DNA Sequence and Mutational Analysis of Rhizobitoxine Biosynthesis Genes in Bradyrhizobium elkanii

We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii. The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of rtxA. A large-deletion mutant of B. elkanii, USDA94Δrtx::Ω1, which lacks rtxA, ORF1 (rtxC), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxA and these ORFs, complemented rhizobitoxine production in USDA94Δrtx::Ω1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the rtxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that rtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.     

Abnormal n-6 fatty acid metabolism in cystic fibrosis is caused by activation of AMP-activated protein kinase

Cystic fibrosis (CF) patients and model systems exhibit consistent abnormalities in PUFA metabolism, including increased metabolism of linoleate to arachidonate. Recent studies have connected these abnormalities to increased expression and activity of the Δ6- and Δ5-desaturase enzymes. However, the mechanism connecting these changes to the CF transmembrane conductance regulator (CFTR) mutations responsible for CF is unknown. This study tests the hypothesis that increased activity of AMP-activated protein kinase (AMPK), previously described in CF bronchial epithelial cells, causes these changes in fatty acid metabolism by driving desaturase expression. Using CF bronchial epithelial cell culture models, we confirm elevated activity of AMPK in CF cells and show that it is due to increased phosphorylation of AMPK by Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ). We also show that inhibition of AMPK or CaMKKβ reduces desaturase expression and reverses the metabolic alterations seen in CF cells. These results signify a novel AMPK-dependent mechanism linking the genetic defect in CF to alterations in PUFA metabolism.     

FADS genetic variants and ω-6 polyunsaturated fatty acid metabolism in a homogeneous island population

Long-chain polyunsaturated fatty acids (PUFA) orchestrate immunity and inflammation through their capacity to be converted to potent inflammatory mediators. We assessed associations of FADS gene cluster polymorphisms and fasting serum PUFA concentrations in a fully ascertained, geographically isolated founder population of European descent. Concentrations of 22 PUFAs were determined by gas chromatography, of which ten fatty acids and five ratios defining FADS1 and FADS2 activity were tested for genetic association against 16 single nucleotide polymorphisms (SNP) in 224 individuals. A cluster of SNPs in tight linkage disequilibrium in the FADS1 gene (rs174537, rs174545, rs174546, rs174553, rs174556, rs174561, rs174568, and rs99780) were strongly associated with arachidonic acid (AA) (P = 5.8 × 10⁻⁷ – 1.7 × 10⁻⁸) among other PUFAs, but the strongest associations were with the ratio measuring FADS1 activity in the ω-6 series (P = 2.11 × 10⁻¹³ – 1.8 × 10⁻²⁰). The minor allele across all SNPs was consistently associated with decreased ω-6 PUFAs, with the exception of dihomo-γ-linoleic acid (DHGLA), where the minor allele was consistently associated with increased levels. Our findings in a geographically isolated population with a homogenous dietary environment suggest that variants in the Δ-5 desaturase enzymatic step likely regulate the efficiency of conversion of medium-chain PUFAs to potentially inflammatory PUFAs, such as AA.     

A Determinant of Substrate Specificity Predicted from the Acyl-Acyl Carrier Protein Desaturase of Developing Cat''s Claw Seed

Cat's claw (Doxantha unguis-cati L.) vine accumulates nearly 80% palmitoleic acid (16:1Δ9) plus cis-vaccenic acid (18:1Δ11) in its seed oil. To characterize the biosynthetic origin of these unusual fatty acids, cDNAs for acyl-acyl carrier protein (acyl-ACP) desaturases were isolated from developing cat's claw seeds. The predominant acyl-ACP desaturase cDNA identified encoded a polypeptide that is closely related to the stearoyl (Δ9–18:0)-ACP desaturase from castor (Ricinis communis L.) and other species. Upon expression in Escherichia coli, the cat's claw polypeptide functioned as a Δ9 acyl-ACP desaturase but displayed a distinct substrate specificity for palmitate (16:0)-ACP rather than stearate (18:0)-ACP. Comparison of the predicted amino acid sequence of the cat's claw enzyme with that of the castor Δ9–18:0-ACP desaturase suggested that a single amino acid substitution (L118W) might account in large part for the differences in substrate specificity between the two desaturases. Consistent with this prediction, conversion of leucine-118 to tryptophan in the mature castor Δ9–18:0-ACP desaturase resulted in an 80-fold increase in the relative specificity of this enzyme for 16:0-ACP. The alteration in substrate specificity observed in the L118W mutant is in agreement with a crystallographic model of the proposed substrate-binding pocket of the castor Δ9–18:0-ACP desaturase.     

