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10200.9402Paradoxical behaviour of pKM101; inhibition of uvr-independent crosslink repair in Escherichia coli by muc gene products. In strains of Escherichia coli deficient in excision repair (uvrA or uvrB), plasmid pKM101 muc+ but not pGW219 mucB::Tn5 enhanced resistance to angelicin monoadducts but reduced resistance to 8-methoxy-psoralen interstrand DNA crosslinks. Thermally induced recA-441 (= tif-1) bacteria showed an additional resistance to crosslinks that was blocked by pKM101. Plasmid-borne muc+ genes also conferred some additional sensitivity to gamma-radiation and it is suggested that a repair step susceptible to inhibition by muc+ gene products and possibly involving double-strand breaks may be involved after both ionizing radiation damage and psoralen crosslinks.19853883148
52810.9247Effect of dimethyl sulphoxide on the expression of nitrogen fixation in bacteria. Storage in dimethyl sulphoxide (DMSO) of Escherichia coli K12 hybrids carrying nif+ genes from Klebsiella pneumoniae can result in selection of a defective nitrogen-fixing phenotype. Similar results are obtained with E. coli K12 hybrids containing the nitrogen-fixing capacity from Rhizobium trifolii. DMSO appears to affect particular inner membrane proteins associated with energy metabolism in E. coli K12 and four chromosomal regions (chlD, chlG, his and unc) are associated with resistance to DMSO.1977332135
55920.9213Coordinated regulation of chemotaxis and resistance to copper by CsoR in Pseudomonas putida. Copper is an essential enzyme cofactor in bacteria, but excess copper is highly toxic. Bacteria can cope with copper stress by increasing copper resistance and initiating chemorepellent response. However, it remains unclear how bacteria coordinate chemotaxis and resistance to copper. By screening proteins that interacted with the chemotaxis kinase CheA, we identified a copper-binding repressor CsoR that interacted with CheA in Pseudomonas putida. CsoR interacted with the HPT (P1), Dimer (P3), and HATPase_c (P4) domains of CheA and inhibited CheA autophosphorylation, resulting in decreased chemotaxis. The copper-binding of CsoR weakened its interaction with CheA, which relieved the inhibition of chemotaxis by CsoR. In addition, CsoR bound to the promoter of copper-resistance genes to inhibit gene expression, and copper-binding released CsoR from the promoter, leading to increased gene expression and copper resistance. P. putida cells exhibited a chemorepellent response to copper in a CheA-dependent manner, and CsoR inhibited the chemorepellent response to copper. Besides, the CheA-CsoR interaction also existed in proteins from several other bacterial species. Our results revealed a mechanism by which bacteria coordinately regulated chemotaxis and resistance to copper by CsoR.202540197389
930.9212Durable broad-spectrum powdery mildew resistance in pea er1 plants is conferred by natural loss-of-function mutations in PsMLO1. Loss-of-function alleles of plant-specific MLO (Mildew Resistance Locus O) genes confer broad-spectrum powdery mildew resistance in monocot (barley) and dicot (Arabidopsis thaliana, tomato) plants. Recessively inherited powdery mildew resistance in pea (Pisum sativum) er1 plants is, in many aspects, reminiscent of mlo-conditioned powdery mildew immunity, yet the underlying gene has remained elusive to date. We used a polymerase chain reaction (PCR)-based approach to amplify a candidate MLO cDNA from wild-type (Er1) pea. Sequence analysis of the PsMLO1 candidate gene in two natural er1 accessions from Asia and two er1-containing pea cultivars with a New World origin revealed, in each case, detrimental nucleotide polymorphisms in PsMLO1, suggesting that PsMLO1 is Er1. We corroborated this hypothesis by restoration of susceptibility on transient expression of PsMLO1 in the leaves of two resistant er1 accessions. Orthologous legume MLO genes from Medicago truncatula and Lotus japonicus likewise complemented the er1 phenotype. All tested er1 genotypes showed unaltered colonization with the arbuscular mycorrhizal fungus, Glomus intraradices, and with nitrogen-fixing rhizobial bacteria. Our data demonstrate that PsMLO1 is Er1 and that the loss of PsMLO1 function conditions durable broad-spectrum powdery mildew resistance in pea.201121726385
