VibD and VibH are required for late steps in vibriobactin biosynthesis in Vibrio cholerae

Vibrio cholerae synthesizes the catechol siderophore vibriobactin. In this report, we present the complete map of a vibriobactin gene region containing two previously unreported vibriobactin biosynthetic genes. vibD encodes a phosphopantetheinyl transferase, and vibH encodes a novel nonribosomal peptide synthase. Both VibD and VibH are required for vibriobactin biosynthesis.     

The ABA1 gene and carotenoid biosynthesis are required for late skotomorphogenic growth in Arabidopsis thaliana

Several plant hormones, including auxin, brassinosteroids and gibberellins, are required for skotomorphogenesis, which is the etiolated growth that seedlings undergo in the absence of light. To examine the growth of abscisic acid (ABA)-deficient mutants in the dark, we analysed several aba1 loss-of-function alleles, which are deficient in zeaxanthin epoxidase. The aba1 mutants displayed a partially de-etiolated phenotype, including reduced hypocotyl growth, cotyledon expansion and the development of true leaves, during late skotomorphogenic growth. In contrast, only small differences in hypocotyl growth were found between wild-type seedlings and ABA-deficient mutants impaired in subsequent steps of the pathway, namely nced3, aba2, aba3 and aao3. Interestingly, phenocopies of the partially de-etiolated phenotype of the aba1 mutants were obtained when wild-type seedlings were dark-grown on medium supplemented with fluridone, an inhibitor of phytoene desaturase, and hence, of carotenoid biosynthesis. ABA supplementation did not restore the normal skotomorphogenic growth of aba1 mutants or fluridone-treated wild-type plants, suggesting a direct inhibitory effect of fluridone on carotenoid biosynthesis. In addition, aba1 mutants showed impaired production of the beta-carotene-derived xanthophylls, neoxanthin, violaxanthin and antheraxanthin. Because fluridone treatment of wild-type plants phenocopied the phenotype of dark-grown aba1 mutants, impaired carotenoid biosynthesis in aba1 mutants is probably responsible for the observed skotomorphogenic phenotype. Thus, ABA1 is required for skotomorphogenic growth, and beta-carotene-derived xanthophylls are putative regulators of skotomorphogenesis.     

SepF, a novel FtsZ-interacting protein required for a late step in cell division

Cell division in nearly all bacteria is initiated by polymerization of the conserved tubulin-like protein FtsZ into a ring-like structure at midcell. This Z-ring functions as a scaffold for a group of conserved proteins that execute the synthesis of the division septum (the divisome). Here we describe the identification of a new cell division protein in Bacillus subtilis. This protein is conserved in Gram positive bacteria, and because it has a role in septum development, we termed it SepF. sepF mutants are viable but have a cell division defect, in which septa are formed slowly and with a severely abnormal morphology. Yeast two-hybrid analysis showed that SepF can interact with itself and with FtsZ. Accordingly, fluorescence microscopy showed that SepF accumulates at the site of cell division, and this localization depends on the presence of FtsZ. Combination of mutations in sepF and ezrA, encoding another Z-ring interacting protein, had a synthetic lethal division effect. We conclude that SepF is a new member of the Gram positive divisome, required for proper execution of septum synthesis.     

A PIP-family protein is required for biosynthesis of tobacco alkaloids

Plants in the Nicotiana genus produce nicotine and related pyridine alkaloids as a part of their chemical defense against insect herbivores. These alkaloids are formed by condensation of a derivative of nicotinic acid, but the enzyme(s) involved in the final condensation step remains elusive. In Nicotiana tabacum, an orphan reductase A622 and its close homolog A622L are coordinately expressed in the root, upregulated by methyl jasmonate treatment, and controlled by the NIC regulatory loci specific to the biosynthesis of tobacco alkaloids. Conditional suppression of A622 and A622L by RNA interference inhibited cell growth, severely decreased the formation of all tobacco alkaloids, and concomitantly induced an accumulation of nicotinic acid β-N-glucoside, a probable detoxification metabolite of nicotinic acid, in both hairy roots and methyl jasmonate-elicited cultured cells of tobacco. N-methylpyrrolinium cation, a precursor of the pyrrolidine moiety of nicotine, also accumulated in the A622(L)-knockdown hairy roots. We propose that the tobacco A622-like reductases of the PIP family are involved in either the formation of a nicotinic acid-derived precursor or the final condensation reaction of tobacco alkaloids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