The Role of Glucosidase I (Cwh41p) in the Biosynthesis of Cell Wall β-1,6-Glucan Is Indirect

CWH41, a gene involved in the assembly of cell wall β-1,6-glucan, has recently been shown to be the structural gene for Saccharomyces cerevisiae glucosidase I that is responsible for initiating the trimming of terminal α-1,2-glucose residue in the N-glycan processing pathway. To distinguish between a direct or indirect role of Cwh41p in the biosynthesis of β-1,6-glucan, we constructed a double mutant, alg5Δ (lacking dolichol-P-glucose synthase) cwh41Δ, and found that it has the same phenotype as the alg5Δ single mutant. It contains wild-type levels of cell wall β-1,6-glucan, shows moderate underglycosylation of N-linked glycoproteins, and grows at concentrations of Calcofluor White (which interferes with cell wall assembly) that are lethal to cwh41Δ single mutant. The strong genetic interactions of CWH41 with KRE6 and KRE1, two other genes involved in the β-1,6-glucan biosynthetic pathway, disappear in the absence of dolichol-P-glucose synthase (alg5Δ). The triple mutant alg5Δcwh41Δkre6Δ is viable, whereas the double mutant cwh41Δkre6Δ in the same genetic background is not. The severe slow growth phenotype and 75% reduction in cell wall β-1,6-glucan, characteristic of the cwh41Δkre1Δ double mutant, are not observed in the triple mutant alg5Δcwh41Δkre1Δ. Kre6p, a putative Golgi glucan synthase, is unstable in cwh41Δ strains, and its overexpression renders these cells Calcofluor White resistant. These results demonstrate that the role of glucosidase I (Cwh41p) in the biosynthesis of cell wall β-1,6-glucan is indirect and that dolichol-P-glucose is not an intermediate in this pathway.     

A High-Throughput Fatty Acid Profiling Screen Reveals Novel Variations in Fatty Acid Biosynthesis in Chlamydomonas reinhardtii and Related Algae

Analysis of fatty acid methyl esters (FAMEs) by gas chromatography (GC) is a common technique for the quantitative and qualitative analysis of acyl lipids. Methods for FAME preparation are typically time-consuming and labor-intensive and require multiple transfers of reagents and products between reaction tubes and autosampler vials. In order to increase throughput and lower the time and materials costs required for FAME preparation prior to GC analysis, we have developed a method in which 10-to-20-mg samples of microbial biomass are transferred to standard GC autosampler vials, transesterified using an emulsion of methanolic trimethylsulfonium hydroxide and hexane, and analyzed directly by GC without further sample handling. This method gives results that are essentially identical to those obtained by the more labor- and material-intensive FAME preparation methods, such as transmethylation with methanolic HCl. We applied this method to the screening of laboratory and environmental isolates of the green alga Chlamydomonas for variations in fatty acid composition. This screening method facilitated two novel discoveries. First, we identified a common laboratory strain of C. reinhardtii, CC-620, completely lacking all ω-3 fatty acids normally found in this organism and showed that this strain contains an inactivating mutation in the CrFAD7 gene, encoding the sole ω-3 desaturase activity in this organism. Second, we showed that some species of Chlamydomonas make Δ6-unsaturated polyunsaturated fatty acids (PUFA) rather than the Δ5 species normally made by the previously characterized laboratory strains of Chlamydomonas, suggesting that there is species-specific variation in the regiospecificity and substrate selectivity of front-end desaturases in this algal genus.     

