芽孢杆菌WL911促低温下小麦生长效应及其功能基因分析

【背景】青藏高原特殊生境孕育着强耐逆性的微生物资源。【目的】探究青藏高原特殊生境的芽孢杆菌对小麦的促生效应。【方法】以分离自青海省海西蒙古族藏族自治州乌兰县的红柳（Tamarix ramosissima）根围菌株WL911为研究对象进行16S rRNA基因及gyrB基因鉴定;测定其低温适生性;以WL911菌悬液（浓度为1×10⁷ cfu/mL）对4℃条件下的小麦（Triticum aestivum）品种“青麦7号”幼苗进行灌根处理,测定其对小麦幼苗的促生效应;采用二代测序平台Illumina HiSeq×10对菌株WL911进行全基因组测序、功能注释及相关功能基因分析。【结果】菌株WL911鉴定为贝莱斯芽孢杆菌（Bacillus velezensis）,可耐受4℃低温;菌株WL911对“青麦7号”幼苗生长具有显著的促生效果,26℃时小麦株高、根长和鲜重分别提高12.22%、50.12%和70.99%;4℃条件下小麦生物量提高的同时,叶绿素含量提高19.72%,H₂O₂、丙二醛（malondialdehyd,MDA）的积累量分别下降23.50%、40.68%,超氧化物歧化酶（superoxide dismutase,SOD）、过氧化物酶（peroxidase,POD）和过氧化氢酶（catalase,CAT）活性分别提高45.82%、17.35%和14.18%;WL911基因组大小为3 915 549 bp,G+C百分含量为46.47%;生物学功能gene ontology （GO）注释到的基因占基因总量的75.74%,包括促生相关功能基因glnB、yclQ和fetB等,可参与合成生长激素、营养元素等途径;存在mnhA、mnhE、pheT、pheS和proV等关键基因参与Na⁺外排机制、编码脯氨酸和酚类化合物等反应;也存在编码冷休克蛋白CspC、CspB和CspD的关键基因cspA。【结论】B.velezensis WL911（登录号为OP874802）是一种优质的生物肥料研发菌株。     

具有ACC脱氨酶活性的绞股蓝内生细菌的分离鉴定及其抑菌促生作用

采用富集筛选法从绞股蓝根中筛选得到6株具有ACC脱氨活性的细菌,其中菌株JDG-6、JDG-7、JDG-14、JDG-16、JDG-23均具有较强的分泌铁载体能力,但菌株JDG-32没有产铁载体能力。抑菌试验结果显示,菌株JDG-6、JDG-7、JDG-14和JDG16对一种或多种供试菌有抑菌作用,其中菌株JDG-14能抑制大肠埃希菌、藤黄八叠球菌和白色念球菌的生长。促生试验表明,菌株JDG-6、JDG-7和JDG-14均能促进水稻幼苗根的伸长,其中菌株JDG-14的促生作用最为明显,与对照组相比水稻幼苗的根长和根鲜重分别增长了26%和21%。通过形态特征观察、生理生化试验以及16S rDNA序列分析,菌株JDG-6、JDG-7、JDG-16和JDG-23属于假单胞杆菌属（Pseudomonas）,菌株JDG-14为木糖葡萄球菌（Staphylococcus xylosus）,而菌株JDG-32为枯草芽胞杆菌（Bacillus subtilis）。菌株JDG-6、JDG-7和JDG-14均具有ACC脱氨酶活性和抑菌活性的促生菌,具有农业应用潜力。     

