Physiological and cellular responses of the 2,4-D degrading bacterium,Burkholderia cepacia YK-2, to the phenoxyherbicides 2,4-D and 2,4,5-T

Our previous research has demonstrated that novel 43-kDa DnaK and 41-kDa GroEL proteins are synthesized in Burkholderia sp. YK-2 in response to sublethal concentrations of 2,4-D stress [Cho et al. (2000) Curr Microbiol 41:33-38]. In this study, we have extended this work to examine the cellular responses of strain YK-2 to stresses induced in response to the phenoxyherbicides 2,4-D or 2,4,5-T. Strain YK-2 exhibited a more sensitive response to 2,4,5-T stress than to 2,4-D stress, as shown in physiological and morphological changes, suggesting a greater cytotoxic effect of 2,4,5-T. SEM analyses revealed the presence of perforations and irregular rod forms with wrinkled surfaces for cells treated with either herbicide. These irregularities were found more frequently for 2,4,5-T-treated cells than for 2,4-D-treated cells. Analysis of cellular fatty acids showed similar effects in the shifts of total cellular fatty acid composition in response to 2,4-D and 2,4,5-T. Strain YK-2 could degrade 2.25 m M 2,4-D completely during 28 h of incubation with transient production of 2,4-dichlorophenol as a metabolite; however, 2,4,5-T was not catabolized at any of the concentrations tested. BIOLOG and 16S rDNA analyses revealed that strain YK-2 was 98% similar to the Burkholderia cepacia species cluster; therefore, we have designated this strain as B. cepacia YK-2.     

Physiological and Cellular Responses of the 2,4-D Degrading Bacterium, Burkholderia cepacia YK-2, to the Phenoxyherbicides 2,4-D and 2,4,5-T

Our previous research has demonstrated that novel 43-kDa DnaK and 41-kDa GroEL proteins are synthesized in Burkholderia sp. YK-2 in response to sublethal concentrations of 2,4-D stress [Cho et al. (2000) Curr Microbiol 41:33–38]. In this study, we have extended this work to examine the cellular responses of strain YK-2 to stresses induced in response to the phenoxyherbicides 2,4-D or 2,4,5-T. Strain YK-2 exhibited a more sensitive response to 2,4,5-T stress than to 2,4-D stress, as shown in physiological and morphological changes, suggesting a greater cytotoxic effect of 2,4,5-T. SEM analyses revealed the presence of perforations and irregular rod forms with wrinkled surfaces for cells treated with either herbicide. These irregularities were found more frequently for 2,4,5-T-treated cells than for 2,4-D-treated cells. Analysis of cellular fatty acids showed similar effects in the shifts of total cellular fatty acid composition in response to 2,4-D and 2,4,5-T. Strain YK-2 could degrade 2.25 mM 2,4-D completely during 28 h of incubation with transient production of 2,4-dichlorophenol as a metabolite; however, 2,4,5-T was not catabolized at any of the concentrations tested. BIOLOG and 16S rDNA analyses revealed that strain YK-2 was 98% similar to the Burkholderia cepacia species cluster; therefore, we have designated this strain as B. cepacia YK-2. Received: 7 February 2002 / Accepted: 7 March 2002     

The sandfly Lutzomyia longipalpis shows specific humoral responses to bacterial challenge

Abstract The presence of immune molecules induced by microorganisms in the haemolymph of Lutzomyia longipalpis sandflies has been investigated. Injections of Escherichia coli and Micrococcus luteus into female sandflies induced anti-bacterial activity in the haemolymph. Inhibition zone assays showed that haemolymph from E.coli and M.luteus injected sandflies differentially inhibited M.luteus growth. This differential effect was specific to M. luteus infection since anti-Zs. coli activity was similar in haemolymph from both E.coli oxM.luteus injected sandflies. Haemolymph following injection of either bacteria showed the induction of a 4kDa peptide. Haemolymph from M.luteus injected sandflies also contained a 33 kDa polypeptide which was absent in haemolymph from E.coli and control uninfected insects. Sandflies, in common with other insects, were shown to possess general and specific humoral immune responses to the presence of microorganisms.     

Testing of 2,4,5-T-amino acid conjugates for mutagenic activity in Salmonella typhimurium strains

Since amino acid conjugates are plant metabolites of the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 5 amino acid conjugates (aspartic acid, glutamic acid, leucine, methionine and tryptophan) of 2,4,5-T were tested for possible mutagenic activity utilizing 5 strains of Salmonella typhimurium (TA97, TA98, TA100, TA1535 and TA1538) with and without rat-liver microsomal and cytosolic enzymes. These compounds did not cause any significant increase in reversions when compared with controls in the presence or absence of the activating system. Further, linear regression analysis showed no significant (p less than 0.05) dose-response relationships. Thus, it was concluded that the tested amino acid conjugates of 2,4,5-T are not mutagens or promutagens in these assays.     

