Characterization and Genetic Mapping of Phosphoglucoisomerase Mutations in Mannitol-Negative Mutants of Pseudomonas aeruginosa

Phosphoglucoisomerase (pgi) mutations in a number of independently isolated mannitol-negative mutants of Pseudomonas aeruginosa PAO1 were mapped on the chromosome by plasmid FP5-mediated conjugation and by cotransduction with the generalized transducing phages G101 and F116L. Mutant allele pgi-9001 exhibited linkage to ilvB, C-9059 (46–85%), car-9003 (93–100%), and pur-9047 (70%), but not with met-9011, in FP5-mediated conjugational crosses. All known pgi mutations and several previously uncharacterized mannitol-negative mutations exhibited transductional linkage to two independent car mutations at frequencies ranging from 13% to 42% and 53% to 99%, in transductional crosses mediated by phages G101 and F116L respectively. These pgi and mannitol-negative mutations also were cotransducible at very low frequencies (<1%) with two independent ilv mutations. Cotransduction of the car and ilv loci could not be detected. These data suggest the location of pgi within the first minute of the P. aeruginosa chromosome closely linked to the car marker and probably between the ilv and car loci. All of the mannitol-negative mutations that exhibited linkage to the car and ilv loci were characterized as pgi mutations by enzyme assays. A phenotypically similar, mannitol-negative mutatant was shown to contain a mutation in glucose-6-phosphate dehydrogenase (zwf-9012) that maps to a different region on the chromosome. Received: 26 April 1996 / Accepted: 10 June 1996     

Investigation of the contribution of RyR target-site mutations in diamide resistance by CRISPR/Cas9 genome modification in Drosophila

Diamide insecticides are used widely against lepidopteran pests, acting as potent activators of insect Ryanodine Receptors (RyRs) and thus inducing muscle contraction and eventually death. However, resistant phenotypes have recently evolved in the field, associated with the emergence of target site resistance mutations (G4946E/V and I4790M). We investigated the frequency of the mutations found in a resistant population of Tuta absoluta from Greece (G4946V ~79% and I4790M ~21%) and the associated diamide resistance profile: there are very high levels of resistance against chlorantraniliprole (9329-fold) and flubendiamide (4969-fold), but moderate levels against cyantraniliprole (191-fold). To further investigate functionally the contribution of each mutation in the resistant phenotype, we used CRISPR/Cas9 to generate genome modified Drosophila carrying alternative allele combinations, and performed toxicity bioassays against all three diamides. Genome modified flies bearing the G4946V mutation exhibited high resistance ratios to flubendiamide (91.3-fold) and chlorantraniliprole (194.7-fold) when compared to cyantraniliprole (5.4-fold). Flies naturally wildtype for the I4790M mutation were moderately resistant to flubendiamide (15.3-fold) but significantly less resistant to chlorantraniliprole (7.5-fold), and cyantraniliprole (2.3-fold). These findings provide in vivo functional genetic confirmation for the role and relative contribution of RyR mutations in diamide resistance and suggest that the mutations confer subtle differences on the relative binding affinities of the three diamides at an overlapping binding site on the RyR protein.     

Enhancement by caffeine of N-methyl-N-nitrosourea-induced mutations and chromosome aberrations in Chinese hamster cells

N-Methyl-N-nitrosourea (MNU) increased the induction of mutations to 8-azaguanine resistance in Chinese hamster cells in a dose-dependent manner. Mutations were only observed with toxic concentrations of MNU. Since a plot of the fraction of cells surviving alkylation against the extent of methylation of DNA exhibited a shoulder it followed that there was a threshold level of DNA reaction which did not lead to mutations possibly due to efficient repair of DNA damage. Post-alkylation incubation in medium containing caffeine decreased cell survival while at the same time it increased the induced mutation frequency. Mutation frequency was increased whether caffeine was present for 48 h or for a further 12 days in the presence of the selective agent 8-azaguanine. MNU caused chromatid aberrations in Chinese hamster cells and these reached a value of 15% of the treated cells by 48 h after methylation. Post-alkylation incubation in caffeine increased the percentage of cells showing chromosomal damage to a maximum of 86% of treated cells by 40 h after alkylation. A large proportion of cells exhibited completely fragmented or shattered chromosomes. The proportion of cells showing the presence of micronuclei also dramatically increased following incubation of methylated cells in caffeine. These results are discussed in terms of the possibility that damage to DNA is responsible for the lethal, mutagenic and cytological effects of MNU in Chinese hamster cells, and that there is a caffeine sensitive step(s) in the repair of the DNA damage which is responsible for these effects.     