50440.9208Activation of Dithiolopyrrolone Antibiotics by Cellular Reductants. Dithiolopyrrolone (DTP) natural products are broad-spectrum antimicrobial and anticancer prodrugs. The DTP structure contains a unique bicyclic ene-disulfide that once reduced in the cell, chelates metal ions and disrupts metal homeostasis. In this work we investigate the intracellular activation of the DTPs and their resistance mechanisms in bacteria. We show that the prototypical DTP holomycin is reduced by several bacterial reductases and small-molecule thiols in vitro. To understand how bacteria develop resistance to the DTPs, we generate Staphylococcus aureus mutants that exhibit increased resistance to the hybrid DTP antibiotic thiomarinol. From these mutants we identify loss-of-function mutations in redox genes that are involved in DTP activation. This work advances the understanding of how DTPs are activated and informs development of bioreductive disulfide prodrugs.202539665630
847250.9207Genetic architecture of resistance to plant secondary metabolites in Photorhabdus entomopathogenic bacteria. BACKGROUND: Entomopathogenic nematodes of the genus Heterorhabditis establish a symbiotic association with Photorhabdus bacteria. Together, they colonize and rapidly kill insects, making them important biological control agents against agricultural pests. Improving their biocontrol traits by engineering resistance to plant secondary metabolites (benzoxazinoids) in Photorhabdus symbiotic bacteria through experimental evolution has been shown to increase their lethality towards benzoxazinoid-defended larvae of the western corn rootworm, a serious crop pest of maize, and it is therefore a promising approach to develop more efficient biocontrol agents to manage this pest. To enhance our understanding of the genetic bases of benzoxazinoid resistance in Photorhabdus bacteria, we conducted an experimental evolution experiment with a phylogenetically diverse collection of Photorhabdus strains from different geographic origins. We cultured 27 different strains in medium containing 6-methoxy-2-benzoxazolinone (MBOA), a highly active benzoxazinoid breakdown product, for 35 24 h-cycles to select for benzoxazinoid-resistant strains. Then, we carried out genome-wide sequence comparisons to uncover the genetic alterations associated with benzoxazinoid resistance. Lastly, we evaluated the resistance of the newly isolated resistant Photorhabdus strains to eight additional bioactive compounds, including 2-benzoxazolinone (BOA), nicotine, caffeine, 6-chloroacetyl-2-benzoxazolinone (CABOA), digitoxin, fenitrothion, ampicillin, and kanamycin. RESULTS: We found that benzoxazinoid resistance evolves rapidly in Photorhabdus in a strain-specific manner. Across the different Photorhabdus strains, a total of nineteen nonsynonymous point mutations, two stop codon gains, and one frameshift were associated with higher benzoxazinoid resistance. The different genetic alterations were polygenic and occurred in genes coding for the EnvZ/OmpR two-component regulatory system, the different subunits of the DNA-directed RNA polymerase, and the AcrABZ-TolC multidrug efflux pump. Apart from increasing MBOA resistance, the different mutations were also associated with cross-resistance to 2-benzoxazolinone (BOA), nicotine, caffeine, and 6-chloroacetyl-2-benzoxazolinone (CABOA) and with collateral sensitivity to fenitrothion, ampicillin, and kanamycin. Targeted mutagenesis will provide a deeper mechanistic understanding, including the relative contribution of the different mutation types. CONCLUSIONS: Our study reveals several genomic features that are associated with resistance to xenobiotics in this important group of biological control agents and enhances the availability of molecular tools to develop better biological control agents, which is essential for more sustainable and ecologically friendly agricultural practices.202541168779
65460.9204Conjugation inhibitors compete with palmitic acid for binding to the conjugative traffic ATPase TrwD, providing a mechanism to inhibit bacterial conjugation. Bacterial conjugation is a key mechanism by which bacteria acquire antibiotic resistance. Therefore, conjugation inhibitors (COINs) are promising compounds in the fight against the spread of antibiotic resistance genes among bacteria. Unsaturated fatty acids (uFAs) and alkynoic fatty acid derivatives, such as 2-hexadecanoic acid (2-HDA), have been reported previously as being effective COINs. The traffic ATPase TrwD, a VirB11 homolog in plasmid R388, is the molecular target of these compounds, which likely affect binding of TrwD to bacterial membranes. In this work, we demonstrate that COINs are