Application of factorial and Doehlert designs for optimization of pectate lyase production by a recombinant Escherichia coli

 The expression of a recombinant pectate lyase from Bacteroides thetaiotaomicron strain 217 was studied in Escherichia coli strain HB101(pBT4). First, two sets of complete 2⁴ factorial designs were used to evaluate the influences of casamino acids, glucose, magnesium, calcium, tetracycline, ampicillin, tryptophan and MOPS buffer on pectate lyase production in a basal medium. While casamino acids, glucose and magnesium were found to be the prevalent factors, the presence of tetracycline, ampicillin and MOPS buffer were necessary for the reproducibility of the process, probably by increasing the plasmid stability. Secondly, application of the Doehlert design, a response-surface methodology, allowed a good prediction of pectate lyase production according to the variation in glucose and magnesium concentrations. This optimization strategy allowed the production of biomass and recombinant pectate lyase respectively to be increased from 0.2 g l^-1 to 1.9 g l^-1 (dry weight) and from 10 units ml^-1 to 210 units ml^-1 within 24 h at 30°C in shake flasks. Received: 26 July 1995/Received revision: 22 January 1996/Accepted: 29 January 1996     

Arg<Superscript>235</Superscript> is an essential catalytic residue of <Emphasis Type="Italic">Bacillus pumilus</Emphasis> DKS1 pectate lyase to degum ramie fibre

After 24 h of incubation with only purified pectate lyase isolated from Bacillus pumilus DKS1 (EF467045), the weight loss of the ramie fibre was found to be 25%. To know the catalytic residue of pectate lyase the pel gene encoding a pectate lyase from the strain Bacillus pumilus DKS1 was cloned in E. coli XL1Blue and expressed in E. coli BL21 (DE3) pLysS. The pel gene was sequenced and showed 1032 bp length. After purification using CM-Sepharose the enzyme showed molecular weight of 35 kDa and maximal enzymatic activity was observed at 60°C and a pH range of 8.5–9.0. Both Ca²⁺ and Mn²⁺ ions were required for activity on Na-pectate salt substrates, while the enzyme was strongly inhibited by Zn²⁺ and EDTA. The deduced nucleotide sequence of the DKS1 pectate lyase (EU652988) showed 90% homology to pectate lyases from Bacillus pumilus SAFR-032 (CP000813). The 3D structure as well as the catalytic residues was predicted using EasyPred software and Catalytic Site Atlas (CSA), respectively. Site directed mutagenesis confirmed that arginine is an essential catalytic residue of DKS1 pectate lyase.     

Survival ofalginate-ecapsulated Pseudomonas fluorescens cells in soil

Alginate-encapsulated and unencapsulated cells of Pseudomonas fluorescens Rsf were introduced into soil microcosms with and without wheat plants to evaluate bacterial survival and colonization of the rhizoplane and rhizosphere. Encapsualtion of cells in alginate amended with skim milk or with skim milk plus bentonite clay significantly enchanced long-term survival of the cells. There was a negligible effect on long-term bacterial survival when cells were encapsulated in alginate amended with TY medium or soil extract, as compared to water. Drying of beads resulted in a significant reduction in bacterial viability. After addition to soil, cells in dried beads increased in numbers and exhibited stable population densities, whereas cells added in moist beads showed stable dynamics at a higher level. Cells encapsulated in dried beads or fresh beads survived better than unencapsulated cells added to soil. Both cells in moist and dried alginate beads also survide a dry/wet cycle in soil, whereas unencapsulated cells were sensitive to these moisture fluctuations. Shortly after inoculation and 63 days after this, cells from moist beads colonized wheat roots at significantly higher levels than unencapsulated cells, whereas cells in dried beads did so at levels similat to unencapsulated cells. Cells in beads initially placed at different distance from developing root mat were able to move towards and colonize the rhizosphere, at levels of roughly 10⁴ to 10⁶ colony-forming units fo P. fluorescens R2f per gram of dry soil. Correspondence to: J. T. Trevors or J. D. van Elsas     

