mutant cells of K. aerogenes resulted in repression of the arylsulfatase synthesis. Thus, we conclude that the folA gene codes a negative regulator for the ats operon.     

Analysis of T4 bacteriophage deletion mutants that lack td and frd genes.

The roles of bacteriophage T4-encoded thymidylate synthase and dihydrofolate reductase as virion structural components have been further investigated. Two mutants, del(63-32)7 and del(63-32)9, bearing deletions in the gene 63 to 32 region of the T4 genome, were characterized by Southern blotting analysis, as well as by enzyme and immunological assays. Our results have confirmed the original report of Homyk and Weil (Virology 61:505-523, 1974) that del7 and del9 each carries a deletion of about 4.0 kilobases, which totally eliminates the frd gene, encoding dihydrofolate reductase, and the td gene, encoding thymidylate synthase. With the well-characterized deletion mutants, along with newly prepared antisera against T4-encoded thymidylate synthase and dihydrofolate reductase, we have reevaluated the experimental results supporting the idea that T4-induced dihydrofolate reductase and thymidylate synthase are essential T4 baseplate components and antigenic determinants of phage particles. These deletion mutant phages are not targets for neutralization by antisera against either dihydrofolate reductase or thymidylate synthase purified from cloned genes. Furthermore, these newly prepared antisera also cannot neutralize the infectivity of T4D. Those results suggest that the phage-neutralizing components in the old antisera used in the earlier studies were not antibodies against either dihydrofolate reductase or thymidylate synthase but were antibodies against minor components of the purified enzyme preparations. Study of the biological properties of the deletion mutants indicates that T4-induced thymidylate synthase and dihydrofolate reductase play significant roles in growth of the phage beyond their known roles in nucleotide biosynthesis, even though they are apparently not essential for phage viability. The deletion mutants should be useful in defining these roles.     

Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli

The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagenized by progressive deletions from both the 5' and the 3' ends and by TAB linker insertion. A number of 5'-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a secondary initiation site. The most likely position of this site at a GTG codon located 35 codons downstream of the normal start site. This product could complement the folC mutation in E. coli strain SF4 as well as a strain deleted in the folC gene. The specific activity of extracts of the mutant enzyme are 4-16% that of the wild type enzyme for the folylpolyglutamate synthetase activity and 6-19% for the dihydrofolate synthetase activity. The relative amount of protein expressed by the mutant, compared to the wild type, in maxicells was comparable to the relative specific activity, suggesting that the kcat of the mutant enzyme is similar to that of the wild type. Mutants with up to 14 amino acids deleted from the carboxy terminal could still complement the folC deletion mutant. Seven out of ten linker insertions dispersed through the coding region of the gene showed complementation of the folC mutation in strain SF4 but none of these insertion mutants were able to complement the strain containing a deleted folC gene. None of the carboxy terminal or linker insertion mutants had a specific activity greater than 0.5% that of the wild type enzyme. The dihydrofolate synthetase and folylpolyglutamate synthetase activities behaved similarly in all mutants, both retaining a large fraction of the wild type activity in the amino terminal deletions and both being very low in the carboxy terminal deletions and linker insertion mutants. These studies are consistent with a single catalytic site for the two activities catalyzed by this enzyme.     

Probing the functional role of phenylalanine-31 of Escherichia coli dihydrofolate reductase by site-directed mutagenesis

The role of Phe-31 of Escherichia coli dihydrofolate reductase in binding and catalysis was probed by amino acid substitution. Phe-31, a strictly conserved residue located in a hydrophobic pocket and interacting with the pteroyl moiety of dihydrofolate (H2F), was replaced by Tyr and Val. The kinetic behavior of the mutant enzymes in general is similar to that of the wild type. The rate-limiting step for both mutant enzymes is the release of tetrahydrofolate (H4F) from the E X NADPH X H4F ternary complex as determined for the wild type. The 2-fold increase in V for the two mutant enzymes arises from faster dissociation of H4F from the enzyme-product complex. The quantitative effect of these mutations is to decrease the rate of hydride transfer, although not to the extent that this step becomes partially rate limiting, but to accelerate the dissociation rates of tetrahydrofolate from product complexes so that the opposing effects are nearly compensating.     

Nucleotide sequence of dihydrofolate reductase genes from trimethoprim-resistant mutants of Escherichia coli. Evidence that dihydrofolate reductase interacts with another essential gene product

Summary We report the construction of recombinant plasmids containing the dihydrofolate reductase structural gene (fol) from several trimethoprim-resistant mutants of Escherichia coli. Strains carrying some of these plasmids produced approximately 6% of their soluble cell protein as dihydrofolate reductase and are therefore excellent sources of the purified enzyme for inhibitor binding or mechanistic studies. The nucleotide sequence of the fol region from each of the plasmids was determined. A plasmid derived from a K_i mutant which produced a dihydrofolate reductase with lowered affinity for trimethoprim contained a mutation in the structural gene that altered the sequence of the polypeptide in a conserved region which is adjacent to the dihydrofolate binding site. Two other independently-isolated mutants which overproduced dihydrofolate reductase had a mutation in the-35 region of the fol promoter. One of them, strain RS35, was also temperature-sensitve for growth in minimal medium. This phenotype was shown to be the result of an additional mutation in a locus unlinked to fol by P1 transduction. The fol regions from two temperature-independent revertants of strain RS35 were sequenced. One of these had a mutation within the dihydrofolate reductase structural gene which altered some properties of the enzyme. This confirmed some previous enzymological data which suggested that some revertants of strain RS35 had mutations in fol (Sheldon 1977). These results suggest that dihydrofolate reductase interacts physically with some other essential gene product in E. coli.     