苯唑西林敏感、mecA基因阳性的猪源金黄色葡萄球菌的流行、分子分型及耐药性研究

[目的] 为了解我国猪源苯唑西林敏感-mecA阳性金黄色葡萄球菌（Oxacillin-susceptible,mecA-positive Staphylococcus aureus,OS-MRSA）的流行情况、菌株分子特征及耐药性,本研究对我国中西部4个省份（甘肃、陕西、河南和广西）的9个规模化养猪场进行鼻腔拭子样本采集。[方法] 运用PCR扩增nuc和mecA基因及苯唑西林耐药性检测对OS-MRSA菌株进行分离鉴定。然后对分离所得的OS-MRSA菌株进行26种毒素编码基因、16种抗生素耐药性以及spa、MLST和SCCmec分型检测。[结果] 结果表明,采集的884份样本中,67份样本7.6%（67/884）分离到金黄色葡萄球菌,包括50株甲氧西林敏感菌株（Methicillin-sensitive Staphylococcus aureus,MSSA）、8株苯唑西林耐受-mecA阳性金黄色葡萄球菌（Oxacillin-resistant mecA-positive,OR-MRSA）和9株OS-MRSA菌株。26种被检毒素编码基因中有9种毒素编码基因被检出,其中hla基因检出率最高,其次为hld、hlb、hlg、sei、sem、seg、sen和seo。此外,67株分离株中仅有16株携带肠毒素编码基因,其中OR-MRSA和OS-MRSA菌株分别占37.5%（6/16）和50.0%（8/16）,且携带毒素编码基因的菌株克隆型均为ST9-t899。16种所测试抗生素中,菌株对12种抗生素表现为耐药,其中MSSA、OR-MRSA和OS-MRSA分离株分别主要对1-8、10-12和7-11种抗生素耐药。所有分离株共有4种克隆型ST398-t571、ST9-t899、ST398-t034和t11241,其中ST9-t899为MRSA菌株唯一克隆型和ST398-t571为MSSA优势克隆型。除4株分离株未检测到SCCmec分型外,IV_b（76.5%,13/17）是MRSA分离株的唯一分型。[结论] 结果表明,我国猪源MRSA分离株对苯唑西林药物敏感性发生了改变,出现了较多的苯唑西林敏感菌株。此外,MSSA和MRSA分离株优势克隆型分别为ST398-t571和ST9-IVb-t899。研究还发现,克隆型与毒素编码基因有显著相关性,携带毒素编码基因的菌株克隆型均为ST9-t899。通过了解我国猪源MSSA、OR-MRSA和OS-MRSA的流行、分子特征和耐药性,可以为我国猪源金黄色葡萄球菌的防控提供数据支持。     

水产源芽胞杆菌的分离鉴定及生物学功能分析

筛选具有较好生物学功能的芽胞杆菌(Bacillus),用以改善池塘养殖过程饲料等有机物降解、抑制水体中的病原菌,对从健康养殖鱼虾塘水体中分离的菌株,进行生化试验和16S rDNA序列分子鉴定;通过产酶、耐酸、耐高温试验,抑菌试验以及安全性试验研究分离菌株的生物学功能。试验共筛选出8株细菌,经生化试验及16S rDNA序列的分子鉴定,确定菌株ZHX17、ZHX18、ZHX31为贝莱斯芽胞杆菌（Bacillus velezensis）,菌株ZHX14、ZHX15为地衣芽胞杆菌（Bacillus licheniformis）、菌株NSX4、NSX7、NSX9为枯草芽胞杆菌（Bacillus subtilis）。试验结果表明,8株芽胞杆菌均具有较强的耐酸、耐高温特性,其中3株贝莱斯芽胞杆菌具有较强的分泌淀粉酶、纤维素酶、蛋白酶的能力,抑菌效果好于其他5株芽胞杆菌。安全性试验结果表明,8株芽胞杆菌对草鱼、罗非鱼相对安全。8株芽胞杆菌同时具备分泌淀粉酶、纤维素酶和蛋白酶3种胞外酶的能力,其中贝莱斯芽胞杆菌具有较强的病原菌抑菌能力,可以作为病原拮抗益生菌做进一步研究。     

