Functions Encoded by Pyrrolnitrin Biosynthetic Genes from Pseudomonas fluorescens

Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity. Recently we described four genes, prnABCD, from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin. In the work presented here, we describe the function of each prn gene product. The four genes encode proteins identical in size and serology to proteins present in wild-type Pseudomonas fluorescens, but absent from a mutant from which the entire prn gene region had been deleted. The prnA gene product catalyzes the chlorination of l-tryptophan to form 7-chloro-l-tryptophan. The prnB gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-l-tryptophan to monodechloroaminopyrrolnitrin. The prnC gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin. The prnD gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. The organization of the prn genes in the operon is identical to the order of the reactions in the biosynthetic pathway.The antibiotic pyrrolnitrin [3-chloro-4-(2′-nitro-3′-chlorophenyl)pyrrole] (PRN) is produced by many pseudomonads and has broad-spectrum antifungal activity (1, 5, 12–14, 17). PRN has been implicated as an important mechanism of biological control of fungal plant pathogens by several Pseudomonas strains (12–14), including P. fluorescens BL915, from which the prn genes were isolated (10).Tryptophan was identified as the precursor for PRN, based on the feeding of cultures with isotopically labeled and substituted tryptophan (2, 7, 8, 17, 25). Biosynthetic pathways were proposed as early as 1967 (7) and have been refined on the basis of tracer studies and the isolation of intermediates (Fig. ​(Fig.1)1) (2, 8, 17, 19, 23, 25). Recently, Hammer et al. (9) described the cloning and characterization of a 5.8-kb DNA region which encodes the PRN biosynthetic pathway. This DNA region confers the ability to produce PRN when expressed heterologously in Escherichia coli and contains four genes, prnABCD, each of which is required for PRN production. In the research described here, we used mutants in which each of the four genes was disrupted and strains which overexpress the individual genes to elucidate the function of each gene product in PRN biosynthesis. Open in a separate windowFIG. 1Biosynthetic pathways for PRN as proposed by van Pée et al. (23) (A) and by Chang et al. (2) (B). The reactions catalyzed by the PRN biosynthetic enzymes encoded by the prnABCD genes are indicated above the appropriate reaction arrows.

Bacterial strains and plasmids.

The bacterial strains and plasmids used in this study are described in Table ​Table1.1. Pseudomonas strains were cultured in Luria-Bertani medium at 28°C. Antibiotics, when used, were added at the following concentrations: tetracycline, 30 μg/ml; and kanamycin, 50 μg/ml. The expression vector pPEH14 consists of the P_tac promoter and rrnB ribosomal terminator from pKK223-3 (Pharmacia, Uppsala, Sweden) cloned into the BglII site of the broad-host-range plasmid pRK290 (4). P_tac is a strong constitutive promoter in Pseudomonas (unpublished data). The PRN biosynthetic genes are the coding regions described by Hammer et al. (9). Each coding region was cloned from the translation initiation codon to the stop codon by PCR with restriction sites added to the ends to facilitate cloning. For prnB, the native GTG initiation codon was changed to ATG. The clones were sequenced after PCR.

TABLE 1

Bacterial strains and plasmids used in this study

Identification and Characterization of aarF, a Locus Required for Production of Ubiquinone in Providencia stuartii and Escherichia coli and for Expression of 2′-N-Acetyltransferase in P. stuartii

Providencia stuartii contains a chromosomal 2′-N-acetyltransferase [AAC(2′)-Ia] involved in the O acetylation of peptidoglycan. The AAC(2′)-Ia enzyme is also capable of acetylating and inactivating certain aminoglycosides and confers high-level resistance to these antibiotics when overexpressed. We report the identification of a locus in P. stuartii, designated aarF, that is required for the expression of AAC(2′)-Ia. Northern (RNA) analysis demonstrated that aac(2′)-Ia mRNA levels were dramatically decreased in a P. stuartii strain carrying an aarF::Cm disruption. The aarF::Cm disruption also resulted in a deficiency in the respiratory cofactor ubiquinone. The aarF locus encoded a protein that had a predicted molecular mass of 62,559 Da and that exhibited extensive amino acid similarity to the products of two adjacent open reading frames of unknown function (YigQ and YigR), located at 86 min on the Escherichia coli chromosome. An E. coli yigR::Kan mutant was also deficient in ubiquinone content. Complementation studies demonstrated that the aarF and the E. coli yigQR loci were functionally equivalent. The aarF or yigQR genes were unable to complement ubiD and ubiE mutations that are also present at 86 min on the E. coli chromosome. This result indicates that aarF (yigQR) represents a novel locus for ubiquinone production and reveals a previously unreported connection between ubiquinone biosynthesis and the regulation of gene expression.     

