Cloning of a gene cluster encoding enzymes responsible for the mevalonate pathway from a terpenoid-antibiotic-producing Streptomyces strain

A gene cluster encoding enzymes responsible for the mevalonate pathway was isolated from Streptomyces griseolosporeus strain MF730-N6, a terpenoid-antibiotic terpentecin producer, by searching a flanking region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene, which had been previously isolated by complementation. By DNA sequencing of an 8.9-kb BamHI fragment, 7 genes encoding geranylgeranyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDPD), phosphomevalonate kinase (PMK), isopentenyl diphosphate (IPP) isomerase, HMG-CoA reductase, and HMG-CoA synthase were suggested to exist in that order. Heterologous expression of these genes in E. coli and Streptomyces lividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate pathway were included in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in E. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. This is the first report, to the best of our knowledge, about eubacterial MK with direct evidence.     

Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin

A gene cluster containing the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer (Y. Hamano, T. Dairi, M. Yamamoto, T. Kawasaki, K Kaneda, T. Kuzuyama, N. Itoh, and H. Seto, Biosci. Biotech. Biochem. 65:1627-1635, 2001). Sequence analysis in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. We constructed two mutants, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, respectively, were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the production of TP. The two cyclase genes were also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC.     

Treatment Default amongst Patients with Tuberculosis in Urban Morocco: Predicting and Explaining Default and Post-Default Sputum Smear and Drug Susceptibility Results

Setting

Public tuberculosis (TB) clinics in urban Morocco.

Objective

Explore risk factors for TB treatment default and develop a prediction tool. Assess consequences of default, specifically risk for transmission or development of drug resistance.

Design

Case-control study comparing patients who defaulted from TB treatment and patients who completed it using quantitative methods and open-ended questions. Results were interpreted in light of health professionals’ perspectives from a parallel study. A predictive model and simple tool to identify patients at high risk of default were developed. Sputum from cases with pulmonary TB was collected for smear and drug susceptibility testing.

Results

91 cases and 186 controls enrolled. Independent risk factors for default included current smoking, retreatment, work interference with adherence, daily directly observed therapy, side effects, quick symptom resolution, and not knowing one’s treatment duration. Age >50 years, never smoking, and having friends who knew one’s diagnosis were protective. A simple scoring tool incorporating these factors was 82.4% sensitive and 87.6% specific for predicting default in this population. Clinicians and patients described additional contributors to default and suggested locally-relevant intervention targets. Among 89 cases with pulmonary TB, 71% had sputum that was smear positive for TB. Drug resistance was rare.

Conclusion

The causes of default from TB treatment were explored through synthesis of qualitative and quantitative data from patients and health professionals. A scoring tool with high sensitivity and specificity to predict default was developed. Prospective evaluation of this tool coupled with targeted interventions based on our findings is warranted. Of note, the risk of TB transmission from patients who default treatment to others is likely to be high. The commonly-feared risk of drug resistance, though, may be low; a larger study is required to confirm these findings.     

Gene cloning and expression of Leifsonia alcohol dehydrogenase (LSADH) involved in asymmetric hydrogen-transfer bioreduction to produce (R)-form chiral alcohols

The gene encoding Leifsonia alcohol dehydrogenase (LSADH), a useful biocatalyst for producing (R)-chiral alcohols, was cloned from the genomic DNA of Leifsonia sp. S749. The gene contained an opening reading frame consisting of 756 nucleotides corresponding to 251 amino acid residues. The subunit molecular weight was calculated to be 24,999, which was consistent with that determined by polyacrylamide gel electrophoresis. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by three column chromatographies. The predicted amino acid sequence displayed 30-50% homology to known short chain alcohol dehydrogenase/reductases (SDRs); moreover, the NADH-binding site and the three catalytic residues in SDRs were conserved. The recombinant E. coli cells which overexpressed lsadh produced (R)-form chiral alcohols from ketones using 2-propanol as a hydrogen donor with the highest level of productivity ever reported and enantiomeric excess (e.e.).     