Antimicrobial characteristic of insoluble alkylpyridinium iodide.

Insoluble and soluble alkylpyridinium iodides (C8 to C18) were synthesized. The insoluble agents were quaternized 4-vinylpyridine-divinylbenzene copolymers. The insoluble agent [C12(50)] that contained 50% divinylbenzene and had a C12 alkyl chain was selected as the most suitable insoluble agent. C12(50) showed poor durability of the antibacterial activity, but C12(50), which had lost the activity, was refreshed by washing with ethanol. This washing became ineffective after a few cycles of antibacterial treatment and refreshment. Such C12(50) recovered the activity upon 1.0 N NaOH treatment. The antibacterial activity of C12(50) depended on its surface area. It showed high antimicrobial activity against gram-positive bacteria and also showed activity against gram-negative bacteria and yeasts. But the activities of C12(50) and laurylpyridinium iodide solution were different against some microbes. The antibacterial activities of the agents were investigated against Escherichia coli and Micrococcus luteus under various conditions. The activity of C12(50) was higher at a higher temperature or at a lower cell concentration. The activity of C12(50) decreased on addition of NaCl, glucose, or bovine albumin to the cell suspension or in 0.01 M sodium-potassium phosphate buffer. C12(50) showed less activity when cells were mixed with dead cells or the supernatant of dead cells killed in an autoclave. The mode of action of the laurylpyridinium iodide solution against E. coli and M. luteus was similar to that of C12(50) except for the influence of E. coli cell concentration.     

Antimicrobial characteristic of insoluble alkylpyridinium iodide

Insoluble and soluble alkylpyridinium iodides (C8 to C18) were synthesized. The insoluble agents were quaternized 4-vinylpyridine-divinylbenzene copolymers. The insoluble agent [C12(50)] that contained 50% divinylbenzene and had a C12 alkyl chain was selected as the most suitable insoluble agent. C12(50) showed poor durability of the antibacterial activity, but C12(50), which had lost the activity, was refreshed by washing with ethanol. This washing became ineffective after a few cycles of antibacterial treatment and refreshment. Such C12(50) recovered the activity upon 1.0 N NaOH treatment. The antibacterial activity of C12(50) depended on its surface area. It showed high antimicrobial activity against gram-positive bacteria and also showed activity against gram-negative bacteria and yeasts. But the activities of C12(50) and laurylpyridinium iodide solution were different against some microbes. The antibacterial activities of the agents were investigated against Escherichia coli and Micrococcus luteus under various conditions. The activity of C12(50) was higher at a higher temperature or at a lower cell concentration. The activity of C12(50) decreased on addition of NaCl, glucose, or bovine albumin to the cell suspension or in 0.01 M sodium-potassium phosphate buffer. C12(50) showed less activity when cells were mixed with dead cells or the supernatant of dead cells killed in an autoclave. The mode of action of the laurylpyridinium iodide solution against E. coli and M. luteus was similar to that of C12(50) except for the influence of E. coli cell concentration.     

小菜蛾溶菌酶Pxlys的基因克隆及其 重组蛋白的抗菌活性分析

【目的】本研究旨在通过研究小菜蛾Plutella xylostella溶菌酶的功能,进一步认识小菜蛾的免疫防御机理,为小菜蛾的生物防治提供新的思路。【方法】利用RACE技术克隆小菜蛾溶菌酶基因。构建原核表达载体pET-29a-Pxlys,利用原核表达系统表达并用镍柱亲和层析纯化重组蛋白Pxlys。利用牛津杯法检测重组蛋白Pxlys对停滞棒杆菌Corynebacterium stationis、藤黄微球菌Micrococcus luteus、金黄色葡萄球菌Staphyloccocus aureus、大肠杆菌Escherichia coli、志贺氏菌Shigella sp.、沙门氏菌Salmonella sp.和苏云金芽胞杆菌Bacillus thuringiensis的抑菌活性,并利用扫描电子显微镜观察重组蛋白Pxlys对停滞棒杆菌和大肠杆菌的溶菌特征。【结果】克隆获得开放阅读框长423 bp的小菜蛾溶菌酶基因Pxlys(GenBank登录号： MN702780)序列,它编码140个氨基酸,相对分子质量为15.79 kD。抑菌试验表明,重组蛋白Pxlys不仅对革兰氏阳性细菌的停滞棒杆菌、藤黄微球菌和金黄色葡萄球菌有较强的抑菌活性(抑菌圈直径分别为20.0±1.1, 19.0±0.5和16.5±0.5 mm),而且对革兰氏阴性细菌大肠杆菌、志贺氏菌和沙门氏菌也有抑菌活性(抑菌圈直径分别为16.3±0.5, 15.0±0.5和14.0±1.1 mm),重组蛋白Pxlys对革兰氏阳性细菌比对革兰氏阴性细菌表现出更强的抑菌活性。另外,重组蛋白Pxlys还表现出对苏云金芽胞杆菌的抑菌活性。扫描电子显微镜下,经重组蛋白Pxlys处理过的停滞棒杆菌和大肠杆菌的溶菌特征不同。【结论】Pxlys具有广谱的抗微生物活性,其对革兰氏阳性细菌和革兰氏阴性细菌的抑菌机理可能存在不同。研究结果为深入研究小菜蛾免疫防御系统提供基础。     