EXPERIMENTAL EVOLUTION OF SENESCENCE: AN ANALYSIS USING A “HETEROGENEITY” MORTALITY MODEL

A long-term laboratory selection experiment has produced replicated populations of fruit flies that differ in mean life span by more than twofold. An analysis of age-specific mortality rates indicated that differences in mean life span have been achieved principally by evolution of patterns of senescence. These results provide empirical confirmation that senescence can be modified within species by appropriate forms of natural selection, which is a fundamental prediction of theories regarding the genetic basis and evolution of senescence. Mortality data were fit to a model that accounts for the leveling off of cohort mortality rates at older ages, but that does not necessarily imply that very old individuals cease to senesce.     

Immortalization of Rat Embryo Fibroblasts by a 3′-Untranslated Region

We have exploited a cross-species expression screen to search for cellular immortalizing activities. A newt blastemal cDNA expression library was transfected into rat embryo fibroblasts and immortal cell lines were selected. This identified a 1-kb cDNA fragment which has a low representation in the cDNA library and is derived from the 3′-UTR of an α-glucosidase-related mRNA. Expression of this sequence in rat embryo fibroblasts has shown that it is active in promoting colony formation and immortalization. It is also able to cooperate with an immortalization-defective deletion mutant of SV40 T antigen, indicating that it can exert its growth-stimulatory activity in the pathway activated by a viral immortalizing oncogene. This is the first example of an immortalizing activity mediated by an RNA sequence, and further analysis of its mechanism should provide new insights into senescence and immortalization.     

Crystalline Cellulose in Hydrated Primary Cell Walls of Three Monocotyledons and One Dicotyledon

The molecular ordering of cellulose, including its crystallinity,in the unlignified primary cell walls of three monocotyledons(Italian ryegrass, pineapple, and onion) and one dicotyledon(cabbage) was characterized by solid-state ¹³C NMR spectroscopy.These species were chosen because their primary cell walls havedifferent non-cellulosic polysaccharides and this may affectthe molecular ordering of cellulose. Values of the proton rotating-framerelaxation [T_1p(H)] and spin-spin relaxation [T₂(H)] time constantsshowed that the cellulose in the cell walls of all four specieswas in a crystalline rather than an amorphous state. Furthermore,a resolution enhancement procedure showed that the triclinic(I) and the monoclinic (I_rß) crystal forms of cellulosewere present in similar proportions in these cell walls. However,the calculated cross-sectional dimensions of the cellulose crystallitesvaried among the cell walls (in the range 2–3 nm): thelargest were in the Italian ryegrass, the smallest were in theonion and cabbage, and those of intermediate size were in thepineapple. The crystallite dimensions may thus be affected bythe non-cellulosic polysaccha-ride compositions of the cellwalls. ⁴Present address: Food Science Postgraduate Programme, Departmentof Chemistry, The University of Auckland, Private Bag 92019,Auckland, New Zealand.     

RGS7 Attenuates Signal Transduction Through the Gαq Family of Heterotrimeric G Proteins in Mammalian Cells

Abstract: The RGS proteins are a recently discovered family of G protein regulators that have been shown to act as GTPase-activating proteins (GAPs) on the G_αi and G_αq subfamilies of the heterotrimeric G proteins. Here, we demonstrate that RGS7 is a potent GAP in vitro on G_αi1 and G_αo heterotrimeric proteins and that RGS7 acts to down-regulate G_αq-mediated calcium mobilization in a whole-cell assay system using a transient expression protocol. This RGS protein and RGS4 are reported to be expressed predominantly in brain, and in situ hybridization studies have revealed similarities in the regional distribution of RGS and G_αq mRNA expression. Our findings provide further evidence to support a functional role for RGS4 and RGS7 in G_αq-mediated signaling in the CNS.     