Suborganellar Localization and Molecular Characterization of Nonproteolytic Degraded Leukoplast Pyruvate Kinase from Developing Castor Oil Seeds

Several genes involved in the ability of Synechococcus sp. PCC 7942 to grow under different CO2 concentrations were mapped in the genomic region of rbcLS (the operon encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase). Insertion of a cartridge encoding kanamycin resistance within open reading frame (ORF) 78, designated ccmJ, located 7 kb upstream of rbcLS, resulted in a kanamycin-resistant, high-CO2-requiring mutant, M3, which does not contain normal carboxysomes. ccmJ shows significant homology to csoS1 encoding a carboxysomal shell polypeptide in Thiobacillus neopolitanus. Analysis of the polypeptide pattern of a carboxysome-enriched fraction indicated several differences between the wild type and the mutant. The amount of the ribulose-1,5-bisphosphate carboxylase/oxygenase subunits was considerably smaller in the carboxysomal fraction of the mutant when compared to the wild type. On the basis of the sequence analyses, ORF286 and ORF466, located downstream of ccmJ, were identified as chlL and chlN, respectively, which are involved in chlorophyll biosynthesis in the dark.     

Overproduction of L-Lysine from methanol by Methylobacillus glycogenes derivatives carrying a plasmid with a mutated dapA gene.

The dapA gene, encoding dihydrodipicolinate synthase (DDPS) partially desensitized to inhibition by L-lysine, was cloned from an L-threonine- and L-lysine-coproducing mutant of the obligate methylotroph Methylobacillus glycogenes DHL122 by complementation of the nutritional requirement of an Escherichia coli dapA mutant. Introduction of the dapA gene into DHL122 and AL119, which is the parent of DHL122 and an L-threonine producing mutant, elevated the specific activity of DDPS 20-fold and L-lysine production 2- to 3-fold with concomitant reduction of L-threonine in test tube cultures. AL119 containing the dapA gene produced 8 g of L-lysine per liter in a 5-liter jar fermentor from methanol as a substrate. Analysis of the nucleotide sequence of the dapA gene shows that it encodes a peptide with an M(r) of 30,664 and that the encoded amino acid sequence is extensively homologous to those of other organisms. In order to study the mutation that occurred in DHL122, the dapA genes of the wild type and AL119 were cloned and sequenced. Comparison of the nucleotide sequences of the dapA genes revealed that the amino acid at residue 88 was F in DHL122 whereas it was L in the wild type and AL119, suggesting that this amino acid alteration that occurred in DHL122 caused the partial desensitization of DDPS to the inhibition by L-lysine. The similarity in the amino acid sequences of DDPS in M. glycogenes and other organisms suggests that the mutation of the dapA gene in DHL122 is located in the region concerned with interaction of the allosteric effector, L-lysine.     

The presence and distribution of reduced folates in Escherichia coli dihydrofolate reductase mutants

Escherichia coli DNA photolyase was overproduced and purified from each of two mutant E. coli strains lacking dihydrofolate reductase. The extent of over-production in the mutants was comparable to that seen in the wild type strain. Examination of the isolated photolyase from these strains revealed that the folate cofactor, 5,10-methenyltetrahydrofolate, was present in these proteins at a level of 60-80% compared to that purified from the wild type strain. Further examination of the dihydrofolate reductase-deficient strains revealed the presence of other tetrahydrofolate derivatives. These findings demonstrate that dihydrofolate reductase is not essential for the production of tetrahydrofolates in E. coli.     

Plasmodium falciparum: induction, selection, and characterization of pyrimethamine-resistant mutants

We have selected eight pyrimethamine resistant mutants of a cloned, drug sensitive, Plasmodium falciparum malaria parasite, strain FCR3. The mutants exhibited resistance to between 10 and 200 times higher concentrations of drug than the wild type parasite. The mutants were selected from cultured parasites that were either unmutagenized or N-methyl-N'-nitro-N-nitrosoguanidine mutagenized. One mutant was shown to contain a mutant dihydrofolate reductase enzyme in parasite extracts that exhibited (1) a five- to ninefold reduction in its binding of methotrexate, (2) an undetectable enzyme activity based on the spectrophotometric conversion of dihydrofolate to tetrahydrofolate, and (3) essentially normal amounts of the parasite's bifunctional thymidylate synthetase-dihydrofolate reductase enzyme. Other mutants exhibited both normal dihydrofolate reductase specific activity and normal enzyme sensitivity to the inhibitory activity of the drug.     

Trimethoprim 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.