家蝇幼虫抗菌相关蛋白/多肽的诱导及抗菌活性分析

对家蝇Musca domestica 3龄幼虫进行针刺、带菌针刺、热激和超声4种处理,并于处理后不同时间分别收集提取家蝇幼虫体内耐热总蛋白,比浊法测定其抗菌活性,经逐步回归分析确定抗菌相关蛋白/多肽。结果表明,4种处理均能诱导家蝇幼虫产生抗菌物质,其中表观分子量为22 kD的蛋白对藤黄微球菌和大肠杆菌均有抗菌作用,50 kD,13 kD,26 kD,7 kD的蛋白抗菌活性具有专一性。还发现一种37 kD的蛋白对抗菌活性有负作用,推测它可能是促进细胞生长的物质。     

Purification and characterization of chlorophenol 4-monooxygenase from Burkholderia cepacia AC1100.

Burkholderia (formerly Pseudomonas) cepacia AC1100 mineralizes the herbicide 2,4,5-trichlorophenoxyacetate (2,4,5-T), and the first intermediate of 2,4,5-T degradation is 2,4,5-trichlorophenol. Chlorophenol 4-monooxygenase activity responsible for 2,4,5-trichlorophenol degradation was detected in the cell extract. The enzyme consisted of two components separated during purification, and both were purified to more than 95% homogeneity. The reconstituted enzyme catalyzed the hydroxylation of several tested chlorophenols with the coconsumption of NADH and oxygen. In addition to chlorophenols, the enzyme also hydroxylated some chloro-p-hydroquinones with the coconsumption of NADH and oxygen. Apparently, the single enzyme was responsible for converting 2,4,5-trichlorophenol to 2,5-dichloro-p-hydroquinone and then to 5-chlorohydroxyquinol (5-chloro-1,2,4-trihydroxybenzene). Component A had a molecular weight of 22,000 and contained flavin adenine dinucleotide. Component A alone catalyzed NADH-dependent cytochrome c reduction, indicating that it had reductase activity. Component B had a molecular weight of 58,000, and no catalytic activity has yet been shown by itself.     

Ethylene-induced Isocoumarin Formation in Carrot Root Tissue

The concentrations of 3-methyl-6-methoxy-8-hydroxy-3,4-dihydroisocoumarin (MMHD) formed in carrot roots inoculated with certain fungi or treated with indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, or 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), were related to the amount of ethylene produced by the root tissue. Ethylene applied exogenously in concentrations above 0.3 ppm induced the formation of MMHD in carrot root discs. Continued production of MMHD required the continued presence of ethylene. The amounts of MMHD in the discs were reduced by CO₂, an inhibitor of ethylene action, and by reduction of the partial pressure of ethylene in fungus-inoculated or 2,4,5-T-treated carrot root discs. The results indicate that ethylene is required for the induction of MMHD formation by carrot root tissue.     

Distribution and effects of 2,4,5-trichlorophenoxyacetic acid in cells of Bacillus megaterium.

Cell death in a resting population of an asporogenous Bacillus megaterium was accelerated by ambient concentrations of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) equal to or greater than 10 mug/ml or 5 mug/mg of cells (dry weight), but only after prolonged exposure. Conversely, populations of growing cells were not markedly influenced even at 100 mug/ml. Effects on cell respiration were not manifest until the ambient concentration reached 1,000 mug of 2,4,5-T/ml, or 500 mug/mg. Cells of B. megaterium did, however, accumulate 2,4,5-T passively to a level approximately twofold above the ambient concentration. Most of the accumulated compound was easily washed from the cells, but, of the firmly bound herbicide, about 0.5 mug/mg of cells (dry weight), nearly 60% by weight, was localized in the protoplast membrane. The foregoing results, obtained with a purified preparation of 2,4,5-T were also elicited by 2,4,5-T analytical standards. The extracted contaminants did not produce the results alone nor did they influence the results when present in combination with 2,4,5-T.     