Δ-6 Desaturase Substrate Competition: Dietary Linoleic Acid (18∶2n-6) Has Only Trivial Effects on α-Linolenic Acid (18∶3n-3) Bioconversion in the Teleost Rainbow Trout

It is generally accepted that, in vertebrates, omega-3 (n-3) and omega-6 (n-6) poly-unsaturated fatty acids (PUFA) compete for Δ-6 desaturase enzyme in order to be bioconverted into long-chain PUFA (LC-PUFA). However, recent studies into teleost fatty acid metabolism suggest that these metabolic processes may not conform entirely to what has been previously observed in mammals and other animal models. Recent work on rainbow trout has led us to question specifically if linoleic acid (LA, 18∶2n-6) and α-linolenic acid (ALA, 18∶3n-3) (Δ-6 desaturase substrates) are in direct competition for access to Δ-6 desaturase. Two experimental diets were formulated with fixed levels of ALA, while LA levels were varied (high and low) to examine if increased availability of LA would result in decreased bioconversion of ALA to its LC-PUFA products through substrate competition. No significant difference in ALA metabolism towards n-3 LC-PUFA was exhibited between diets while significant differences were observed in LA metabolism towards n-6 LC-PUFA. These results are evidence for minor if any competition between substrates for Δ-6 desaturase, suggesting that, paradoxically, the activity of Δ-6 desaturase on n-3 and n-6 substrates is independent. These results call for a paradigm shift in the way we approach teleost fatty acid metabolism. The findings are also important with regard to diet formulation in the aquaculture industry as they indicate that there should be no concern for possible substrate competition between 18∶3n-3 and 18∶2n-6, when aiming at increased n-3 LC-PUFA bioconversion in vivo.     

ssb Gene Duplication Restores the Viability of ΔholC and ΔholD Escherichia coli Mutants

The HolC-HolD (χψ) complex is part of the DNA polymerase III holoenzyme (Pol III HE) clamp-loader. Several lines of evidence indicate that both leading- and lagging-strand synthesis are affected in the absence of this complex. The Escherichia coli ΔholD mutant grows poorly and suppressor mutations that restore growth appear spontaneously. Here we show that duplication of the ssb gene, encoding the single-stranded DNA binding protein (SSB), restores ΔholD mutant growth at all temperatures on both minimal and rich medium. RecFOR-dependent SOS induction, previously shown to occur in the ΔholD mutant, is unaffected by ssb gene duplication, suggesting that lagging-strand synthesis remains perturbed. The C-terminal SSB disordered tail, which interacts with several E. coli repair, recombination and replication proteins, must be intact in both copies of the gene in order to restore normal growth. This suggests that SSB-mediated ΔholD suppression involves interaction with one or more partner proteins. ssb gene duplication also suppresses ΔholC single mutant and ΔholC ΔholD double mutant growth defects, indicating that it bypasses the need for the entire χψ complex. We propose that doubling the amount of SSB stabilizes HolCD-less Pol III HE DNA binding through interactions between SSB and a replisome component, possibly DnaE. Given that SSB binds DNA in vitro via different binding modes depending on experimental conditions, including SSB protein concentration and SSB interactions with partner proteins, our results support the idea that controlling the balance between SSB binding modes is critical for DNA Pol III HE stability in vivo, with important implications for DNA replication and genome stability.     

Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as “transceptors” with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.     