Effect of N concentration and N source on root colonization by Pseudomonas fluorescens 2-79RLI

Wheat (Triticum aestivum L.) was grown in nutrient solution with low or high N supply (NH₄NO₃ as N source). To further evaluate the influence of N form and its interaction with the nutrient solution pH, wheat plants were grown with NH ₄ ⁺ or NO ₃ ^- either in an conventional nutrient solution or in a nutrient solution in which the pH was maintained at pH 6.5 using a pH-stat system. The nutrient solution was inoculated with Pseudomonas fluorescens 2-79RLI, a genetically modified bacterium that contains lux genes activated by a ribosomal promoter. Cell numbers and physiological status of P. fluorescens 2-79RLI (length of the lag phase of bioluminescence) in the rhizosphere were determined at the root tip and in the lateral root zone. Nitrogen deficiency decreased both plant growth and root colonization by P. fluorescens 2-79RLI at the root tip while it had no effect on root colonization in the lateral root zone. The physiological status of P. fluorescens 2-79RLI was not affected by nitrogen deficiency. Ammonium nutrition increased root colonization by P. fluorescens 2-79RLI at the root tip and in the lateral root zone when the pH of the nutrient solution was allowed to change according to the N form provided. Under these conditions, the physiological status of P. fluorescens 2-79RLI was higher in the lateral root zone than at the root tip. In contrast, N source had no effect on root colonization or physiological status of P. fluorescens 2-79RLI in the nutrient solution maintained at pH 6.5. It is concluded that the stimulation of root colonization by NH ₄ ⁺ in the nutrient solution, not maintained at a constant pH, may be due to increased leakage of solutes into the rhizosphere as a result of impaired exudate retention by high H⁺ concentration in the rhizosphere or the apoplast. This revised version was published online in June 2006 with corrections to the Cover Date.     

Colonization of sorghum and wheat by seed inoculation with <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis>

Colonization of sorghum and wheat after seed inoculation with Gluconacetobacter diazotrophicus strains PAL 5 and UAP 5541/pRGS561 (containing the marker gene gusA) was studied by colony counting and microscopic observation of plant tissues. Inoculum levels as low as 10² CFU per seed were enough for root colonization and further spreading in aerial tissues. Rhizoplane colonization was around 7 log CFU g^?1 (fresh weight). G. diazotrophicus was found inside sorghum and wheat roots with populations higher than 5 log CFU g^?1 (fresh weight). Stem colonization remained stable for 30 days post inoculation with endophyte concentrations from 4 to 5 log CFU g^?1 (fresh weight) (in both plants). Population in leaves decreased continuously being undetectable after 17 days post inoculation.     

Partial purification and properties of pectin lyase from Penicillium expansum

A pectin lyase, poly(methoxygalacturonide) lyase, EC 4.2.2.10, from a culture filtrate of Penicillium expansum was partially purified 33-fold with 7.3% yield. The enzyme was monomeric with a molecular mass of 36.5 kDa. The enzyme did not contain pectate lyase activity and degraded citrus and apple pectin best at pH 7.0 and 40 to 45°C. The K _m for citrus pectin was 9 mg ml^-1.     

