草酸青霉YTY及其突变体解磷能力、pH和分泌有机酸的关系

本研究利用离子束-UV复合诱变草酸青霉YTY选育高效解磷突变体,分析了出发菌株YTY及其突变体解磷过程中的解磷能力、pH和有机酸的变化及其相关性,探讨草酸青霉的解磷机理。结果表明: 离子束-UV复合诱变选育获得5株高效解磷突变株P9-8、P9-9、P15-4、P15-6和P15-7,解磷能力均较YTY提高60%以上。解磷过程中突变株解磷能力及解磷速率均高于YTY,而pH显著低于YTY,同时,突变体分泌有机酸种类及含量发生了不同程度的变化,突变体和YTY均可分泌乳酸、乙酸和草酸,P9-8还产生了柠檬酸。皮尔逊相关分析显示,YTY及5株突变株的解磷能力和pH值呈显著负相关;YTY及其突变体(P15-4除外)的解磷能力与有机酸浓度和pH呈显著相关。分泌有机酸和降低环境pH值可能是草酸青霉解磷的内在机制,离子束-UV复合诱变可引起草酸青霉YTY有机酸分泌种类和分泌量的变化,还可能诱发YTY启动其他H⁺释放途径降低pH参与解磷。本研究为高效解磷青霉的开发及青霉解磷机理阐明提供了生物材料及理论依据。     

农杆菌介导海洋草酸青霉转化体系及聚酮合酶Pks生物学功能*

为研究南海柳珊瑚共附生草酸青霉SCSGAF0023的聚酮合酶(PKS)生物学功能,采用农杆菌介导法构建草酸青霉SCSGAF0023的Pks敲除株ΔPks,比较野生菌株及ΔPks的生长发育及环境适应性差异。以草酸青霉SCSGAF0023分生孢子为受体,p0380-hygB为双元载体,成功实现草酸青霉SCSGAF0023的遗传转化。结果表明:农杆菌浓度为OD₆₀₀=0.5,在200μmol/L 乙酰丁香酮(AS)诱导下与10⁷个/ml草酸青霉SCSGAF0023孢子于25℃共孵育时转化效率最高。基于上述转化体系,成功获得Pks敲除株ΔPks,并首次证实Pks正向调控草酸青霉SCSGAF0023产孢,但不影响其对环境的适应性。这为进一步系统研究真菌PKSs及聚酮化合物对真菌生长发育与环境适应性的影响提供素材。     

A metal-independent hydrolase from a Penicillium oxalicum strain able to use phosphonoacetic acid as the only phosphorus source

A Penicillium oxalicum strain was capable of the phosphate-sensitive utilization of phosphonoacetic acid as the sole source of phosphorus. A carbon-to-phosphorus bond-cleavage enzyme yielding acetic acid and inorganic phosphate was detected and characterized in extracts from cells grown on this phosphonate. Contrary to bacterial phosphonoacetate hydrolases, the fungal enzyme neither required nor was stimulated by divalent cations.     

~(32)P示踪法研究溶磷真菌对磷肥转化固定和有效性的影响

采用³²P示踪技术,研究了溶磷青霉菌P8对肥料磷与土壤有效磷的转化、固定和有效性的影响.结果表明,溶磷青霉菌菌剂能够增加玉米、花生的生物量,促进作物对土壤和肥料磷素的吸收;溶磷菌剂具有防止有效磷转化为难溶Ca₁₀-P的作用,增加有效态磷(Ca₂-³²P、Ca₈-³²P)的比例.随时间延长,施入的³²P转化为Ca₁₀-P的数量(或比例)逐渐增加,但是相对于未接种菌剂处理,接种青霉菌菌剂的土壤磷和肥料磷转化为Ca₁₀-P比例最低.溶磷青霉菌菌剂不仅能够防止有效磷向难溶磷Ca₁₀-P的转化,而且其效果能够维持较长时间.     