abundantly incorporated into Escherichia coli membranes, replacing palmitic acid as the major component of the membrane. We also show that TrwD binds palmitic acid, thus facilitating its interaction with the membrane. Our findings also suggest that COINs bind TrwD at a site that is otherwise occupied by palmitic acid. Accordingly, molecular docking predictions with palmitic acid indicated that it shares the same binding site as uFAs and 2-HDA, although it differs in the contacts involved in this interaction. We also identified 2-bromopalmitic acid, a palmitate analog that inhibits many membrane-associated enzymes, as a compound that effectively reduces TrwD ATPase activity and bacterial conjugation. Moreover, we demonstrate that 2-bromopalmitic and palmitic acids both compete for the same binding site in TrwD. Altogether, these detailed findings open up a new avenue in the search for effective synthetic inhibitors of bacterial conjugation, which may be pivotal for combating multidrug-resistant bacteria.201830201608
65370.9201Connecting Algal Polysaccharide Degradation to Formaldehyde Detoxification. Formaldehyde is a toxic metabolite that is formed in large quantities during bacterial utilization of the methoxy sugar 6-O-methyl-d-galactose, an abundant monosaccharide in the red algal polysaccharide porphyran. Marine bacteria capable of metabolizing porphyran must therefore possess suitable detoxification systems for formaldehyde. We demonstrate here that detoxification of formaldehyde in the marine Flavobacterium Zobellia galactanivorans proceeds via the ribulose monophosphate pathway. Simultaneously, we show that the genes encoding the key enzymes of this pathway are important for maintaining high formaldehyde resistance. Additionally, these genes are upregulated in the presence of porphyran, allowing us to connect porphyran degradation to the detoxification of formed formaldehyde.202235561127
60780.9199A novel copper-sensing two-component system for inducing Dsb gene expression in bacteria. In nature, bacteria must sense copper and tightly regulate gene expression to evade copper toxicity. Here, we identify a new copper-responsive two-component system named DsbRS in the important human pathogen Pseudomonas aeruginosa; in this system, DsbS is a sensor histidine kinase, and DsbR, its cognate response regulator, directly induces the transcription of genes involved in protein disulfide bond formation (Dsb) (i.e., the dsbDEG operon and dsbB). In the absence of copper, DsbS acts as a phosphatase toward DsbR, thus blocking the transcription of Dsb genes. In the presence of copper, the metal ion directly binds to the sensor domain of DsbS, and the Cys82 residue plays a critical role in this process. The copper-binding behavior appears to inhibit the phosphatase activity of DsbS, leading to the activation of DsbR. The copper resistance of the dsbRS knock-out mutant is restored by the ectopic expression of the dsbDEG operon, which is a DsbRS major target. Strikingly, cognates of the dsbRS-dsbDEG pair are widely distributed across eubacteria. In addition, a DsbR-binding site, which contains the consensus sequence 5'-TTA-N(8)-TTAA-3', is detected in the promoter region of dsbDEG homologs in these species. These findings suggest that the regulation of Dsb genes by DsbRS represents a novel mechanism by which bacterial cells cope with copper stress.202236546013
60990.9197A metazoan ortholog of SpoT hydrolyzes ppGpp and functions in starvation responses. In nutrient-starved bacteria, RelA and SpoT proteins have key roles in reducing cell growth and overcoming stresses. Here we identify functional SpoT orthologs in metazoa (named Mesh1, encoded by HDDC3 in human and Q9VAM9 in Drosophila melanogaster) and reveal their structures and functions. Like the bacterial enzyme, Mesh1 proteins contain an active site for ppGpp hydrolysis and a conserved His-Asp-box motif for Mn(2+) binding. Consistent with these structural data, Mesh1 efficiently catalyzes hydrolysis of guanosine 3',5'-diphosphate (ppGpp) both in vitro and in vivo. Mesh1 also suppresses SpoT-deficient lethality and RelA-induced delayed cell growth in bacteria. Notably, deletion of Mesh1 (Q9VAM9) in Drosophila induces retarded body growth and impaired starvation resistance. Microarray analyses reveal that the amino acid-starved Mesh1 null mutant has highly downregulated DNA and protein synthesis-related genes and upregulated stress-responsible genes. These data suggest that metazoan SpoT orthologs have an evolutionarily conserved function in starvation responses.201020818390