Agglutination, Adherence, and Root Colonization by Fluorescent Pseudomonads

Two fractions of agglutination activity towards fluorescent pseudomonads were detected in root washes of potato, tomato, wheat, and bean. High-molecular-mass (>10⁶ Da) components in crude root washes agglutinated only particular saprophytic, fluorescent Pseudomonas isolates. Ion-exchange treatment of the crude root washes resulted in preparations of lower-molecular-mass (10⁵ to 10⁶ Da) fractions which agglutinated almost all Pseudomonas isolates examined. Also, components able to suppress agglutination reactions of pseudomonads with the lower-molecular-mass root components were detected in crude root washes of all crops studied. Pseudomonas isolates were differentially agglutinated by both types of root components. The involvement of these two types of root components in short-term adherence and in colonization was studied in potato, tomato, and grass, using Pseudomonas isolates from these crops. Short-term adherence of isolates to roots was independent of their agglutination with either type of root components. With agglutination-negative mutants, the high-molecular-mass components seemed to be involved in adherence of Pseudomonas putida Corvallis to roots of all crops studied. Short-term adherence to roots of four Pseudomonas isolates could be influenced by addition of both crude and ion-exchange-treated root washes, depending on their agglutination phenotype with these root wash preparations. Potato root colonization by 10 different isolates from this crop, over a period of 7 days, was not correlated with their agglutination phenotype. Agg^- mutants of P. putida Corvallis were not impaired in root colonization. It is concluded that the root agglutinins studied can be involved in short-term adherence of pseudomonads to roots but do not play a decisive role in their root colonization.     

Tryptophanyl-tRNA synthetase is found closely associated with and stimulates DNA polymerase α-like activity from wheat embryos

DNA polymerase α-like from wheat embryos is found to purify closely associated with a tryptophanyl-tRNA synthetase activity. No other aminoacyl-tRNA synthetases were present. A purified preparation of wheat tryptophanyl-tRNA synthetase free of polymerase activity was able to stimulate plant DNA polymerase of the α-like type, while the γ-like polymerase from wheat embryos was not affected by the enzyme. We have not been able to find a diadenosine 5′, 5′′′-P¹,P⁴-tetraphosphate binding activity associated to the polymerase-synthetase complex. We have also observed a specific inhibition by beef tRNA^Trp of DNA polymerase α-like activity, while other tRNAs will not change the enzyme activity.     

Transient Activation of Plasmalemma K Efflux and H Influx in Tobacco by a Pectate Lyase Isozyme from Erwinia chrysanthemi

A purified pectate lyase isozyme derived from Erwinia chrysanthemi induced rapid net K⁺ efflux and H⁺ influx in suspension-cultured tobacco cells. Comparable fluxes of other ions (Na⁺, Cl⁻) were not observed. The K⁺ efflux/H⁺ influx response began within 15 minutes after addition of enzyme to cell suspensions and continued for approximately 1 hour after which cells resumed the net H⁺ efflux exhibited prior to enzyme treatment. The response was not prolonged by a second enzyme dose 1 hour after the first. The K⁺/H⁺ response was characterized by saturation at low enzymic activity (2 × 10⁻³ units per milliliter), and inhibition by the protonophore, carbonyl cyanide m-chlorophenylhydrazone, and was not associated with membrane leakiness caused by structural cell wall damage. The total K⁺ loss and H⁺ uptake induced by enzyme was one-fourth to one-third that induced by Pseudomonas syringae pv. pisi and did not reduce cell viability. These results indicate that pectate lyase induces a K⁺ efflux/H⁺ influx response in tobacco similar to but of shorter duration than that induced by P. syringae pv. pisi during the hypersensitive response. Pectate lyase or other cell wall degrading enzymes may therefore influence the induction of hypersensitivity.     

Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris

The methylotrophic yeast Pichia pastoris is an attractive heterologous protein expression host, mainly for genes from higher eukaryotes. However, no successful examples for the expression of bacterial gene encoding pectate lyase in P. pastoris have been reported. The present study reports for the first time the cloning and functional expression of the bacterial Bacillus subtilis gene encoding alkaline pectate lyase in P. pastoris. A molecular weight of 43,644 Da was calculated from the deduced amino acid sequence. A pectate lyase activity as high as 100 U/ml was attained in the fermentation broth of P. pastoris GS 115, which was about 10 times higher than when the gene is expressed in Escherichia coli. The recombinant pectate lyase was purified to homogeneity and maximal activity of the enzyme was observed at 65 °C, and pH 9.4. The recombinant enzyme showed a wider pH and thermal stability spectrum than the purified pectate lyase from B. subtilis WSHB04-02. Pectate lyase activity slightly increased in the presence of Mg²⁺ (ion) but decreased in the presence of other metal ions. Analysis of polygalacturonic acid degradation products by electrospray ionization-mass spectrometry revealed that the degradation products were unsaturated trigalacturonic acid and unsaturated bigalacturonic acid, which confirms that the enzyme catalyzes a trans-elimination reaction.     