Glutamic-dehydrogenase activity inhibition by phenoxyacilic acids

Summary Experiments were carried out on the effects of some phenoxyacilic acids, used as herbicides, on the glutamic-dehydrogenase (GLDH) activity. 2,4-D,2,4,5-T, MCPA and Dichlorprop inhibited GLDH activity in a competitive way. Phenoxyacilic acids were ranked in the following order of affinity for the enzyme on the basis of the values of the inhibition constants: 2,4,5-T>2,4-D>MCPA>Dichlorprop; on the basis of chemical structure, it was established that the carboxyl dissociation degree is instrumental in linking the herbicide with the active site of the enzyme.     

Purification and Properties of Component B of 2,4,5-Trichlorophenoxyacetate Oxygenase from Pseudomonas cepacia AC1100

Pseudomonas cepacia AC1100 degrades 2,4,5-trichlorophenoxyacetate (2,4,5-T), an herbicide and chlorinated aromatic compound. Although some progress has been made in understanding 2,4,5-T degradation by AC1100 by molecular analysis, little is known about the biochemistry involved. Enzymatic activity converting 2,4,5-T to 2,4,5-trichlorophenol in the presence of NADH and O(inf2) was detected in cell extracts of AC1100. Phenyl agarose chromatography of the ammonium sulfate-fractionated cell extracts yielded no active single fractions, but the mixing of two fractions, named component A and component B, resulted in the recovery of enzyme activity. Component B was further purified to homogeneity by hydroxyapatite and DEAE chromatographies. Component B had a native molecular weight of 140,000, and it was composed of two 49-kDa (alpha)-subunits and two 24-kDa (beta)-subunits. Component B was red, and its spectrum in the visible region had maxima at 430 and 560 nm (shoulder), whereas upon reduction it had maxima at 420 (shoulder) and 530 nm. Each mole of (alpha)(beta) heterodimer contained 2.9 mol of iron and 2.1 mol of labile sulfide. These properties suggest strong similarities between component B and the terminal oxygenase components of the aromatic ring-hydroxylating dioxygenases. Component A was highly purified but not to homogeneity. The reconstituted 2,4,5-T oxygenase, consisting of components A and B, converted 2,4,5-T quantitatively into 2,4,5-trichlorophenol and glyoxylate with the coconsumption of NADH and O(inf2).     

Two novel insect defensins from larvae of the cupreous chafer, Anomala cuprea: purification, amino acid sequences and antibacterial activity.

A humoral immune response in larvae of the coleopteran insect, Anomala cuprea has been examined for exploring the molecular basis of host-pathogen interactions. The antibacterial activity against the Gram-positive strain, Micrococcus luteus was detected at a low level in absence of injection. The activity increased strikingly in the hemolymph of the larvae challenged with Escherichia coli, showing the fluctuating profile through a time course, which consists of the static induction phase, the production phase rising to a maximum level, and the reduction phase extending over a long duration. Two peptides were purified and characterized by reverse-phase HPLC, Edman degradation and mass spectrometry. They were isoforms, composed of similar sequences with two amino acid substitutions in 43 residues, and novel members of the insect defensins, cysteine-rich antibacterial peptides. Anomala defensins A and B showed potent activity against Gram-positive bacteria, with slight differences in activity against a few strains of tested bacteria. Anomala defensin B was active at high concentration of 40 microM against the Gram-negative strain, Xenorhabdus japonicus, a pathogen toward the host, A. cuprea larvae.     

Raoultella planticola, a new strain degrading 2,4,5-trichlorophenoxyacetic acid

A new strain that degrades the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was isolated from soil, which was exposed to factors related to the petrochemical industry. According to its physiological, biochemical, cultural, and morphological traits, together with the sequence of the 16S rRNA gene, the strain was identified as Raoultella planticola 33-4ch. The strain could consume 2,4,5-T as a sole source of carbon and energy. The amount of 2,4,5-T in the culture medium decreased by 51% after five days of incubation. Raoultella planticola 33-4ch consumes 2,4,5-T to produce 4-chlorophenoxyacetic, phenoxyacetic, and 3-methyl-2,6-dioxo-4-hexenoic acids.     