Phenotypic Suppression of Methicillin Resistance in Staphylococcus aureus by Mutant Noninducible Penicillinase Plasmids

Methicillin (intrinsic) resistance of Staphylococcus aureus was suppressed almost completely by regulatory gene (penI₁) mutations of penicillinase plasmids that made penicillinase production strictly noninducible. Methicillin resistance was restored by secondary regulatory gene mutations that altered the noninducible phenotype or by complementation with a compatible plasmid that did not bear the noninducible mutation. No evidence was obtained for genetic linkage between a penicillinase plasmid and the gene for methicillin resistance. We suggest, therefore, that the mutant noninducible repressor acted in trans by binding to a site on the methicillin resistance determinant. This hypothesis would imply an appreciable degree of homology between penicillinase plasmids and methicillin resistance genes.     

Disruption of Lysosome Function Promotes Tumor Growth and Metastasis in Drosophila

Lysosome function is essential to many physiological processes. It has been suggested that deregulation of lysosome function could contribute to cancer. Through a genetic screen in Drosophila, we have discovered that mutations disrupting lysosomal degradation pathway components contribute to tumor development and progression. Loss-of-function mutations in the Class C vacuolar protein sorting (VPS) gene, deep orange (dor), dramatically promote tumor overgrowth and invasion of the Ras^V12 cells. Knocking down either of the two other components of the Class C VPS complex, carnation (car) and vps16A, also renders Ras^V12 cells capable for uncontrolled growth and metastatic behavior. Finally, chemical disruption of the lysosomal function by feeding animals with antimalarial drugs, chloroquine or monensin, leads to malignant tumor growth of the Ras^V12 cells. Taken together, our data provide evidence for a causative role of lysosome dysfunction in tumor growth and invasion and indicate that members of the Class C VPS complex behave as tumor suppressors.     

Inhibition of repair of UV-damaged DNA by caffeine and mutation induction in Chinese hamster cells

The effect of caffeine on UV-irradiated Chinese hamster cells in vitro was studied on the cellular and molecular levels. Caffeine (1 mM) was shown to decrease the colony-forming ability and the frequencies of spontaneous and UV-induced mutations in Chinese hamster cells. The effect of caffeine in reducing the frequency of UV-induced mutations was demonstrated only if caffeine was present in the culture medium during the first post-irradiation cell division. Using alkaline sucrose gradient centrifugation, both parental and newly synthesized DNA in UV-irradiated and unirradiated cells were studied in the presence and absence of caffeine. Caffeine affected the sedimentation profile of DNA synthesized in UV-irradiated cells but not in unirradiated cells. Caffeine had no apparent effect on the incorporation of [³H]-thymidine into DNA of control or UV-irradiated cells, nor on the small amount of excision of UV-induced pyrimidine dimers. These results may be interpreted by a hypothesis that caffeine inhibits a certain S-phase specific, post-replication, dark-repair mechanism. The hamster and perhaps other rodent cells exposed to low doses of UV are capable of DNA replication, by-passing the non-excised pyrimidine dimers. This postulated repair process probably involves de novo DNA synthesis to seal the gaps in the nascent strand. This repair may be also responsible for the enzymatic production of mutations.     

Design,synthesis and antiplasmodial activity of novel imidazole derivatives based on 7-chloro-4-aminoquinoline

A series of short chain 4-aminoquinoline-imidazole derivatives have been synthesized in one pot two step multicomponent reaction using van leusen standard protocol. The diethylamine function of chloroquine is replaced by substituted imidazole derivatives containing tertiary terminal nitrogen. All the synthesized compounds were screened against the chloroquine sensitive (3D7) and chloroquine resistant (K1) strains of Plasmodium falciparum. Some of the compounds (6, 8, 9 and 17) in the series exhibited comparable activity to CQ against K1 strain of P. falciparum. All the compounds displayed resistance factor between 0.09 and 4.57 as against 51 for CQ. Further, these analogues were found to form a strong complex with hematin and inhibit the β-hematin formation, therefore these compounds act via heme polymerization target.     