不同作物对旱地农田残留硝态氮的利用差异

在黄土高原南部娄土上,通过2a田间试验研究了小麦和苜蓿对土壤中不同累积量的残留硝态氮的利用差异。研究包括0—3 m土壤残留硝态氮累积量(设N1、N2、N3、N4、N5和N6共6个水平,残留硝态氮量依次增加)和作物种类(冬小麦和苜蓿)2个因素,分别采用冬小麦-夏休闲-冬小麦和苜蓿连作种植方式。结果表明,不施用氮肥条件下,冬小麦-休闲-冬小麦轮作周期与苜蓿连作2a内,土壤残留硝态氮的消长有明显差异。在第1季小麦生长期间,小麦的氮素携出量(63.9—130.3 kg/hm2)、氮素携出量占播前残留硝态氮量的比例(18%—27%)及氮素携出量占该生长季硝态氮减少量的比例(29%—62%)均显著高于同期的苜蓿处理。在第2个生长季内,苜蓿的氮素携出量是小麦当季氮素携出量的近6倍,但由于苜蓿固氮作用强烈,至第2生长季结束后,0—3 m土壤硝态氮量与苜蓿播前相比平均只减少了72.4 kg/hm2,而麦田0—3 m土壤硝态氮量与小麦播前相比减少了158.3 kg/hm2。在短期内如果通过种植作物消耗土壤剖面的残留硝态氮,冬小麦比苜蓿更有优势。第1季小麦氮素携出量与小麦播前0—2 m(r=0.920**)和0—3 m(r=0.857*)土层残留硝态氮量呈显著或极显著正相关,与0—1 m土层残留硝态氮量没有显著相关性;第1生长季苜蓿氮素携出量与播前0—1 m土壤硝态氮累积量呈显著正相关关系(r=0.846*),而与0—2 m和0—3 m土壤硝态氮累积量的相关性并不显著。小麦比苜蓿能利用更深土层中的硝态氮。随着播前0—3 m土壤残留硝态氮的增加,小麦和苜蓿地上部氮素携出量呈增加的趋势,硝态氮表观损失也显著增加。     

Neuroblastoma cell adenylate cyclase: direct activation by adenosine and prostaglandins.

—Adenylate cyclase activity of permeabilized neuroblastoma cells was measured by the conversion of [α³²P]ATP into labelled cyclic AMP. Adenosine (10^?6 - 10^?4m ) induced a dose-dependent increase in cyclic AMP formation. This effect could not be accounted for either by an adenosine-induced inhibition of the phosphodiesterase activity present in the enzyme preparation, or by a direct conversion of adenosine into cyclic AMP. This indicates that the observed increase in cyclic AMP accumulation reflected an activation of adenylate cyclase. Adenosine is partially metabolized during the course of incubation with the enzyme preparation. However, none of the identified non-phosphorylated adenosine metabolites were able to induce an adenylate cyclase activation. This suggests that adenosine itself is the stimulatory agent. The apparent K_m of the adenylate cyclase for adenosine was 5 ± 10^?6-10^?5m . Maximal activation represented 3-4 times the basal value (10-100 pmol cyclic AMP formed/10 min/mg protein). The adenosine effect was stereospecific, since structural analogues of adenosine were inactive. Adenosine increased the maximal velocity of the adenylate cyclase reaction. The stimulatory effect of adenosine was inhibited by theophylline. Prostaglandin PGE₁ had a stimulatory effect much more pronounced than that of adenosine (6-10-fold the basal value at 10^?6m ). Dopamine and norepinephrine induced a slight adenylate cyclase activation which was not potentiated by adenosine. It is concluded that adenosine is able to activate directly neuroblastoma cell adenylate cyclase. It seems very likely that such a direct activation is also present in intact nervous tissue and account, at least partly, for the observed cyclic AMP accumulation in response to adenosine.     