338100.9193Repair by genetic recombination in bacteria: overview. DNA molecules that have been damaged in both strands at the same level are not subject to repair by excision but instead can be repaired through recombination with homologous molecules. Examples of two-strand damage include postreplication gaps opposite pyrimidine dimers, two-strand breaks produced by X-rays, and chemically induced interstrand cross-links. In ultraviolet-irradiated bacteria, the newly synthesized DNA is of length equal to the interdimer spacing. With continued incubation, this low-molecular-weight DNA is joined into high-molecular-weight chains (postreplication repair), a process associated with sister exchanges in bacteria. Recombination is initiated by pyrimidine dimers opposite postreplication gaps and by interstrand cross-links that have been cut by excision enzymes. The free ends at the resulting gaps presumably initiate the exchanges. Postreplication repair in Escherichia coli occurs in recB- AND RECC but is greatly slowed in recF- mutants. RecB and recC are the structural genes for exonuclease V, which digests two-stranded DNA by releasing oligonucleotides first from one strand and then from the other. The postreplication sister exchanges in ultra-violet-irradiated bacteria result in the distribution of pyrimidine dimers between parental and daughter strands, indicating that long exchanges involving both strands of each duplex occur. The R1 restriction endonuclease from E. COli has been used to cut the DNA of a bacterial drug-resistance transfer factor with one nuclease-sensitive site, and also DNA from the frog Xenopus enriched for ribosomal 18S and 28S genes. The fragments were annealed with the cut plasmid DNA and ligated, producing a new larger plasmid carrying the eukaryotic rDNA and able to infect and replicate in E. coli.19751103833
57110.9190Functional analysis of NtMPK2 uncovers its positive role in response to Pseudomonas syringae pv. tomato DC3000 in tobacco. Mitogen-activated protein kinase cascades are highly conserved signaling modules downstream of receptors/sensors and play pivotal roles in signaling plant defense against pathogen attack. Extensive studies on Arabidopsis MPK4 have implicated that the MAP kinase is involved in multilayered plant defense pathways. In this study, we identified tobacco NtMPK2 as an ortholog of AtMPK4. Transgenic tobacco overexpressing NtMPK2 markedly enhances resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) virulent and avirulent strains. Transcriptome analysis of NtMPK2-dependent genes shows that possibly the basal resistance system is activated by NtMPK2 overexpression. In addition to NtMPK2-mediated resistance, multiple pathways are involved in response to the avirulent bacteria based on analysis of Pst-responding genes, including SA and ET pathways. Notably, it is possible that biosynthesis of antibacterial compounds is responsible for inhibition of Pst DC3000 avirulent strain when programmed cell death processes in the host. Our results uncover that NtMPK2 positively regulate tobacco defense response to Pst DC3000 and improve our understanding of plant molecular defense mechanism.201626482478
340120.9189Study of MFD-type repair in locus determining resistance of Escherichia coli to streptomycin. The yield of induced mutations to streptomycin resistance (Str) in E. coli, UV-irradiated and temporarily incubated in liquid medium not permitting protein synthesis, depends upon the conditions of preirradiation growth and preirradiation treatment of the bacteria, i.e. on their physiological state at the moment of irradiation. This fact is not readily reconciled with a model postulating mutation production in the structural genes of E. coli during excision repair. A preferred explanation is offered, based on the assumption that the efficiency of mutagenesis at the rpsL (strA) locus is determined by interference of antimutagenic (generalized excision repair and MFD) and promutagenic (mutation fixation of excision repair) events. The participation of macromolecular syntheses in Str mutation fixation is suggested.19863537780