Mutational analysis of plant cap-binding protein eIF4E reveals key amino acids involved in biochemical functions and potyvirus infection

The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against several potyviruses in plants. In lettuce, the recessive resistance genes mo1¹ and mo1² against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo1¹ or mo1² varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast. However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be distinct from its physiological function in cellular mRNA translation.     

Potential Application of N-Carbamoyl-β-Alanine Amidohydrolase from Agrobacterium tumefaciens C58 for β-Amino Acid Production

An N-carbamoyl-β-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (βcar_At) has been characterized. βcar_At is most active at 30°C and pH 8.0 with N-carbamoyl-β-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn²⁺, Ni²⁺, and Co²⁺. The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-α-, -β-, -γ-, and -δ-amino acids, with the greatest catalytic efficiency for N-carbamoyl-β-alanine. βcar_At also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the β-amino acids taurine and ciliatine, respectively. βcar_At is able to produce monosubstituted β²- and β³-amino acids, showing better catalytic efficiency (k_cat/K_m) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-β-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make βcar_At an outstanding candidate for application in the biotechnology industry.     

High DDT resistance without apparent association to kdr and Glutathione-S-transferase (GST) gene mutations in Aedes aegypti population at hotel compounds in Zanzibar

Global efforts to control Aedes mosquito-transmitted pathogens still rely heavily on insecticides. However, available information on vector resistance is mainly restricted to mosquito populations located in residential and public areas, whereas commercial settings, such as hotels are overlooked. This may obscure the real magnitude of the insecticide resistance problem and lead to ineffective vector control and resistance management. We investigated the profile of insecticide susceptibility of Aedes aegypti mosquitoes occurring at selected hotel compounds on Zanzibar Island. At least 100 adults Ae. aegypti females from larvae collected at four hotel compounds were exposed to papers impregnated with discriminant concentrations of DDT (4%), permethrin (0.75%), 0.05 deltamethrin (0.05%), propoxur (0.1%) and bendiocarb (0.1%) to determine their susceptibility profile. Allele-specific qPCR and sequencing analysis were applied to determine the possible association between observed resistance and presence of single nucleotide polymorphisms (SNPs) in the voltage-gated sodium channel gene (VGSC) linked to DDT/pyrethroid cross-resistance. Additionally, we explored the possible involvement of Glutathione-S-Transferase gene (GSTe2) mutations for the observed resistance profile. In vivo resistance bioassay indicated that Ae. aegypti at studied sites were highly resistant to DDT, mortality rate ranged from 26.3% to 55.3% and, moderately resistant to deltamethrin with a mortality rate between 79% to and 100%. However, genotyping of kdr mutations affecting the voltage-gated sodium channel only showed a low frequency of the V1016G mutation (n = 5; 0.97%). Moreover, for GSTe2, seven non-synonymous SNPs were detected (L111S, C115F, P117S, E132A, I150V, E178A and A198E) across two distinct haplotypes, but none of these were significantly associated with the observed resistance to DDT. Our findings suggest that cross-resistance to DDT/deltamethrin at hotel compounds in Zanzibar is not primarily mediated by mutations in VGSC. Moreover, the role of identified GSTe2 mutations in the resistance against DDT remains inconclusive. We encourage further studies to investigate the role of other potential insecticide resistance markers.     