A role for carbon catabolite repression in the metabolism of phosphonoacetate by Agromyces fucosus Vs2

A strain of Agromyces fucosus, designated Vs2, metabolized a range of organophosphonate compounds as sole phosphorus sources for growth and metabolized phosphonoacetate as a sole carbon, energy and phosphorus source for growth. With phosphonoacetate as the sole phosphorus source and a pyruvate carbon source, transient phosphate release to the medium was observed, in contrast to cultures grown with glucose and phosphonoacetate, where no phosphate release to the medium was observed. Carbon catabolite repression, specifically by means of inducer exclusion of phosphonoacetate, was proposed as the mechanism responsible, and phosphonoacetate hydrolase enzyme assays carried out on cell extracts confirmed that induced phosphonoacetate hydrolase activities were indeed higher in cells grown on pyruvate with phosphonoacetate as sole phosphorus source. This phenomenon has not previously been demonstrated in vivo, and must represent a significant metabolic control of organophosphonate metabolism. The catabolite repression phenomenon was also evident when A. fucosus grew on 2-aminoethylphosphonate as sole phosphorus source, allowing demonstration of a third mode of control for biodegradation of this compound. Excision of stained zymogram gel pieces, followed by tryptic digestion and mass spectrometric analysis, allowed the identification of phosphonoacetate hydrolase-derived peptides.     

Cytosolic CP bond cleavage activity in bacterial cells isolated from soil

Three kinds of bacteria (CP1, CP9 and CP10), able to accumulate inorganic phosphate (Pi) in a growth medium containing phosphonoacetate as a sole source of phosphorus, were isolated from two hundred soil samples. CP bond cleavage activity in these strains was determined using extracts prepared from cells grown on a medium containing phosphonoacetate. The activity was not found in cell extracts of CP1. Cell extracts prepared from CP9 catalyzed the liberation of Pi only from phosphonoacetate and 2-aminoethylphosphonate. The cell size of CP10 was abnormally large compared with that of CP1 and CP9, and the extracts of CP10 catalyzed the cleavage of CP bonds in methylphosphonate, phosphonoacetate, phenylphosphonate, 2-amino-ethylphosphonate, 2-amino-4-phosphonobutyrate, glyphosate and in phosphonomycine.     

A comparison of three bacterial phosphonoacetate hydrolases from different environmental sources

Cleavage of the carbon–phosphorus bond of the xenobiotic phosphonoacetate by phosphonoacetate hydrolase represents a novel route for the microbial metabolism of organophosphonates, and is unique in that it is substrate-inducible and its expression is independent of the phosphate status of the cell. The enzyme has previously only been demonstrated in cell extracts of Pseudomonas fluorescens 23F. Phosphonoacetate hydrolase activity is now reported in extracts of environmental Curtobacterium sp. and Pseudomonas sp. isolates capable of the phosphate-insensitive mineralization of phosphonoacetate as the sole source of carbon, energy and phosphorus at concentrations up to 40 mmol l⁻¹ and 100 mmol l⁻¹, respectively. The enzymes in both strains were similarly inducible by phosphonoacetate and had a unique specificity for this substrate. However, they differed significantly from each other, and from the previously described Ps. fluorescens 23F enzyme, in respect of their apparent molecular masses, temperature optima, thermostability, sensitivity to inhibition by chelating agents and by structural analogues of phosphonoacetate, and in their affinities for the substrate.     

纤维二糖水解酶N-糖基化对其在草酸青霉中的分泌和酶活影响*

目的:纤维素酶水解天然纤维素产生易被微生物利用的葡萄糖是进行生物炼制的关键。丝状真菌分泌的纤维素酶大多数是经过糖基化修饰的,研究丝状真菌纤维二糖水解酶(Cel7A)的催化功能域N-糖基化修饰对其分泌及酶活的影响,有助于优化纤维素酶的表达。方法:利用定点突变将草酸青霉和深绿木霉Cel7A催化功能域的N-糖基化位点去除,构建突变体PoCel7A^*和TaCel7A^*。以草酸青霉为宿主构建分泌表达PoCel7A^*、TaCel7A和TaCel7A^*的重组菌,检测N-糖基化去除对Cel7A分泌和酶活力的影响。结果:PoCel7A催化功能域的N-糖基化去除对其蛋白分泌和酶活力无影响。TaCel7A催化功能域的N-糖基化去除不影响其蛋白分泌;但突变体的pNPCase、FPase和Avicelase酶活力分别下降了21.2%,15.2%和17.6%。去除Cel7A催化功能域N-糖基化,加强了细胞内UPR响应。外源蛋白TaCel7A和TaCel7A^*的表达也加强了胞内UPR响应。结论:不仅可以为丝状真菌Cel7A的酶工程改造提供理性设计思路,而且为进一步了解糖基化在纤维素酶降解纤维素过程中的作用及机理奠定一定基础。     