红树林土壤中产聚羟基脂肪酸酯细菌的分离及其评估

[背景] 细菌能通过合成聚羟基脂肪酸酯（Polyhydroxyalknoates,PHA）在细胞内储存物质和能量,提高对环境的适应能力。在红树林中,由于土壤周期性受海水浸没,形成营养物质种类丰富和含量波动大的特殊生境,为细菌进化出特殊的PHA合成途径提供了条件。[目标] 为了增加对红树林产PHA细菌资源的了解,获得产PHA细菌,使用纯培养方法分离和鉴定细菌,并评估菌株的产PHA能力。[方法] 采集红树植物海桑根系和红树滩涂土壤样品,连续5周培养、分离纯化获得细菌菌株;通过16S rRNA基因相似性及系统进化分析鉴定细菌分类地位,利用PHA合成酶基因（phaC）鉴定细菌合成PHA的能力;通过基因组草图测序,分析细菌的phaC基因种类、代谢通路及系统进化关系;通过气相色谱分析细菌产PHA的累积量及组成。[结果] 从红树林土壤样品中分离得到97株细菌,其中13株带有phaC基因,包括坚强芽孢杆菌（Cytobacillus firmus）、弯曲芽孢杆菌（Bacillus flexus）、除烃海杆菌（Marinobacter hydrocarbonoclasticus）和酯香微杆菌（Microbacterium esteraromaticum）。B. flexus MN15-19以丙酮酸盐为碳源,可累积细胞干重11%的PHA,同时具有固碳功能的还原性三羧酸循环通路,有开发成为固碳产PHA工程菌株的潜力。酯香微杆菌可产PHA,但是其phaC基因结构特殊,基因组注释未能识别出任何已知phaC基因。[结论] 研究发现红树林土壤可培养细菌中存在未知的PHA合成途径,说明红树林生态系统中的细菌具有资源挖掘的重要价值。     

一株高效甘蔗内生固氮细菌GXS16的鉴定及其对甘蔗的促生长作用

[背景] 我国甘蔗生产中氮肥过量施用严重,导致生产成本居高不下,充分发挥甘蔗与内生固氮菌的联合固氮作用,减少氮肥施用量,对促进我国甘蔗产业可持续发展具有重要意义。[目的] 筛选优势甘蔗内生固氮菌,对其基本特性、联合固氮效率及促生长功能进行评价。[方法] 从甘蔗根系分离到一株内生固氮菌GXS16,利用乙炔还原法测定固氮酶活性,通过PCR扩增nifH基因确定菌株为固氮菌;通过形态观察、Biolog检测和16S rRNA基因序列分析等对菌株进行分类;通过接种盆栽甘蔗检测菌株的促生长作用,采用¹⁵N同位素稀释法检测菌株相对固氮效率。[结果] 菌株GXS16固氮酶活性为2.42μmol-C₂H₄/（h·mL）,根据菌株培养性状和菌体形态观察、Biolog检测、16S rRNA、nifH、acdS基因序列分析结果,菌株GXS16属于伯克氏菌属（Burkholderia）;菌株GXS16还具有1-氨基环丙烷-1-羧酸脱氨酶（1-Aminocyclopropane-1-Carboxylate Deaminase,ACC）活性及合成生长素吲哚乙酸（Indoleacetic Acid,IAA）、降解无机磷的功能;接种GXS16处理甘蔗植株的株高比对照增长15%以上,干重增长20%以上,¹⁵N同位素测定显示甘蔗根、茎、叶从空气中获得氮的百分比分别为7.69%、15.64%和8.72%,效率显著优于模式菌株G.diazotrophicus PAL5。[结论] Burkholderia sp.GXS16是一株高效甘蔗内生固氮菌,具有良好应用前景。     

多株功能菌种混合培养条件的响应面优化及其生长关系

对3种具有土壤中多种重金属污染修复和病虫害防治功能的光合细菌（Photosynthetic bacteria,PSB,包括Rhodobacter sphaeroides H菌株、Rhodopseudomonas palustris N菌株和Rhodospirillium rubrum M菌株）、枯草芽胞杆菌（Bacillus subtilis, BS）和植物乳酸杆菌（Lactobacillus plantarum, LP）进行混合培养,利用单因素和响应面试验优化培养基配方以及培养条件。确定功能菌混合培养的最佳条件：以光合细菌培养基为基础培养基,外加碳源为10 g/L蔗糖、氮源为1.25 g/L硫酸铵、培养温度为31.5 ℃、初始pH值为7.1、接种量为接种后的初始培养活菌数1.90×10⁹cfu/mL、培养时间为28.0 h。通过功能菌的加性模型法、生长动力学以及混合培养的生长关系分析表明,光合细菌、枯草芽胞杆菌和植物乳酸杆菌的生长均存在互惠互利关系。     