SNARE proteins are required for macroautophagy

Macroautophagy mediates the degradation of long-lived proteins and organelles via the de novo formation of double-membrane autophagosomes that sequester cytoplasm and deliver it to the vacuole/lysosome; however, relatively little is known about autophagosome biogenesis. Atg8, a phosphatidylethanolamine-conjugated protein, was previously proposed to function in autophagosome membrane expansion, based on the observation that it mediates liposome tethering and hemifusion in vitro. We show here that with physiological concentrations of phosphatidylethanolamine, Atg8 does not act as a fusogen. Rather, we provide evidence for the involvement of exocytic Q/t-SNAREs in autophagosome formation, acting in the recruitment of key autophagy components to the site of autophagosome formation, and in regulating the organization of Atg9 into tubulovesicular clusters. Additionally, we found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 transport. Thus, multiple SNARE-mediated fusion events are likely to be involved in autophagosome biogenesis.     

Mu-opioid receptor and CREB activation are required for nicotine reward

Environmental cues associated with nicotine delivery are an important part of the stimulus that sustains smoking behavior and is often coupled with craving and relapse; however, the neuronal circuitry and molecular substrates underlying this process are still poorly understood. Exposure to an environment previously associated with rewarding properties of nicotine results in an increase of CREB phosphorylation similar to that seen following nicotine administration, and this response is absent in MOR(-/-) mice. Moreover, a single administration of an opioid receptor antagonist, naloxone, blocks both the conditioned molecular response (CREB phosphorylation) and the conditioned behavioral response (nicotine reward) in a place preference paradigm. Lastly, repeated nicotine administration results in increased expression of MORs. However, this effect, along with rewarding properties of nicotine, is blocked in mice with a targeted disruption in the CREB gene. Together, pharmacologic and genetic manipulations indicate that phosphorylation of CREB and upregulation of functional MORs are required for nicotine-conditioned reward.     

Identification of a Saccharopolyspora erythraea gene required for the final hydroxylation step in erythromycin biosynthesis.

In analyzing the region of the Saccharopolyspora erythraea chromosome responsible for the biosynthesis of the macrolide antibiotic erythromycin, we identified a gene, designated eryK, located about 50 kb downstream of the erythromycin resistance gene, ermE. eryK encodes a 44-kDa protein which, on the basis of comparative analysis, belongs to the P450 monooxygenase family. An S. erythraea strain disrupted in eryK no longer produced erythromycin A but accumulated the B and D forms of the antibiotic, indicating that eryK is responsible for the C-12 hydroxylation of the macrolactone ring, one of the last steps in erythromycin biosynthesis.     

Gamma-aminobutyric acid mediates the nicotine biosynthesis in tobacco under flooding stress

Gamma-aminobutyric acid (GABA) is a four-carbon non-protein amino acid conserved from bacteria to plants and vertebrates. Increasing evidence supports a regulatory role for GABA in plant development and the plant′s response to environmental stress. The biosynthesis of nicotine, the main economically important metabolite in tobacco, is tightly regulated. GABA has not hitherto been reported to function in nicotine biosynthesis. Here we found that water flooding treatment (hypoxia) markedly induced the accumulation of GABA and stimulated nicotine biosynthesis. Suppressing GABA accumulation by treatment with glutamate decarboxylase inhibitor impaired flooding-induced nicotine biosynthesis, while exogenous GABA application directly induced nicotine biosynthesis. Based on these results, we propose that GABA triggers nicotine biosynthesis in tobacco seedlings subjected to flooding. Our results provide insight into the molecular mechanism of nicotine biosynthesis in tobacco plants exposed to environmental stress.     