The Small Molecule GMX1778 Is a Potent Inhibitor of NAD+ Biosynthesis: Strategy for Enhanced Therapy in Nicotinic Acid Phosphoribosyltransferase 1-Deficient Tumors

GMX1777 is a prodrug of the small molecule GMX1778, currently in phase I clinical trials for the treatment of cancer. We describe findings indicating that GMX1778 is a potent and specific inhibitor of the NAD⁺ biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Cancer cells have a very high rate of NAD⁺ turnover, which makes NAD⁺ modulation an attractive target for anticancer therapy. Selective inhibition by GMX1778 of NAMPT blocks the production of NAD⁺ and results in tumor cell death. Furthermore, GMX1778 is phosphoribosylated by NAMPT, which increases its cellular retention. The cytotoxicity of GMX1778 can be bypassed with exogenous nicotinic acid (NA), which permits NAD⁺ repletion via NA phosphoribosyltransferase 1 (NAPRT1). The cytotoxicity of GMX1778 in cells with NAPRT1 deficiency, however, cannot be rescued by NA. Analyses of NAPRT1 mRNA and protein levels in cell lines and primary tumor tissue indicate that high frequencies of glioblastomas, neuroblastomas, and sarcomas are deficient in NAPRT1 and not susceptible to rescue with NA. As a result, the therapeutic index of GMX1777 can be widended in the treatment animals bearing NAPRT1-deficient tumors by coadministration with NA. This provides the rationale for a novel therapeutic approach for the use of GMX1777 in the treatment of human cancers.The cyanoguanidinopyridine GMX1778 (previously known as CHS828) is the active form of the prodrug GMX1777 and has potent antitumor activity in vitro and in vivo against cell lines derived from several different tumor origins (11). The antitumor activity of GMX1778 has been widely studied since its discovery (1, 11, 19-21, 24), but positive identification of the molecular target and the mechanism of action of GMX1778 has been elusive. Here, we demonstrate that GMX1778 exerts its antitumor activity via its potent and selective antagonism of NAD⁺ biosynthesis. GMX1777 is currently being assessed in phase I clinical trials for treatment of patients with refractory solid tumors.The pyridine nucleotide NAD⁺ plays a major role in the regulation of several essential cellular processes (7, 22, 25, 38). In addition to being a biochemical cofactor for enzymatic redox reactions involved in cellular metabolism, including ATP production, NAD⁺ is important in diverse cellular pathways responsible for calcium homeostasis (17), gene regulation (5), longevity (18), genomic integrity (33), and apoptosis (36). Cancer cells exhibit a significant dependence on NAD⁺ for support of the high levels of ATP production necessary for rapid cell proliferation. They also consume large amounts of this cofactor via reactions that utilize poly(ADP) ribosylation, including DNA repair pathways (10, 37, 39).In eukaryotes, the biosynthesis of NAD⁺ occurs via two biochemical pathways: the de novo pathway, in which NAD⁺ synthesis occurs through the metabolism of l-tryptophan via the kynurenine pathway, and the salvage pathway. The NAD⁺ salvage pathway can use either nicotinamide (niacinamide) (NM) or nicotinic acid (niacin) (NA) (via the Preiss-Handler pathway) as a substrate for NAD⁺ production. Saccharomyces cerevisiae species predominantly use NA as the substrate for NAD⁺ biosynthesis, through the deamidation of NM by the nicotinamidase PNC1 (25). However, mammalian cells do not express a nicotinamidase enzyme and use NM as the preferred substrate for the NAD⁺ salvage pathway. The mammalian NAD⁺ biosynthesis salvage pathway using NM is composed of NA phosphoribosyltransferase (NAMPT), which is the rate-limiting and penultimate enzyme that catalyzes the phosphoribosylation of NM to produce nicotinamide mononucleotide (NMN) (27, 29). NMN is subsequently converted to NAD⁺ by NMN adenyltransferases (NMNAT). The gene encoding NAMPT was originally identified as encoding a cytokine named pre-B-cell colony-enhancing factor (PBEF1) (30). NAMPT was also identified as a proposed circulating adipokine named visfatin (thought to be secreted by fat cells) and was suggested to function as an insulin mimetic; however, this role of NAMPT currently remains controversial (8). In mice, NAMPT has been shown to act as a systemic NAD⁺ biosynthetic enzyme that regulates insulin secretion from β cells (28). The molecular structure of NAMPT from human (15), rat (16) and mouse (35) tissue, containing either NMN or the inhibitor APO866, have been determined by X-ray crystallography. These structures revealed that NAMPT is a dimeric type II phosphoribosyltransferase.Here, we report that the anticancer compound GMX1778 is a specific inhibitor of NAMPT in vivo and in vitro and is itself a substrate for the enzyme. Phosphoribosylated GMX1778 inhibits NAMPT as potently as GMX1778 but is preferentially retained within cells. Finally, we have identified a novel anticancer strategy utilizing NA rescue of GMX1778 cytotoxicity to increase the therapeutic index of GMX1777 activity in tumors that are deficient in NA phosphoribosyltransferase 1 (NAPRT1).     