基于基因组的一株土壤固氮菌分离菌株鉴定及其促生作用

[目的] 为获得高效固氮菌株,充分研究利用土壤固氮菌资源。[方法] 选取固氮能力较高的紫色土发育水稻土,采用富集纯化法分离固氮微生物菌株。通过16S rRNA基因系统发育分析和全基因组相关指数比较对新分离菌株进行物种鉴定。采用乙炔还原法和¹⁵N₂示踪法定量测定新分离菌株的固氮能力,通过培养特性和接种效果初步研究固氮菌株的促生作用。[结果] 从紫色土发育水稻土中分离得到1株可在无氮培养基上快速生长的菌株P208。基于16S rRNA基因和基因组92个核心基因的系统发育分析结果表明,新分离菌株P208与Azotobacter chroococcum IAM 12666^T（=ATCC 9043^T）系统发育距离最近（16S rRNA基因相似度为99.79%）。菌株P208与A.chroococcum ATCC 9043^T的基因组平均核苷酸一致性（ANI）、平均氨基酸一致性（AAI）和数字DNA-DNA杂交值（dDDH）高于物种分类阈值（ANI>95%-96%,AAI>95%-96%,dDDH>70%）,最大唯一匹配指数（MUMi）低于物种分类阈值（<0.33）,得出新分离菌株P208为褐球固氮菌（A.chroococcum）。A.chroococcum P208固氮活性为模式菌株A.chroococcum ATCC 9043^T的2.61倍。除固氮能力外,A.chroococcum P208具有IAA生成、溶磷活性和铁载体生成等促进植物生长潜力的培养特性,室内培养条件下接种A.chroococcum P208能够促进水稻、小麦幼苗根系的生长。[结论] 从固氮能力较强的水稻土中分离纯化得到1株具有较强固氮、促生潜力的固氮菌,具有潜在的开发应用价值,可为研究利用生物固氮提供微生物资源。     

基于16S rRNA基因高通量测序研究狞猫肠道微生物多样性

[目的]研究狞猫肠道微生物多样性特征。[方法]对7只健康成年野生狞猫（2只雄性,5只雌性;2只来自济南野生动物园,5只来自威海野生动物园）粪便微生物16S rRNA基因V3-V4区进行高通量测序,对狞猫肠道微生物多样性进行研究。[结果] 7只狞猫共获得肠道微生物16S rRNA基因V3-V4区有效序列1458741条,平均208392条,序列平均长度433 bp。通过以97%的序列相似性进行分类,获得操作分类单元（OTU）平均 233个。经序列比对和分类鉴定,这些OTU都属于细菌域,包括13个门,26个纲,43个目,75个科,119个属。其中,丰度最高的细菌门是厚壁菌门（Firmicutes,平均占61.7%）、放线菌门（Actinobacteria,12.42%）、拟杆菌门（Bacteroidetes,7.79%）、梭杆菌门（Fusobacteroidetes,7.79%）和变形菌门（Proteobacteria,7.53%）。丰度最高的科依次是消化链球菌科（Peptostreptococcaceae,平均占16.15%）,梭菌科I（Clostridiaceae_I,14.78%）,毛螺菌科（Lachnospiraceae,13.13%）,红蝽杆菌科（Coriobacteriaceae,12.31%）等。丰度最高的属是柯林斯氏菌属（Collinsella,11.44%）,艰难梭菌属（Peptoclostridium,10.91%）,狭窄梭菌属1（Clostridium_sensu_stricto_1,10.3%）,拟杆菌属（Bacteroides,7.41%）,消化链球菌属（Peptostreptococcus,5.21%）等。7只狞猫的肠道微生物中有平均15.35%的OTU没有归类到属。样本间聚类分析结果表明,来自同一动物园的样本聚为一支。[结论]本文通过高通量测序技术研究了狞猫肠道微生物的组成和多样性特征,为狞猫的救护饲养和消化生理学研究提供基础数据。     