Quinones as hydrogen carriers for a late step in anaerobic heme biosynthesis in Escherichia coli.

A late step in anaerobic heme synthesis, the oxidation of protoprophyrinogen with fumarate as electron acceptor, was studied in extracts and particles of Escherichia coli mutants deficient in quinones or cytochromes. Mutants specifically deficient in menaquinone did not couple protoporphyrinogen oxidation to fumarate reduction, whereas mutants containing menaquinone but deficient in either ubiquinone or cytochromes exhibited this activity. These findings indicate that this coupled reaction is dependent upon menaquinone as hydrogen carrier but independent of ubiquinone and cytochromes. Other characteristics of this coupled reaction were also studied. The activity was located exclusively in the membrane fraction of cell-free extracts. Coproporphyrinogen III could not replace protoporphyrinogen as substrate. Methylene blue, triphenyl tetrazolium and nitrate, but not nitrite, could replace fumarate as anaerobic hydrogen acceptor. These findings have implications for the mechanism and regulation of microbial heme and chlorophyll synthesis and for the physiology of cytochrome synthesis in anaerobic microorganisms.     

Identification of Escherichia coli ubiB, a gene required for the first monooxygenase step in ubiquinone biosynthesis

It was recently discovered that the aarF gene in Providencia stuartii is required for coenzyme Q (CoQ) biosynthesis. Here we report that yigR, the Escherichia coli homologue of aarF, is ubiB, a gene required for the first monooxygenase step in CoQ biosynthesis. Both the P. stuartii aarF and E. coli ubiB (yigR) disruption mutant strains lack CoQ and accumulate octaprenylphenol. Octaprenylphenol is the CoQ biosynthetic intermediate found to accumulate in the E. coli strain AN59, which contains the ubiB409 mutant allele. Analysis of the mutation in the E. coli strain AN59 reveals no mutations within the ubiB gene, but instead shows the presence of an IS1 element at position +516 of the ubiE gene. The ubiE gene encodes a C-methyltransferase required for the synthesis of both CoQ and menaquinone, and it is the 5' gene in an operon containing ubiE, yigP, and ubiB. The data indicate that octaprenylphenol accumulates in AN59 as a result of a polar effect of the ubiE::IS1 mutation on the downstream ubiB gene. AN59 is complemented by a DNA segment containing the contiguous ubiE, yigP, and ubiB genes. Although transformation of AN59 with a DNA segment containing the ubiB coding region fails to restore CoQ biosynthesis, transformation with the ubiE coding region results in a low-frequency but significant rescue attributed to homologous recombination. In addition, the fre gene, previously considered to correspond to ubiB, was found not to be involved in CoQ biosynthesis. The ubiB gene is a member of a predicted protein kinase family of which the Saccharomyces cerevisiae ABC1 gene is the prototypic member. The possible protein kinase function of UbiB and Abc1 and the role these polypeptides may play in CoQ biosynthesis are discussed.     

Misaminoacylation and transamidation are required for protein biosynthesis in Lactobacillus bulgaricus

Aminoacylation studies with Lactobacillus bulgaricus show that this organism possesses glutamyl-tRNA synthetase activity; however, glutamyl-tRNA synthetase activity cannot be demonstrated. Instead, Glu-tRNAGln, which is formed by glutamyl-tRNA synthetase, is amidated by a specific amidotransferase to Gln-tRNAGln. The amide donor in this reaction is glutamine. Thus, Gln-tRNAGln in this organism is not formed by direct glutaminylation of tRNAGln, but instead by a pathway which involves misaminoacylation and transamidation.     