Analysis of the Lactobacillus Metabolic Pathway

We performed analyses of the phenotypic and genotypic relationships focusing on biosyntheses of amino acids, purine/pyrimidines, and cofactors in three Lactobacillus strains. We found that Lactobacillus fermentum IFO 3956 perhaps synthesized para-aminobenzoate (PABA), an intermediate of folic acid biosynthesis, by an alternative pathway.The biosynthetic pathways of primary metabolites have been established with model microorganisms such as Escherichia coli and Saccharomyces cerevisiae. For a long time, the biosynthetic routes established were believed to be common to all microorganisms. However, we now realize that some microorganisms possess alternative biosynthetic pathways since the genome database has enabled us to determine the presence or absence of orthologs of the genes responsible for known biosynthetic pathways. These surveys were one of the triggers to find the 2-C-methyl-d-erythritol 4-phosphate pathway (6) for isopentenyl diphosphate biosynthesis and the futalosine pathway (4) for menaquinone biosynthesis. As exemplified by the discovery of these pathways, microorganisms are expected to have additional alternative pathways for the biosynthesis of primary metabolites.Lactobacilli are Gram-positive lactic acid-producing bacteria with low G+C contents and are utilized in the food industry (7, 15). These bacteria are known to have mutations in many primary metabolic pathways and require rich media containing various amino acids and nucleobases for their growth. After the whole-genome sequence of Lactobacillus plantarum WCFS1 was determined in 2003 (5), phenotypic and genotypic analysis of the primary metabolic pathway in Lactobacillus strains commenced (1, 2, 8, 11, 12, 14). All of these analyses, however, were performed with a database of the known biosynthetic pathways. We are interested in an alternative pathway for biosynthesis of primary metabolites in microorganisms. Considering that some Lactobacillus strains do not possess some of the orthologs of the known biosynthetic pathways and that the genome sizes of Lactobacillus strains are relatively large (1.8 to 3.4 Mb) compared to those of the symbiotic bacteria, such as Mycoplasma strains (0.6 to 1.4 Mb) (http://www.genome.jp/kegg/catalog/org_list.html), we suspected the presence of an alternative primary metabolic pathway in Lactobacillus strains. In this paper, we examined the phenotypic and genotypic relationships in Lactobacillus fermentum IFO 3956 (genome size, 2.1 Mb) (10), Lactobacillus reuteri JCM 1112 (2.0 Mb) (10), and Lactobacillus brevis ATCC 367 (2.3 Mb) (9), all of which showed relatively good growth in LSP medium (3a) (20 g/liter glucose, 3.1 g/liter KH₂PO₄, 1.5 g/liter K₂HPO₄, 2 g/liter diammonium hydrogen citrate, 10 g/liter potassium acetate, 1 g/liter calcium lactate, 0.02 g/liter NaCl, 1 g/liter Tween 80, 0.5 g/liter MgSO₄·7H₂O, 0.05 g/liter MnSO₄·5H₂O, 0.5 g/liter CoSO₄).As for the amino acid, purine/pyrimidine, and vitamin (thiamine, nicotinate, pantothenate, riboflavin, and vitamin B₆) biosynthetic pathways, the phenotypes of the three strains were essentially in agreement with the genotype (see Tables S1, S2, and S3 in the supplemental material) by the single-omission growth test, although we found several discrepancies, such as a prototrophic phenotype despite the absence of ortholog genes and an auxotrophic phenotype despite the presence of ortholog genes. However, these discrepancies were limited to one of the steps of the established biosynthetic pathway. In contrast, we observed a discrepancy between the phenotype and genotype for the biosynthesis of folic acid. Neither L. fermentum IFO 3956 nor L. reuteri JCM 1112 required folic acid for their growth, in contrast to L. brevis ATCC 367, which was auxotrophic for folic acid. The former two strains did not possess orthologs of pabA, -B, and -C (13), which were involved in the conversion of chorismate into para-aminobenzoate (PABA), an intermediate of folic acid biosynthesis. Therefore, we investigated the biosynthesis of PABA in L. fermentum IFO 3956 in more detail. Although pabA, -B, and -C were absent in strain IFO 3956, we found an ortholog of FolP (LAF_1336; EC 2.5.1.15), which catalyzes the formation of 7,8-dihydropteroate from PABA and 6-hydroxymethyl-dihydropterin diphosphate. Therefore, we examined if LAF_1336 showed the expected enzyme activity. We constructed a ΔfolP E. coli mutant by homologous recombination with the Lambda Red system (3) (see Table S4 and Fig. S1 in the supplemental material). The constructed ΔfolP E. coli mutant required folic acid for its growth (see Fig. S2 in the supplemental material) and was used in complementation experiments. The ΔfolP E. coli mutant transformed with a plasmid carrying a folP gene cloned from E. coli was able to grow reasonably in the absence of folic acid. Moreover, the ΔfolP E. coli mutant harboring a plasmid carrying LAF_1336 was also able to grow without folic acid (see Fig. S2 in the supplemental material), demonstrating that LAF_1336 complemented the folP defect.We examined LAF_1336 using PABA as the substrate by two strategies. First, we constructed a ΔfolP ΔpabABC E. coli mutant for in vivo analysis. The ΔfolP E. coli mutant was used as the starting strain, and pabA, pabB, and pabC were successively disrupted by homologous recombination. The growth of the constructed mutant, in which PABA was not supplied from chorismate, was completely dependent on the presence of folic acid. When pUC118-FolP, carrying the E. coli folP gene, was introduced into the ΔfolP ΔpabABC E. coli mutant, the transformant was able to grow in medium containing PABA as expected (Table ​(Table1).1). The growth of the ΔfolP ΔpabABC E. coli mutant transformed with pUC118-1336 carrying LAF_1336 was also completely dependent on the presence of PABA. These results clearly suggested that LAF_1336 used PABA as the substrate for the formation of folic acid via 7,8-dihydropteroate.