Mechanism of Caffeine Enhancement of Mutations Induced by Sublethal Ultraviolet Dosages

Certain chemical compounds increase mutation frequency of Escherichia coli B/r significantly when used in conjunction with nonlethal ultraviolet (UV) dosages. Studies were done to elucidate the mechanism of this enhancing mutational effect. Dark survival curves showed that 500 μg of caffeine per ml in the postirradiation medium markedly decreased survival to 60 ergs/mm² of UV in strain B/r. Caffeine did not markedly decrease survival to UV in strain B/r WP-2 hcr⁻. At least 90% of the mutations induced to streptomycin resistance by UV and 85% of those induced by UV with caffeine could be photoreversed. Experiments with thymine analogues suggested that thymine dimerization at the streptomycin locus was the primary premutational photoproduct induced by sublethal UV dosages. Caffeine did not interfere with the photoreversal of induced mutants, indicating that it probably does not bind to the photoreactivating enzyme or to a UV-induced lesion in the DNA. Addition of DNA or irradiated DNA with 500 μg of caffeine per ml resulted in no loss of the caffeine activity. The excision of UV-induced thymine-containing dimers from E. coli B/r T⁻ was investigated in the presence and absence of caffeine. Our results indicated that caffeine prevents excision of thymine dimers, presumably by binding to the excising enzyme. This binding results in an impairment of repair, which produces the increase in mutant numbers.     

Pyrethroid-Resistance and Presence of Two Knockdown Resistance (kdr) Mutations,F1534C and a Novel Mutation T1520I,in Indian Aedes aegypti

Background

Control of Aedes aegypti, the mosquito vector of dengue, chikungunya and yellow fever, is a challenging task. Pyrethroid insecticides have emerged as a preferred choice for vector control but are threatened by the emergence of resistance. The present study reports a focus of pyrethroid resistance and presence of two kdr mutations—F1534C and a novel mutation T1520I, in Ae. aegypti from Delhi, India.

Methodology/Principal Findings

Insecticide susceptibility status of adult-female Ae. aegypti against DDT (4%), deltamethrin (0.05%) and permethrin (0.75%) was determined using WHO''s standard insecticide susceptibility kit, which revealed resistance to DDT, deltamethrin and permethrin with corrected mortalities of 35%, 72% and 76% respectively. Mosquitoes were screened for the presence of kdr mutations including those reported earlier (I1011V/M, V1016G/I, F1534C, D1794Y and S989P), which revealed the presence of F1534C and a novel mutation T1520I. Highly specific PCR-RFLP assays were developed for genotyping of these two mutations. Genotyping using allele specific PCR and new PCR-RFLP assays revealed a high frequency of F1534C (0.41–0.79) and low frequency of novel mutation T1520I (0.13). The latter was observed to be tightly linked with F1534C and possibly serve as a compensatory mutation. A positive association of F1534C mutation with DDT and deltamethrin resistance in Ae. aegypti was established. However, F1534C-kdr did not show significant protection against permethrin.

Conclusions/Significance

The Aedes aegypti population of Delhi is resistant to DDT, deltamethrin and permethrin. Two kdr mutations, F1534C and a novel mutation T1520I, were identified in this population. This is the first report of kdr mutations being present in the Indian Ae. aegypti population. Highly specific PCR-RFLP assays were developed for discrimination of alleles at both kdr loci. A positive association of F1534C mutation with DDT and deltamethrin resistance was confirmed.     

Short-Term Responders of Non–Small Cell Lung Cancer Patients to EGFR Tyrosine Kinase Inhibitors Display High Prevalence of TP53 Mutations and Primary Resistance Mechanisms

Non–small cell lung cancer (NSCLC) with activating EGFR mutations in exon 19 and 21 typically responds to EGFR tyrosine kinase inhibitors (TKI); however, for some patients, responses last only a few months. The underlying mechanisms of such short responses have not been fully elucidated. Here, we sequenced the genomes of 16 short-term responders (SR) that had progression-free survival (PFS) of less than 6 months on the first-generation EGFR TKI and compared them to 12 long-term responders (LR) that had more than 24 months of PFS. All patients were diagnosed with advanced lung adenocarcinoma and harbored EGFR 19del or L858R mutations before treatment. Paired tumor samples collected before treatment and after relapse (or at the last follow-up) were subjected to targeted next-generation sequencing of 416 cancer-related genes. SR patients were significantly younger than LR patients (P < .001). Collectively, 88% of SR patients had TP53 variations compared to 13% of LR patients (P < .001). Additionally, 37.5% of SR patients carried EGFR amplifications compared to 8% of LR patients. Other potential primary resistance factors were also identified in the pretreatment samples of 12 SR patients (75%), including PTEN loss; BIM deletion polymorphism; and amplifications of EGFR, ERBB2, MET, HRAS, and AKT2. Comparatively, only three LR patients (25%) were detected with EGFR or AKT1 amplifications that could possibly exert resistance. The diverse preexisting resistance mechanisms in SR patients revealed the complexity of defining treatment strategies even for EGFR-sensitive mutations.     