Antibacterial activity in the hemolymph of myriapods (Arthropoda)

The hemolymphs of two diplopod (Chicobolus sp. and Rhapidostreptus virgator) and two chilopod species (Lithobius forficatus and Scolopendra cingulata) were tested for the presence of antibacterial substances using Petri dish tests. The native hemolymph of all species had substances acting on living Micrococcus luteus, whereas only Rhapidostreptus, Scolopendra, and Lithobius were effective against lyophilized Micrococcus. The antibacterial activity against living Micrococcus increased after inoculation with bacteria (Enterobacter cloacae beta-12) in Chicobolus and Rhapidostreptus and also against lyophilized Micrococcus in the latter. Thus, these effects appear to be inducible. None of the myriapods tested had any bacteriostatic effect on Escherichia coli D-31 whereas the growth of gram-negative E. cloacae was inhibited. The antibacterial substances in the diplopod species were unstable when heated but were resistant to freezing. At least two antibacterial substances (a lysozyme-like one and another substance) are considered to occur in Myriapoda.     

Sequence analysis of a gene cluster involved in metabolism of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia AC1100.

Burkholderia cepacia AC1100 utilizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. PT88 is a chromosomal deletion mutant of B. cepacia AC1100 and is unable to grow on 2,4,5-T. The nucleotide sequence of a 5.5-kb chromosomal fragment from B. cepacia AC1100 which complemented PT88 for growth on 2,4,5-T was determined. The sequence revealed the presence of six open reading frames, designated ORF1 to ORF6. Five polypeptides were produced when this DNA region was under control of the T7 promoter in Escherichia coli; however, no polypeptide was produced from the fourth open reading frame, ORF4. Homology searches of protein sequence databases were performed to determine if the proteins involved in 2,4,5-T metabolism were similar to other biodegradative enzymes. In addition, complementation studies were used to determine which genes were essential for the metabolism of 2,4,5-T. The first gene of the cluster, ORF1, encoded a 37-kDa polypeptide which was essential for complementation of PT88 and showed significant homology to putative trans-chlorodienelactone isomerases. The next gene, ORF2, was necessary for complementation and encoded a 47-kDa protein which showed homology to glutathione reductases. ORF3 was not essential for complementation; however, both the 23-kDa protein encoded by ORF3 and the predicted amino acid sequence of ORF4 showed homology to glutathione S-transferases. ORF5, which encoded an 11-kDa polypeptide, was essential for growth on 2,4,5-T, but the amino acid sequence did not show homology to those of any known proteins. The last gene of the cluster, ORF6, was necessary for complementation of PT88, and the 32-kDa protein encoded by this gene showed homology to catechol and chlorocatechol-1,2-dioxygenases.     

Raoultella planticola, a new strain degrading 2,4,5-trichlorophenoxyacetic acid

A new strain that degrades the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was isolated from soil, which was exposed to factors related to the petrochemical industry. According to its physiological, biochemical, cultural, and morphological traits, together with the sequence of the 16S rRNA gene, the strain was identified as Raoultella planticola 33-4ch. The strain could consume 2,4,5-T as a sole source of carbon and energy. The amount of 2,4,5-T in the culture medium decreased by 51% after five days of incubation. Raoultella planticola 33-4ch consumes 2,4,5-T to produce 4-chlorophenoxyacetic, phenoxyacetic, and 3-methyl-2,6-dioxo-4-hexenoic acids.     

Involvement of apolipophorin III in antibacterial defense of Galleria mellonella larvae

Apolipophorin III (apoLp-III) is an abundant hemolymph protein involved in lipid transport and immune response in insects. We investigated involvement of apoLp-III in the antibacterial response in Galleria mellonella larvae. Immune challenge with Gram-negative (Escherichia coli, Klebsiella pneumoniae) and Gram-positive (Micrococcus luteus) bacteria led to an increase in the level of apoLp-III in G. mellonella hemolymph, 0.5-2h and 8h after treatment, respectively. ApoLp-III purified from larval hemolymph as well as that present in hemolymph extracts adsorbed on the surface of different bacteria. The adsorption capacity of apoLp-III on bacterial cells prompted us to investigate the effect of this phenomenon on bacterial growth. Our results demonstrate antibacterial activity of apoLp-III against selected Gram-positive and Gram-negative bacteria in vitro. Among bacteria tested, Salmonella typhimurium and K. pneumoniae were the most sensitive to apoLp-III. LIVE/DEAD staining of bacteria incubated with purified apoLp-III revealed their growth inhibition; however, neither morphological changes in the cell shape nor formation of cell aggregates was noticed. The results suggest that apoLp-III is a multifunctional protein in G. mellonella hemolymph.