一株高效溶磷伯克霍尔德菌的筛选鉴定及对马尾松幼苗的促生作用

采用标准稀释平板法从马尾松根际土中分离溶磷细菌,利用钼锑抗比色法测定溶磷细菌的溶磷特性;通过分析溶磷菌的溶磷能力与发酵液pH的关系,以及液相色谱-质谱 (HPLC-MS)联用对发酵液中有机酸的测定,探究其溶磷机制;通过对接种溶磷菌马尾松盆栽苗生长、生理、土壤养分和土壤酶活性的测定,明确溶磷菌对马尾松生长和生理的影响。结果表明: 由马尾松根际土壤中共筛选到溶磷细菌16株,其中菌株WJ27溶磷效果最优,液体培养5 d时的溶磷量达411.98 mg·L^-1。经过表型观察、生理生化鉴定和系统发育树分析,发现菌株WJ27属于伯克霍尔德菌属;其对不同磷源的溶磷特性存在差异,溶磷能力依次为: Ca₃(PO₄)₂(220.85 mg·L^-1)＞AlPO₄(182.33 mg·L^-1)＞FePO₄·2H₂O(129.19 mg·L^-1)＞CaHPO₄·2H₂O (115.23 mg·L^-1)。胞外有机酸测定结果表明,该菌株通过分泌柠檬酸、丙二酸等有机酸降低发酵液中pH,进而发挥溶磷作用;盆栽试验结果表明,接种菌株WJ27对马尾松幼苗生长、生理、土壤养分和土壤酶活性有积极作用。与对照相比,接种WJ27的马尾松的苗高、主根长、侧根数量、地上部(茎、枝、叶)鲜重、干重和根系鲜重、干重分别增加了14.3%、36.9%、56.1%、44.7%、60.0%、158.3%和100.0%;叶绿素b、总叶绿素、地上部可溶性蛋白和可溶性糖、根系活力和根系可溶性蛋白分别增加了145.8%、45.2%、206.3%、59.4%、80.5%和260.0%;根系超氧化物歧化酶、过氧化物酶和地上部过氧化氢酶的活性分别提高了71.2%、197.5%和36.6%;根际土壤中速效氮、速效钾、速效磷含量和土壤脲酶、过氧化氢酶、磷酸酶活性分别较对照土壤显著增加18.1%、17.0%、11.9%和34.3%、45.5%、62.6%。说明接种WJ27可以改善土壤养分和土壤酶活性,进而促进马尾松幼苗的生长。     

Detection of a novel carbon-phosphorus bond cleavage activity in cell-free extracts of an environmental Pseudomonas fluorescens isolate.

Cell-free extracts of Pseudomonas fluorescens strain 23F catalyzed the hydrolysis of phosphonoacetate to acetate and inorganic phosphate; the products were detected in almost equimolar quantities. The stable in vitro activity responsible was distinct from phosphonoacetaldehyde hydrolase and appears to represent a novel mode of carbon-phosphorus bond cleavage.     

Phosphate-independent utilization of phosphonoacetic acid as sole phosphorus source by a psychrophilic strain of Geomyces pannorum P15

A psychrophilic fungal strain of Geomyces pannorum P15 was screened for its ability to utilize a range of synthetic and natural organophosphonate compounds as the sole source of phosphorus, nitrogen, or carbon. Only phosphonoacetic acid served as a phosphorus source for microbial growth in phosphate-independent manner. Substrate metabolism did not lead to extracellular release of inorganic phosphate. No phosphonate metabolizing enzyme activity was detectable in cell-free extracts prepared from Geomyces biomass pregrown on 2 mmol/L phosphonoacetic acid.     

Detection of phosphonoacetate degradation and phnA genes in soil bacteria from distinct geographical origins suggest its possible biogenic origin

Phosphonoacetate is regarded as an antiviral xenobiotic whose mineralization can be catalysed by an enzyme, phosphonoacetate hydrolase, encoded by the phnA gene. To date the enzyme's activity has been detected in only a limited number of bacteria. Its expression has been shown to occur in a manner independent of the phosphate status of the cell, in direct contrast to the general rule of organophosphonate metabolism being under the control of the pho regulon. In this study the environmental occurrence of the phnA gene was evaluated by polymerase chain reaction amplification of DNA extracts obtained directly from various soil environments. Sensitivity of this method was improved such that a positive result was routinely obtained with soil spiked with as few as 6 colony-forming units (cfu) per gram of soil of Pseudomonas fluorescens 23F (phnA(+)). When total DNA from a variety of Northern Irish, Greek and Bolivian soils was tested, all were positive for phnA. Bacteria capable of utilizing phosphonoacetate as sole carbon, energy and phosphorus source, with the release of essentially equimolar concentrations of phosphate to the culture supernatant, were isolated from all soil samples tested. Analysis of three such isolates revealed all to be species of Pseudomonas sensu stricto, possessing phosphonoacetate hydrolase activity in cell-free extracts. Sequence determination of the phnA gene revealed a similarity of the putative protein sequences at levels of 98.3-99.3% between the Pseudomonas strains. This is the first study to use molecular methods to investigate the distribution of a gene encoding organophosphonate metabolism, and indicates that the phnA gene is ubiquitous within soils from geographically distinct regions. Such an observation supports the proposition that phosphonoacetate is a compound that may also have a biogenic origin.     