Knock-down of both eIF4E1 and eIF4E2 genes confers broad-spectrum resistance against potyviruses in tomato

Background

The eukaryotic translation initiation factor eIF4E plays a key role in plant-potyvirus interactions. eIF4E belongs to a small multigenic family and three genes, eIF4E1, eIF4E2 and eIF(iso)4E, have been identified in tomato. It has been demonstrated that eIF4E-mediated natural recessive resistances against potyviruses result from non-synonymous mutations in an eIF4E protein, which impair its direct interaction with the potyviral protein VPg. In tomato, the role of eIF4E proteins in potyvirus resistance is still unclear because natural or induced mutations in eIF4E1 confer only a narrow resistance spectrum against potyviruses. This contrasts with the broad spectrum resistance identified in the natural diversity of tomato. These results suggest that more than one eIF4E protein form is involved in the observed broad spectrum resistance.

Methodology/Principal Findings

To gain insight into the respective contribution of each eIF4E protein in tomato-potyvirus interactions, two tomato lines silenced for both eIF4E1 and eIF4E2 (RNAi-4E) and two lines silenced for eIF(iso)4E (RNAi-iso4E) were obtained and characterized. RNAi-4E lines are slightly impaired in their growth and fertility, whereas no obvious growth defects were observed in RNAi-iso4E lines. The F1 hybrid between RNAi-4E and RNAi-iso4E lines presented a pronounced semi-dwarf phenotype. Interestingly, the RNAi-4E lines silenced for both eIF4E1 and eIF4E2 showed broad spectrum resistance to potyviruses while the RNAi-iso4E lines were fully susceptible to potyviruses. Yeast two-hybrid interaction assays between the three eIF4E proteins and a set of viral VPgs identified two types of VPgs: those that interacted only with eIF4E1 and those that interacted with either eIF4E1 or with eIF4E2.

Conclusion/Significance

These experiments provide evidence for the involvement of both eIF4E1 and eIF4E2 in broad spectrum resistance of tomato against potyviruses and suggest a role for eIF4E2 in tomato-potyvirus interactions.     

The protein patterns of intracytoplasmic membranes and reaction center particles isolated fromRhodopseudomonas capsulata

Intracytoplasmic membranes of wild type strain 37 b 4 and mutant strains A1a car^-bchl^-, A1a car^-bchl⁺ ofRhodopseudomonas capsulata were isolated. The membrane proteins were solubilized and separated by polyacrylamide gel electrophoresis (methods of Takayamaet al., 1964; Weber and Osborn, 1969). The band patterns were compared with each other. From the strain A1a car^-bchl⁺ reaction center particles were isolated by treatment of membrane with Triton X-100 followed by sucrose density gradient centrifugation. The reaction center particles were found to be enriched in reaction center bacteriochlorophyll. This pigment shows a reversible bleaching at 855 nm and a blue shift at 798 nm. The light harvesting bacteriochlorophyll portion of this fraction was 14–22% of the total bacteriochlorophyll content. The three main proteins of the reaction center particles amount to about 80% of the total protein of the particles. The molecular weights of the main proteins were estimated to be 32000, 27500 and 22500 daltons.     

Identifying Loci Contributing to Natural Variation in Xenobiotic Resistance in Drosophila