Modulation of nutrient acquisition and polyamine pool in salt-stressed wheat (Triticum aestivum L.) plants inoculated with arbuscular mycorrhizal fungi

Two wheat (Triticum aestivum L.) cultivars, Sids 1 and Giza 168, were grown under non-saline or saline conditions (4.7 and 9.4 dS m^?1) with and without arbuscular mycorrhizal fungi (AMF) inoculation. Salt stress considerably decreased root colonization, plant productivity and N, P, K⁺, Fe, Zn and Cu concentrations, while it increased Na⁺ level, particularly in Giza 168. Mycorrhizal colonization significantly enhanced plant productivity and N, P, K⁺, Fe, Zn and Cu acquisition, while it diminished Na⁺ uptake, especially in Sids 1. Salinity increased putrescine level in Giza 168, however, values of spermidine and spermine increased in Sids 1 and decreased in Giza 168. Mycorrhization changed the polyamine balance under saline conditions, an increase in putrescine level associated with low contents of spermidine and spermine in Giza 168 was observed, while Sids 1 showed a decrease in putrescine and high increase in spermidine and spermine. Moreover, mycorrhizal inoculation significantly reduced the activities of diamine oxidase and polyamine oxidase in salt-stressed wheat plants. Modulation of nutrient acquisition and polyamine pool can be one of the mechanisms used by AMF to improve wheat adaptation to saline soils. This is the first report dealing with mycorrhization effect on diamine oxidase and polyamine oxidase activities under salt stress.     

Production of L-tartaric acid by immobilized bacterial cells Nocardia tartaricans

Nocardia tartaricans converted sodium cis-epoxysuccinate to L-tartrate. The highest cis-epoxysuccinate hydrolase activity (37.7 U mg^–1) was obtained with 0.02% (w/v) sodium deoxycholate, but this inactivated the cells. Immobilized N. tartaricans in pectate gel showed higher enzyme activity (51 U mg ^–1) compare to the free cells (8.9 U mg ^–1). After 450 days, the immobilized cells still possessed 0.65 U mg ^–1, i.e. 30% of the initial enzyme activity.     

蜡样芽孢杆菌B3-7在大田小麦根部的定殖动态及其对小麦纹枯病的防治效果

为了阐明蜡样芽孢杆菌B3-7在大田条件下的生态适应性以及对于小麦纹枯病的生防效果,通过利用绿色荧光蛋白编码基因gfp标记生防菌株B3-7,室内比较了GFP标记菌株和原始出发菌株在菌落形态、生长特性,生物薄膜产生以及在小麦根部定殖等方面的特性,结果发现GFP标记菌株和出发菌株在上述特性方面无明显差别。在此基础上,大田条件下测定了GFP标记菌株在小麦根部的定殖动态和对于小麦纹枯病的生防效果。结果发现,GFP标记菌株在小麦根部能够长期定殖,其存在量在小麦分蘖期最大,每克根重达到105CFU,拔节期后,该细菌数量一直维持在104CFU之上。同时发现,生防菌株能够有效降低小麦纹枯病的严重度和提高罹病小麦的产量。小麦分蘖期、孕穗期和灌浆期生防菌对于小麦纹枯病的防治效果分别达到60%、34%,34%,小麦成熟后产量提高13%—15%。结果表明,B3-7在大田条件下具有较好的生态适应性和防治小麦纹枯病的能力。     