806130.9188A two-component small multidrug resistance pump functions as a metabolic valve during nicotine catabolism by Arthrobacter nicotinovorans. The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [(14)C]nicotine was added to the growth medium the bacteria exported the (14)C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria. Efflux of [(14)C]nicotine-derived radioactivity from bacteria was also inhibited in a pmfR : cmx strain with downregulated nepAB expression. Because of low amine oxidase levels in the pmfR : cmx strain, gamma-N-methylaminobutyrate, the methylamine precursor, accumulated. Complementation of this strain with the nepAB genes, carried on a plasmid, restored the efflux of nicotine breakdown products. Both NepA and NepB were required for full export activity, indicating that they form a two-component efflux pump. NepAB may function as a metabolic valve by exporting methylamine, the end product of nicotine catabolism, and, in conditions under which it accumulates, the intermediate gamma-N-methylaminobutyrate.200717464069
108140.9187RtcB2-PrfH Operon Protects E. coli ATCC25922 Strain from Colicin E3 Toxin. In the bid to survive and thrive in an environmental setting, bacterial species constantly interact and compete for resources and space in the microbial ecosystem. Thus, they have adapted to use various antibiotics and toxins to fight their rivals. Simultaneously, they have evolved an ability to withstand weapons that are directed against them. Several bacteria harbor colicinogenic plasmids which encode toxins that impair the translational apparatus. One of them, colicin E3 ribotoxin, mediates cleavage of the 16S rRNA in the decoding center of the ribosome. In order to thrive upon deployment of such ribotoxins, competing bacteria may have evolved counter-conflict mechanisms to prevent their demise. A recent study demonstrated the role of PrfH and the RtcB2 module in rescuing a damaged ribosome and the subsequent re-ligation of the cleaved 16S rRNA by colicin E3 in vitro. The rtcB2-prfH genes coexist as gene neighbors in an operon that is sporadically spread among different bacteria. In the current study, we report that the RtcB2-PrfH module confers resistance to colicin E3 toxicity in E. coli ATCC25922 cells in vivo. We demonstrated that the viability of E. coli ATCC25922 strain that is devoid of rtcB2 and prfH genes is impaired upon action of colicin E3, in contrast to the parental strain which has intact rtcB2 and prfH genes. Complementation of the rtcB2 and prfH gene knockout with a high copy number-plasmid (encoding either rtcB2 alone or both rtcB2-prfH operon) restored resistance to colicin E3. These results highlight a counter-conflict system that may have evolved to thwart colicin E3 activity.202235742896
339150.9186Multiple mechanisms of resistance to cisplatin toxicity in an Escherichia coli K12 mutant. The mechanisms underlying cellular resistance to the antitumor drug cis-diamminedichloro-platinum(II) (CDDP) were studied in Escherichia coli K12. A bacterial strain (MC4100/DDP) was selected from the MC4100 wild-type strain after growth for four cycles in CDDP. MC4100/DDP bacteria showed a high level of resistance and exhibited various modifications including (1) a decrease in drug uptake and platinum/DNA binding which only partly contributed to resistance, (2) an increase in glutathione content not involved in the resistant phenotype, (3) an increase in DNA repair capacity. Resistance was unmodified by introducing a uvrA mutation which neutralizes the excision-repair pathway. In contrast, it was abolished by deletion of the recA gene which abolishes recombination and SOS repair but also by a mutation in the recA gene leading to RecA co-protease minus (no SOS induction). RecA protein was unchanged in MC4100/DDP but the expression of RecA-dependent gene(s) was required for CDDP resistance. The regulation of genes belonging to the SOS regulon was analysed in MC4100/DDP by monitoring the expression of sfiA and recA::lacZ gene fusions after UV irradiation. These gene fusions were derepressed faster and the optimal expression was obtained for a lower number of UV lesions in MC4100/DDP, suggesting a role of RecA co-protease activity in the mechanism of resistance to CDDP in this E. coli strain.19947974517