A complete enzymatic capacity for biosynthesis of docosahexaenoic acid (DHA, 22 : 6n–3) exists in the marine Harpacticoida copepod Tigriopus californicus

The long-standing paradigm establishing that global production of Omega-3 (n–3) long-chain polyunsaturated fatty acids (LC-PUFA) derived almost exclusively from marine single-cell organisms, was recently challenged by the discovery that multiple invertebrates possess methyl-end (or ωx) desaturases, critical enzymes enabling the biosynthesis of n–3 LC-PUFA. However, the question of whether animals with ωx desaturases have complete n–3 LC-PUFA biosynthetic pathways and hence can contribute to the production of these compounds in marine ecosystems remained unanswered. In the present study, we investigated the complete enzymatic complement involved in the n–3 LC-PUFA biosynthesis in Tigriopus californicus, an intertidal harpacticoid copepod. A total of two ωx desaturases, five front-end desaturases and six fatty acyl elongases were successfully isolated and functionally characterized. The T. californicus ωx desaturases enable the de novo biosynthesis of C₁₈ PUFA such as linoleic and α-linolenic acids, as well as several n–3 LC-PUFA from n–6 substrates. Functions demonstrated in front-end desaturases and fatty acyl elongases unveiled various routes through which T. californicus can biosynthesize the physiologically important arachidonic and eicosapentaenoic acids. Moreover, T. californicus possess a Δ4 desaturase, enabling the biosynthesis of docosahexaenoic acid via the ‘Δ4 pathway’. In conclusion, harpacticoid copepods such as T. californicus have complete n–3 LC-PUFA biosynthetic pathways and such capacity illustrates major roles of these invertebrates in the provision of essential fatty acids to upper trophic levels.     

DNA Methylation in an Enhancer Region of the FADS Cluster Is Associated with FADS Activity in Human Liver

Levels of omega-6 (n-6) and omega-3 (n-3), long chain polyunsaturated fatty acids (LcPUFAs) such as arachidonic acid (AA; 20∶4, n-6), eicosapentaenoic acid (EPA; 20∶5, n-3) and docosahexaenoic acid (DHA; 22∶6, n-3) impact a wide range of biological activities, including immune signaling, inflammation, and brain development and function. Two desaturase steps (Δ6, encoded by FADS2 and Δ5, encoded by FADS1) are rate limiting in the conversion of dietary essential 18 carbon PUFAs (18C-PUFAs) such as LA (18∶2, n-6) to AA and α-linolenic acid (ALA, 18∶3, n-3) to EPA and DHA. GWAS and candidate gene studies have consistently identified genetic variants within FADS1 and FADS2 as determinants of desaturase efficiencies and levels of LcPUFAs in circulating, cellular and breast milk lipids. Importantly, these same variants are documented determinants of important cardiovascular disease risk factors (total, LDL, and HDL cholesterol, triglycerides, CRP and proinflammatory eicosanoids). FADS1 and FADS2 lie head-to-head (5′ to 5′) in a cluster configuration on chromosome 11 (11q12.2). There is considerable linkage disequilibrium (LD) in this region, where multiple SNPs display association with LcPUFA levels. For instance, rs174537, located ∼15 kb downstream of FADS1, is associated with both FADS1 desaturase activity and with circulating AA levels (p-value for AA levels = 5.95×10⁻⁴⁶) in humans. To determine if DNA methylation variation impacts FADS activities, we performed genome-wide allele-specific methylation (ASM) with rs174537 in 144 human liver samples. This approach identified highly significant ASM with CpG sites between FADS1 and FADS2 in a putative enhancer signature region, leading to the hypothesis that the phenotypic associations of rs174537 are likely due to methylation differences. In support of this hypothesis, methylation levels of the most significant probe were strongly associated with FADS1 and, to a lesser degree, FADS2 activities.     

The β Subunit of the Heterotrimeric G Protein Triggers the Kluyveromyces lactis Pheromone Response Pathway in the Absence of the γ Subunit