磷酸氢二铵对糙皮侧耳菌丝生长及基质降解酶活性的影响

氮源种类和浓度均会对糙皮侧耳Pleurotus ostreatus菌丝长势、生长速率以及代谢产生影响,而氮匮乏与氮过量积累都会对糙皮侧耳的生长产生不利影响。为了研究氮源对糙皮侧耳菌丝生长和基质降解酶活性的影响,本研究选取磷酸氢二铵[(NH4)2HPO4]作为唯一氮源,通过在培养基中添加不同浓度的磷酸氢二铵检测糙皮侧耳菌丝生长速率和木质纤维素降解酶的活性,筛选出糙皮侧耳生长的最适磷酸氢二铵浓度。结果表明,当磷酸氢二铵的添加浓度区间为10–20 mmol/L时菌丝长势最好。以缺氮时的酶活作为对照,当磷酸氢二铵的添加浓度为10 mmol/L时,羧甲基纤维素酶的活力最强,显著高于对照组(P<0.05);当添加浓度为5 mmol/L时,滤纸纤维素酶的活力最强,显著高于对照组(P<0.05),而10 mmol/L浓度下的酶活力比5 mmol/L时稍有下降,但二者之间无显著性差异(P>0.05);当添加浓度为40 mmol/L时,木聚糖酶的活力最强,显著高于对照组(P<0.05);当添加浓度为10mmol/L时,漆酶的活力最强,显著高于对照组(P<0.05)。基质降解酶...     

In vitro characterization of a phosphate starvation-independent carbon-phosphorus bond cleavage activity in Pseudomonas fluorescens 23F.

A novel, metal-dependent, carbon-phosphorus bond cleavage activity, provisionally named phosphonoacetate hydrolase, was detected in crude extracts of Pseudomonas fluorescens 23F, an environmental isolate able to utilize phosphonoacetate as the sole carbon and phosphorus source. The activity showed unique specificity toward this substrate; its organic product, acetate, was apparently metabolized by the glyoxylate cycle enzymes of the host cell. Unlike phosphonatase, which was also detected in crude extracts of P. fluorescens 23F, phosphonoacetate hydrolase was inducible only in the presence of its sole substrate and did not require phosphate starvation.     

Plant growth-promoting Pseudomonas bearing catabolic plasmids: Naphthalene degradation and effect on plants

Two IncP-9 naphthalene degradative plasmids pOV17 and pBS216 were transferred into plant growth-promoting Pseudomonas which were represented by species P. aureofaciens, P. chlororaphis, P. fluorescens, and P. putida. The strains with the same plasmid differed significantly by their growth parameters, stability of the plasmid and plant protective effect from naphthalene action. Strains P. putida 53a(pOV17) and P. chlororaphis PCL1391(pOV17) demonstrated higher population number in the rhizosphere. Moreover, they protected the mustard plants from naphthalene toxic influence more effectively than the wild type strain P. aureofaciens OV17(pOV17). The activity of catechol-2,3-dioxygenase in the strains with the plasmid pOV17 was higher than that in strains with the plasmid pBS216. The strain P. putida 53a(pOV17) with high catechol-2,3-dioxygenase activity has been demonstrated to have the best protective effect. The strain P. putida 53a(pBS216) without catechol dioxygenases activities did not have protective effect but suppressed the plant germination.     