过表达基因elo1和ole1增强光滑球拟酵母Δmed15B菌株的低pH耐受能力

[目的] 研究中介体亚基Med15B（ORF CAGL0H06215g）介导的脂肪酸代谢影响光滑球拟酵母（Candida glabrata）耐受低pH胁迫的生理机制。[方法] 在菌株Δmed15B中过量表达脂肪酸延伸酶基因elo1（ORF CAGL0L08184g）和Δ9去饱和酶基因ole1（ORF CAGL0I00418g）,构建过表达菌株Δmed15B/elo1-ole1,然后与菌株Δmed15B和亲本菌株ATCC55对比分析基因elo1和ole1的表达水平、脂肪酸组分比例、细胞膜完整性和耐受能力在pH 2.0和pH 6.0条件下的差异。[结果] 发现,在pH 2.0条件下过表达菌株Δmed15B/elo1-ole1：（1）基因elo1和ole1表达水平比菌株Δmed15B分别上调了4.1倍和3.3倍,与亲本菌株ATCC55相比分别上调了2.5倍和2.2倍;（2）脂肪酸平均链长延长至17.4,高于菌株Δmed15B的16.8和亲本菌株ATCC55的17.1;不饱和脂肪酸与饱和脂肪酸的比值比菌株Δmed15B和亲本菌株ATCC55分别提高了53.1%和41.5%;（3）碘化丙啶（PI）染色细胞数占总检测细胞数的比例,与菌株Δmed15B和亲本菌株ATCC55比较分别下降了60.8%和37.7%;（4）细胞半抑制pH（胁迫）（IC₅₀）值达到pH 2.3,比菌株Δmed15B的pH 3.7和亲本菌株ATCC55的pH 3.1具有更强的耐受能力。[结论] 在菌株Δmed15B中过表达基因elo1和ole1能通过提高长链脂肪酸和不饱和脂肪酸含量,增强细胞对低pH胁迫的耐受能力。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

Interconnection of parameters of regulation system of lipid peroxidation and of morphophysiological parameters of mouse liver

A complex analysis of seasonal fluctuations of the mean group parameters of the system of regulation of lipid peroxidation has been performed in liver of Balb/c mice. Association of lipid characteristics and morphophysiological parameters is studied in the Balb/c mouse liver. An inter-connection is revealed between the liver index and the amount of lysoforms of phospholipids, the scale and character of the interconnection differing essentially depending on proportion of phos-phatidylcholine in mouse liver phospholipids.     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate, N-(β-acetoxyethyl) piperidinium iodomethylate (“piperidylcholine”), and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic hydrolysis parameters—activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholinesterase substrate—N-(β-acetoxyethyl) piperidinium.     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation     

Modulation of allostery of pyruvate kinase by shifting of an ensemble of microstates

Since the introduction of the concepts of allostery about four decades ago, much advancement has been made in elucidating the structure-function correlation in allostery. However, there are still a number of issues that remain unresolved. In this review we used mammalian pyruvate kinase (PK) as a model system to understand the role of protein dynamics in modulating cooperativity. PK has a triosephosphate isomerase (TIM)(α/β)₈ barrel structural motif. PK is an ideal system to address basic questions regarding regulatory mechanisms about this common (α/β)₈ structural motif. The simplest model accounting for all of the solution thermodynamic and kinetic data on ligand-enzyme interactions involves two conformational states, inactive E^T and active E^R. These conformational states are represented by domain movements. Further studies provide the first evidence for a differential effect of ligand binding on the dynamics of the structural elements, not major secondary structural changes. These data are consistent with our model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the inactive E^T and active E^R represent the two extreme end states. Sequence differences and ligands can modulate the distribution of states leading to alterations of functions. The future work includes: defining the network of functionally connected residues; elucidating the chemical principles governing the sequence differences which affect functions; and probing the nature of mutations on the stability of the secondary structural elements, which in turn modulate allostery.