The Kluyveromyces lactis heterotrimeric G protein is a canonical Gαβγ complex; however, in contrast to Saccharomyces cerevisiae, where the Gγ subunit is essential for mating, disruption of the KlGγ gene yielded cells with almost intact mating capacity. Expression of a nonfarnesylated Gγ, which behaves as a dominant-negative in S. cerevisiae, did not affect mating in wild-type and ΔGγ cells of K. lactis. In contrast to the moderate sterility shown by the single ΔKlGα, the double ΔKlGα ΔKlGγ mutant displayed full sterility. A partial sterile phenotype of the ΔKlGγ mutant was obtained in conditions where the KlGβ subunit interacted defectively with the Gα subunit. The addition of a CCAAX motif to the C-end of KlGβ, partially suppressed the lack of both KlGα and KlGγ subunits. In cells lacking KlGγ, the KlGβ subunit cofractionated with KlGα in the plasma membrane, but in the ΔKlGα ΔKlGγ strain was located in the cytosol. When the KlGβ-KlGα interaction was affected in the ΔKlGγ mutant, most KlGβ fractionated to the cytosol. In contrast to the generic model of G-protein function, the Gβ subunit of K. lactis has the capacity to attach to the membrane and to activate mating effectors in absence of the Gγ subunit.     

Accelerated Recovery of Mitochondrial Membrane Potential by GSK-3β Inactivation Affords Cardiomyocytes Protection from Oxidant-Induced Necrosis

Loss of mitochondrial membrane potential (ΔΨ_m) is known to be closely linked to cell death by various insults. However, whether acceleration of the ΔΨ_m recovery process prevents cell necrosis remains unclear. Here we examined the hypothesis that facilitated recovery of ΔΨ_m contributes to cytoprotection afforded by activation of the mitochondrial ATP-sensitive K⁺ (mK_ATP) channel or inactivation of glycogen synthase kinase-3β (GSK-3β). ΔΨ_m of H9c2 cells was determined by tetramethylrhodamine ethyl ester (TMRE) before or after 1-h exposure to antimycin A (AA), an inducer of reactive oxygen species (ROS) production at complex III. Opening of the mitochondrial permeability transition pore (mPTP) was determined by mitochondrial loading of calcein. AA reduced ΔΨ_m to 15±1% of the baseline and induced calcein leak from mitochondria. ΔΨ_m was recovered to 51±3% of the baseline and calcein-loadable mitochondria was 6±1% of the control at 1 h after washout of AA. mK_ATP channel openers improved the ΔΨ_m recovery and mitochondrial calcein to 73±2% and 30±7%, respectively, without change in ΔΨ_m during AA treatment. Activation of the mK_ATP channel induced inhibitory phosphorylation of GSK-3β and suppressed ROS production, LDH release and apoptosis after AA washout. Knockdown of GSK-3β and pharmacological inhibition of GSK-3β mimicked the effects of mK_ATP channel activation. ROS scavengers administered at the time of AA removal also improved recovery of ΔΨ_m. These results indicate that inactivation of GSK-3β directly or indirectly by mK_ATP channel activation facilitates recovery of ΔΨ_m by suppressing ROS production and mPTP opening, leading to cytoprotection from oxidant stress-induced cell death.     

Intragenic Suppressor of a Plug Deletion Nonmotility Mutation in PotB,a Chimeric Stator Protein of Sodium-Driven Flagella

The torque of bacterial flagellar motors is generated by interactions between the rotor and the stator and is coupled to the influx of H⁺ or Na⁺ through the stator. A chimeric protein, PotB, in which the N-terminal region of Vibrio alginolyticus PomB was fused to the C-terminal region of Escherichia coli MotB, can function with PomA as a Na⁺-driven stator in E. coli. Here, we constructed a deletion variant of PotB (with a deletion of residues 41 to 91 [Δ41–91], called PotBΔL), which lacks the periplasmic linker region including the segment that works as a “plug” to inhibit premature ion influx. This variant did not confer motile ability, but we isolated a Na⁺-driven, spontaneous suppressor mutant, which has a point mutation (R109P) in the MotB/PomB-specific α-helix that connects the transmembrane and peptidoglycan binding domains of PotBΔL in the region of MotB. Overproduction of the PomA/PotBΔL(R109P) stator inhibited the growth of E. coli cells, suggesting that this stator has high Na⁺-conducting activity. Mutational analyses of Arg109 and nearby residues suggest that the structural alteration in this α-helix optimizes PotBΔL conformation and restores the proper arrangement of transmembrane helices to form a functional channel pore. We speculate that this α-helix plays a key role in assembly-coupled stator activation.