Effect of mannan oligosaccharide elicitor and ferulic acid on enhancement of laccases production in liquid cultures of basidiomycetes

The effects of mannan oligosaccharides preparation (MO), as elicitor, and ferulic acid inducer for enhancement in laccases production in liquid cultures of three strains of basidiomycetes, Pycnoporus sanguineus, Coriolopsis polyzona and Pleurotus ostreatus was studied using a full factorial 3² experimental design. MO, either individually or combined with ferulic acid, enhanced laccases levels in the three different strains of the white-rot fungi. The enhancement of laccases production was species specific with the highest increase in liquid cultures of P. sanguineus (88-fold) followed by P. ostreatus (3-fold) and C. polyzona (2-fold). Separate additions of 75 mg/l of MO to the cultures of P. sanguineus and P. ostreatus caused the increase while a combined addition of 150 mg/l of MO and 1 mM ferulic acid resulted in the optimal production of laccases in the cultures of C. polyzona.     

青海岩羊源小反刍兽疫病毒N、F基因的遗传进化分析

2021年1月19日,青海省海西州都兰县巴隆乡伊克高里村发生岩羊不明原因的死亡,表现为离群独处、卧地不起、体质虚弱、觅食困难、肛门周围黑色附着物等现象。通过临床症状、病理学解剖及实时荧光RT-PCR诊断,为小反刍兽疫病毒(PPRV)感染。采用RT-PCR技术从病死岩羊病料组织中扩增出了PPRV N、F基因的部分片段。采用MegAlig、NT1和MEGA6.0软件对岩羊PPRV株N、F基因序列进行了比对和分析,绘制系统进化树,结果显示：此次岩羊感染的PPRV株N、F基因片段与新疆株(China/Xinjiang/2015/16)序列片段的同源性分别为99.43%和99.73%。遗传进化分析,该病原属于基因Ⅳ系。在N、F基因核酸序列水平上,与国内新疆地区分离毒株亲缘关系最近,同在一个小的分支;与国外毒株相比,N基因与塞内加尔和尼日利亚分离毒株亲缘关系较远,F基因与国外分离毒株亲缘关系较远。综上所述,青海岩羊源PPRV株属于基因Ⅳ系,与当前我国流行的野毒株属于同一个谱系。     

红花尔基樟子松优良抗旱菌树组合的筛选

为筛选红花尔基樟子松（Pinus sylvestris var.mongolica）优良抗旱菌树组合,采用樟子松林下5个优势外生菌根真菌菌株为接种体,分别对5个月龄樟子松实生苗进行人工接种,接种8个月后观察菌根侵染情况及菌根形态,并在非干旱胁迫和干旱胁迫条件下测定不同菌树组合的生长和生理生化指标。结果表明：5个乡土菌种均能成功侵染樟子松并形成典型的外生菌根;干旱胁迫下,菌根化樟子松的各项生长指标均显著高于对照（P<0.05）,且接种粘盖牛肝菌（Suillus bovinus）具有最高的菌根侵染率、苗高、地上及地下生物量和根茎比;外生菌根共生体可通过提高植物SOD活性与POD活性,同时降低MDA含量来提高樟子松对干旱的耐受力;干旱胁迫下,所有接种处理苗木的萎蔫时间较对照处理均推迟,推迟时间最长的是粘盖牛肝菌接种处理,较对照推迟96.3 h;另外,接种处理均能显著延长宿主临界致死时间,尤其是接种粘盖牛肝菌可延长113.8 h。因此,可以认为粘盖牛肝菌与樟子松是一个较为理想的抗旱菌树组合。     

RT-TGGE as a guide for the successful isolation of phosphonoacetate degrading bacteria

AIMS: Use of molecular techniques for the isolation of bacteria capable of phosphonoacetate mineralization as carbon, phosphorus and energy source. METHODS AND RESULTS: RNA extracts obtained at three different stages of an enrichment selecting for phosphonoacetate degrading bacteria were reverse transcribed using 16S rRNA-specific primers, amplified and analysed by temperature gradient gel electrophoresis (TGGE). This information was used to devise a strategy for the isolation of members of the enrichment that were otherwise difficult to obtain in pure culture. We were able to pull out, in total, four out of the six main microbial cultures that were detected by TGGE. Two of the isolates belonging to Mycobacterium and Agromyces genera were for the first time shown to grow in the presence of phosphonoacetate as sole carbon, phosphorus and energy source releasing almost equimolar levels of inorganic phosphate into the culture medium, and they were shown to exhibit phosphonoacetate hydrolase activity in vitro. CONCLUSIONS: The ubiquity of pseudomonad in degradation processes is more likely a consequence of our ignorance of bacterial requirements and physiology, rather than their possession of unique metabolic properties. SIGNIFICANCE AND IMPACT OF THE STUDY: RT-TGGE analysis can be used to guide the successful isolation of micro-organisms difficult to obtain by culture-dependent methods alone.