Quinones as hydrogen carriers for a late step in anaerobic heme biosynthesis in Escherichia coli

A late step in anaerobic heme synthesis, the oxidation of protoporphyrinogen with fumarate as electron acceptor, was studied in extracts and particles of Escherichia coli mutants deficient in quinones or cytochromes. Mutants specifically deficient in menaquinone did not couple protoporphyrinogen oxidation to fumarate reduction, whereas mutants containing menaquinone but deficient in either ubiquinone or cytochromes exhibited this activity. These findings indicate that this coupled reaction is dependent upon menaquinone as hydrogen carrier but independent of ubiquinone and cytochromes. Other characteristics of this coupled reaction were also studied. The activity was located exclusively in the membrane fraction of cell-free extracts. Coproporphyrinogen III could not replace protoporphyrinogen as substrate. Methylene blue, triphenyl tetrazolium and nitrate, but not nitrite, could replace fumarate as anaerobic hydrogen acceptor. These findings have implications for the mechanism and regulation of microbial heme and chlorophyll synthesis and for the physiology of cytochrome synthesis in anaerobic microorganisms.     

Jasmonate mediates salt-induced nicotine biosynthesis in tobacco(Nicotiana tabacum L.)

Jasmonate(JA),as an important signal,plays a key role in multiple processes of plant growth,development and stress response.Nicotine and related pyridine alkaloids in tobacco(Nicotiana tabacum L.) are essential secondary metabolites.Whether environmental factors control nicotine biosynthesis and the underlying mechanism remains previously unreported.Here,we applied physiological and biochemical approaches to investigate how salt stress affects nicotine biosynthesis in tobacco.We found that salt stress induced the biosynthesis of JA,which subsequently triggered the activation of JA-responsive gene expression and,ultimately,nicotine synthesis.Bioinformatics analysis revealed the existence of many Nt MYC2a-recognized G-box motifs in the promoter regions of Nt LOX,Nt AOS,Nt AOC and Nt OPR genes.Applying exogenous JA increased nicotine content,while suppressing JA biosynthesis reduced nicotine biosynthesis.Salt treatment could not efficiently induce nicotine biosynthesis in transgenic anti-COI1 tobacco plants.These results demonstrate that JA acts as the essential signal which triggers nicotine biosynthesis in tobacco after salt stress.     

ENaC proteins are required for NGF-induced neurite growth

Neurite growth is required for nervous system development and repair. Multiple signals, including neurotrophic factors and intact mechanosensing mechanisms, interact to regulate neurite growth. Degenerin/epithelial Na⁺ channel (DEG/ENaC) proteins have been identified as putative mechanosensors in sensory neurons. Recently, others have shown that the neurotrophic factor NGF stimulates expression of acid-sensing ion channel molecules, which are members of the DEG/ENaC family. However, it is unknown whether NGF regulates ENaC expression or whether ENaC expression is required for neurite formation. Therefore, the aims of the present study were to determine whether ENaC expression is 1) regulated by NGF and 2) required for NGF-induced neurite growth in pheochromocytoma PC-12 cells. We found NGF-induced expression of - and -subunits of ENaC, but not -ENaC. Tyrosine kinase A (TrkA) receptor blockade abolished NGF-induced - and -ENaC expression and neurite formation. NGF-induced neurite formation was inhibited by disruption of ENaC expression using 1) pharmacological blockade with benzamil, a specific ENaC inhibitor; 2) small interfering RNA; and 3) dominant-negative ENaC molecules. These data indicate NGF-TrkA regulation of ENaC expression may be required for neurite growth and may suggest a novel role for DEG/ENaC proteins in neuronal remodeling and differentiation. mechanosensation; degenerins; neurotrophins; tyrosine kinase A; pheochromocytoma cells     

The v-ATPase V0 subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila

The V(0) complex forms the proteolipid pore of an ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been proposed largely based on yeast vacuolar fusion experiments. We have isolated mutations in the largest V(0) component vha100-1 in flies in an unbiased genetic screen for synaptic malfunction. The protein is only required in neurons, colocalizes with markers for synaptic vesicles as well as active zones, and interacts with t-SNAREs. Loss of vha100-1 leads to vesicle accumulation in synaptic terminals, suggesting a deficit in release. The amplitude of spontaneous release events and release with hypertonic stimulation indicate normal levels of neurotransmitter loading, yet mutant embryos display severe defects in evoked synaptic transmission and FM1-43 uptake. Our data suggest that Vha100-1 functions downstream of SNAREs in synaptic vesicle fusion.