Development and function ofAzospirillum-inoculated roots

Summary The surface distribution ofAzospirillum on inoculated roots of maize and wheat is generally similar to that of other members of the rhizoplane microflora. During the first three days, colonization takes place mainly on the root elongation zone, on the base of root hairs and, to a lesser extent, on the surface of young root hairs.Azospirillum has been found in cortical tissues, in regions of lateral root emergence, along the inner cortex, inside xylem vessels and between pith cells. Inoculation of several cultivars of wheat, corn, sorghum and setaria with several strains ofAzospirillum caused morphological changes in root starting immediately after germination. Root length and surface area were differentially affected according to bacterial age and inoculum level. During the first three weeks after germination, the number of root hairs, root hair branches and lateral roots was increased by inoculation, but there was no change in root weight. Root biomass increased at later stages. Cross-sections of inoculated corn and wheat root showed an irregular arrangement of cells in the outer layers of the cortex. These effects on plant morphology may be due to the production of plant growth-promoting substances by the colonizing bacteria or by the plant as a reaction to colonization. Pectic enzymes may also be involved. Morphological changes had a physiological effect on inoculated roots. Specific activities of oxidative enzymes, and lipid and suberin content, were lower in extracts of inoculated roots than in uninoculated controls. This suggests that inoculated roots have a larger proportion of younger roots. The rate of NO^– ₃, K⁺ and H₂PO^– ₄ uptake was greater in inoculated seedlinds. In the field, dry matter, N, P and K accumulated at faster rates, and water content was higher inAzospirillum-inoculated corn, sorghum, wheat and setaria. The above improvements in root development and function lead in many cases to higher crop yield.     

Expression and thermostability of Paenibacillus campinasensis BL11 pectate lyase and its applications in bast fibre processing

An open reading frame (ORF) with 963 nucleotides from Paenibacillus campinasensis BL11 was cloned and expressed in Escherichia coli. It encodes a pectate lyase (EC 4.2.2.2) of 35.6 kDa, denominated Pel‐BL11. The recombinant Pel‐BL11 was fused with His‐tag and purified. An optimal activity of 1623 IU mg^?1 was exhibited at 50°C, pH 10. Significant activities of Pel‐BL11 are demonstrated between 40 and 70°C and from a pH of 7–11. The observed half‐lives are 103 min at 70°C and 288 min at 40°C. Compared to other published acid and alkaline pectate lyases, Pel‐BL11 demonstrated exceptional thermostability and wider pH adaptability. Temperature effects on the cleavage of the pectate α‐1,4‐glycosidic bond by Pel‐BL11 were examined. Continuous cleavage occurred for the first 3 h at 30 and 50°C. However, at 70°C, the majority of the cleavage occurred during the first 10 min. Weight loss in gampi and paper mulberry fibres after enzyme treatment validate the potential of this treatment in fibre degumming.     

The phosphatase activity of the plasma membrane Ca pump. Activation by acidic lipids in the absence of Ca increases the apparent affinity for Mg

The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg²⁺ and K⁺. Micromolar concentrations of Ca²⁺ inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca²⁺ concentrations. Acidic lipids increased 5-10 fold the pNPPase activity either in the presence or in the absence of Ca²⁺. The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K⁺ but the apparent affinity of the enzyme for Mg²⁺ increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg²⁺. Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca²⁺ free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca²⁺ and the activation improves the interaction of the enzyme with Mg²⁺.     

The IAA-oxidase system of citrus roots

Summary The activity of indoleacetic acid oxidase in citrus plants (roots of mature trees and roots and stems of seedlings) was demonstrated. The interrelationships between 2,4-dichlorophenol, Mn⁺⁺ and the enzymatic activity were examined. The presence of Mn⁺⁺ was not found necessary, even in dialyzed enzyme reaction mixtures. However, Mn⁺⁺ promoted the enzymatic activity at the very high concentration of 10^-1M. The inhibitory effect induced by excessive amounts of 2,4-dichlorophenol (10^-3 M) was removed in the presence of Mn⁺⁺.The enzyme showed a specificity to IAA as compared with other synthetic auxins in a wheat coleoptile bioassay. Moreover, the enzyme acted selectively on some endogenous auxins found in citrus tissues.The existence of endogenous inhibitors in stems and cofactors in roots was also demonstrated.This study is part of a project carried out under grants authorized by Public Law 480 for the U.S. Department of Agriculture (10-CR-22, FG-Is-136). The financial support provided is gratefully acknowledged.