Natural populations exhibit a great deal of interindividual genetic variation in the response to toxins, exemplified by the variable clinical efficacy of pharmaceutical drugs in humans, and the evolution of pesticide resistant insects. Such variation can result from several phenomena, including variable metabolic detoxification of the xenobiotic, and differential sensitivity of the molecular target of the toxin. Our goal is to genetically dissect variation in the response to xenobiotics, and characterize naturally-segregating polymorphisms that modulate toxicity. Here, we use the Drosophila Synthetic Population Resource (DSPR), a multiparent advanced intercross panel of recombinant inbred lines, to identify QTL (Quantitative Trait Loci) underlying xenobiotic resistance, and employ caffeine as a model toxic compound. Phenotyping over 1,700 genotypes led to the identification of ten QTL, each explaining 4.5–14.4% of the broad-sense heritability for caffeine resistance. Four QTL harbor members of the cytochrome P450 family of detoxification enzymes, which represent strong a priori candidate genes. The case is especially strong for Cyp12d1, with multiple lines of evidence indicating the gene causally impacts caffeine resistance. Cyp12d1 is implicated by QTL mapped in both panels of DSPR RILs, is significantly upregulated in the presence of caffeine, and RNAi knockdown robustly decreases caffeine tolerance. Furthermore, copy number variation at Cyp12d1 is strongly associated with phenotype in the DSPR, with a trend in the same direction observed in the DGRP (Drosophila Genetic Reference Panel). No additional plausible causative polymorphisms were observed in a full genomewide association study in the DGRP, or in analyses restricted to QTL regions mapped in the DSPR. Just as in human populations, replicating modest-effect, naturally-segregating causative variants in an association study framework in flies will likely require very large sample sizes.     

Generation and Molecular Characterization of New Temperature-Sensitive Plasmids Intended for Genetic Engineering of Pasteurellaceae

Temperature-sensitive (TS) plasmids were generated through chemical mutagenesis of a derivative of the streptomycin resistance parent plasmid pD70, isolated from Mannheimia hemolytica serotype 1. Three TS plasmids which failed to replicate at or above 42°C in M. hemolytica but which were fully functional below 31°C were selected for further analysis. Two of the TS plasmids were shown by sequencing to possess unique single-base-pair mutations. The third TS plasmid contained a unique base pair substitution and a second mutation that had been previously identified. These mutations were clustered within a 200-bp region of the presumed plasmid origin of replication. Site-directed single-nucleotide substitutions were introduced into the wild-type pD70 origin of replication to confirm that mutations identified by sequencing had conferred thermoregulated replication. Deletion analysis on the wild-type pD70 plasmid replicon revealed that approximately 720 bp are necessary for plasmid maintenance. Replication of the TS plasmids was thermoregulated in Pasteurella multocida and Haemophilus somnus as well. To consistently transform H. somnus with TS plasmid, in vitro DNA methylation with commercially available HhaI methyltransferase was necessary to protect against the organism's restriction enzyme HsoI (recognition sequence 5′-GCGC-3′) characterized herein.     

Conversion of nicotinic acid to trigonelline is catalyzed by N-methyltransferase belonged to motif B′ methyltransferase family in Coffea arabica

Trigonelline (N-methylnicotinate), a member of the pyridine alkaloids, accumulates in coffee beans along with caffeine. The biosynthetic pathway of trigonelline is not fully elucidated. While it is quite likely that the production of trigonelline from nicotinate is catalyzed by N-methyltransferase, as is caffeine synthase (CS), the enzyme(s) and gene(s) involved in N-methylation have not yet been characterized. It should be noted that, similar to caffeine, trigonelline accumulation is initiated during the development of coffee fruits. Interestingly, the expression profiles for two genes homologous to caffeine synthases were similar to the accumulation profile of trigonelline. We presumed that these two CS-homologous genes encoded trigonelline synthases. These genes were then expressed in Escherichiacoli, and the resulting recombinant enzymes that were obtained were characterized. Consequently, using the N-methyltransferase assay with S-adenosyl[methyl-¹⁴C]methionine, it was confirmed that these recombinant enzymes catalyzed the conversion of nicotinate to trigonelline, coffee trigonelline synthases (termed CTgS1 and CTgS2) were highly identical (over 95% identity) to each other. The sequence homology between the CTgSs and coffee CCS1 was 82%. The pH-dependent activity curve of CTgS1 and CTgS2 revealed optimum activity at pH 7.5. Nicotinate was the specific methyl acceptor for CTgSs, and no activity was detected with any other nicotinate derivatives, or with any of the typical substrates of B′-MTs. It was concluded that CTgSs have strict substrate specificity. The K_m values of CTgS1 and CTgS2 were 121 and 184 μM with nicotinic acid as a substrate, and 68 and 120 μM with S-adenosyl-l-methionine as a substrate, respectively.