TABLE 1.

Growth of the ΔfolP ΔpabABC mutant and its transformant harboring the E. coli folP gene or L. fermentum LAF_1336 gene^a

Using the MaxEnt model for assessing the impact of climate change on the Aurasian Aleppo pine distribution in Algeria

In the context of recent climate changes and their impact on the vegetation cover in the southern shore of the Mediterranean, this paper deals with the potential distribution in the present and the future of the Aleppo pine in the north‐east of Algeria. Accordingly, this study focuses on modelling of the spatiotemporal distribution of the Aurasian Aleppo pine, by means of MaxEnt program. The models developed with MaxEnt showed good predictivity with AUC > 0.80 for the RCP 4.5 and RCP 8.5 scenarios projected, respectively, for 2055 and 2085. The results showed that the annual thermal amplitude followed by altitude appear to be the main factors of the spatiotemporal distribution of Aleppo pine in the study area. Under current conditions, only 20% of the territory seems favourable to the presence of the Aleppo pine. However, future predictions expect an extension of the areas classified as “moderately favourable” to the Aleppo pine. In response to climate changes, the Aurasian Aleppo pine may display two contrasted tendencies: a progressive evolution in the north, and a regressive evolution in the south of the Aurès region as well as a displacement of suitable areas for Aleppo pine to the north.