48160.9186Priming of the Arabidopsis pattern-triggered immunity response upon infection by necrotrophic Pectobacterium carotovorum bacteria. Boosted responsiveness of plant cells to stress at the onset of pathogen- or chemically induced resistance is called priming. The chemical β-aminobutyric acid (BABA) enhances Arabidopsis thaliana resistance to hemibiotrophic bacteria through the priming of the salicylic acid (SA) defence response. Whether BABA increases Arabidopsis resistance to the necrotrophic bacterium Pectobacterium carotovorum ssp. carotovorum (Pcc) is not clear. In this work, we show that treatment with BABA protects Arabidopsis against the soft-rot pathogen Pcc. BABA did not prime the expression of the jasmonate/ethylene-responsive gene PLANT DEFENSIN 1.2 (PDF1.2), the up-regulation of which is usually associated with resistance to necrotrophic pathogens. Expression of the SA marker gene PATHOGENESIS RELATED 1 (PR1) on Pcc infection was primed by BABA treatment, but SA-defective mutants demonstrated a wild-type level of BABA-induced resistance against Pcc. BABA primed the expression of the pattern-triggered immunity (PTI)-responsive genes FLG22-INDUCED RECEPTOR-LIKE KINASE 1 (FRK1), ARABIDOPSIS NON-RACE SPECIFIC DISEASE RESISTANCE GENE (NDR1)/HAIRPIN-INDUCED GENE (HIN1)-LIKE 10 (NHL10) and CYTOCHROME P450, FAMILY 81 (CYP81F2) after inoculation with Pcc or after treatment with purified bacterial microbe-associated molecular patterns, such as flg22 or elf26. PTI-mediated callose deposition was also potentiated in BABA-treated Arabidopsis, and BABA boosted Arabidopsis stomatal immunity to Pcc. BABA treatment primed the PTI response in the SA-defective mutants SA induction deficient 2-1 (sid2-1) and phytoalexin deficient 4-1 (pad4-1). In addition, BABA priming was associated with open chromatin configurations in the promoter region of PTI marker genes. Our data indicate that BABA primes the PTI response upon necrotrophic bacterial infection and suggest a role for the PTI response in BABA-induced resistance.201322947164
126170.9184Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum. Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite D,L-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of L-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.200515668756
558180.9184Thiamine pyrophosphate riboswitches are targets for the antimicrobial compound pyrithiamine. Thiamine metabolism genes are regulated in numerous bacteria by a riboswitch class that binds the coenzyme thiamine pyrophosphate (TPP). We demonstrate that the antimicrobial action of the thiamine analog pyrithiamine (PT) is mediated by interaction with TPP riboswitches in bacteria and fungi. For example, pyrithiamine pyrophosphate (PTPP) binds the TPP riboswitch controlling the tenA operon in Bacillus subtilis. Expression of a TPP riboswitch-regulated reporter gene is reduced in transgenic B. subtilis or Escherichia coli when grown in the presence of thiamine or PT, while mutant riboswitches in these organisms are unresponsive to these ligands. Bacteria selected for PT resistance bear specific mutations that disrupt ligand binding to TPP riboswitches and derepress certain TPP metabolic genes. Our findings demonstrate that riboswitches can serve as antimicrobial drug targets and expand our understanding of thiamine metabolism in bacteria.200516356850
9991190.9183A bifunctional dihydrofolate synthetase--folylpolyglutamate synthetase in Plasmodium falciparum identified by functional complementation in yeast and bacteria. Folate metabolism in the human malaria parasite Plasmodium falciparum is an essential activity for cell growth and replication, and the target of an important class of therapeutic agents in widespread use. However, resistance to antifolate drugs is a major health problem in the developing world. To date, only two activities in this complex pathway have been targeted by antimalarials. To more fully understand the mechanisms of antifolate resistance and to identify promising targets for new chemotherapies, we have cloned genes encoding as yet uncharacterised enzymes in this pathway. By means of complementation experiments using 1-carbon metabolism mutants of both Escherichia coli and Saccharomyces cerevisiae, we demonstrate here that one of these parasite genes encodes both dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) activities, which catalyse the synthesis and polyglutamation of folate derivatives, respectively. The malaria parasite is the first known example of a eukaryote encoding both DHFS and FPGS activities in a single gene. DNA sequencing of this gene in antifolate-resistant strains of P. falciparum, as well as drug-inhibition assays performed on yeast and bacteria expressing PfDHFS--FPGS, indicate that current antifolate regimes do not target this enzyme. As PfDHFS--FPGS harbours two activities critical to folate metabolism, one of which has no human counterpart, this gene product offers a novel chemotherapeutic target with the potential to deliver a powerful blockage to parasite growth.200111223131