Kainate-Induced Apoptosis in Cultured Murine Cerebellar Granule Cells Elevates Expression of the Cell Cycle Gene Cyclin D1

Abstract: Recent evidence suggests that neuronal apoptosis is the consequence of an inappropriate reentry into the cell cycle. Expression of the cell cycle gene cyclin D1, a G1-phase cell cycle regulator, was examined in primary cultures of murine cerebellar granule cells (CGCs) during kainate (KA)-mediated apoptosis. Using cultures of CGCs, we found that a 24-h exposure to KA (1–3,000 µ M ) induced a concentration-dependent cell death with neurons exhibiting characteristic apoptotic morphology and extensive labeling using the terminal transferase-mediated nick end-DNA labeling (TUNEL) method. KA induced a time- and concentration-dependent increase in expression of cyclin D1 as determined by immunocytochemistry and western blot analysis. KA-induced apoptosis and cyclin D1 expression exhibited a similar concentration dependence and were significantly attenuated by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (50 µ M ), indicating a KA receptor-mediated effect. Here we present evidence for the first time that KA-induced apoptosis in cultured CGCs involves the induction of cyclin D1, suggesting its involvement in excitotoxic receptor-mediated apoptosis.     

Mechanotransduction molecules in the plant gravisensory response: Amyloplast/statolith membranes contain a β1 integrin-like protein

Summary It has been hypothesized that the sedimentation of amyloplasts within root cap cells is the primary event in the plant gravisensory-signal transduction cascade. Statolith sedimentation, with its ability to generate weighty mechanical signals, is a legitimate means for organisms to discriminate the direction of the gravity vector. However, it has been demonstrated that starchless mutants with reduced statolith densities maintain some ability to sense gravity, calling into question the statolith sedimentation hypothesis. Here we report on the presence of a ₁ integrin-like protein localized inside amyloplasts of tobacco NT-1 suspension culture, callus cells, and whole-root caps. Two different antibodies to the ₁ integrin, one to the cytoplasmic domain and one to the extracellular domain, localize in the vicinity of the starch grains within amyloplasts of NT-1. Biochemical data reveals a 110-kDa protein immunoprecipitated from membrane fractions of NT-1 suspension culture indicating size homology to known ₁ integrin in animals. This study provides the first direct evidence for the possibility of integrin-mediated signal transduction in the perception of gravity by higher plants. An integrin-mediated pathway, initiated by starch grain sedimentation within the amyloplast, may provide the signal amplification necessary to explain the gravitropic response in starch-depleted cultivars.Abbreviations BA 6-benzylaminopurine - ETOH ethyl alcohol - LP liquid propane - LR London Resin - PBST phosphate-buffered saline with Tween - TEM transmission electron microscopy - OSM optical-sectioning microscopy     

Membrane fractionation and enrichment of callose synthase from pollen tubes of Nicotiana alata Link et Otto

The callose synthase (UDP-glucose: 1,3-β-d-glucan 3-β-d-glucosyl transferase; EC 2.4.1.34) enzyme (CalS) from pollen tubes of Nicotiana alata Link et Otto is responsible for developmentally regulated deposition of the cell wall polysaccharide callose. Membrane preparations from N. alata pollen tubes grown in liquid culture were fractionated by density-gradient centrifugation. The CalS activity sedimented to the denser regions of the gradient, approximately 1.18 g · ml⁻¹, away from markers for Golgi, endoplasmic reticulum and mitochondria, and into fractions enriched in ATPase activity and in membranes staining with phosphotungstic acid at low pH. This suggests that pollen-tube CalS is localised in the plasma membrane. Callose synthase activity from membranes enriched by downward centrifugation was solubilised with digitonin, which gave a 3- to 4-fold increase in enzyme activity, and the solubilised activity was then enriched a further 10-fold by product entrapment. The complete procedure gave final CalS specific activities up to 1000-fold higher than those of pollen-tube homogenates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that several polypeptides co-fractionated with CalS activity through purification, with a polypeptide of 190 kDa being enriched in product-entrapment pellets. Received: 24 September 1997 / Accepted: 12 November 1997