温度和湿度对高寒草甸凋落物分解的影响

多数研究发现增温增湿加快了凋落物失重率,但对如何影响凋落物分解过程中CO₂和可溶性有机碳（DOC）释放的影响研究较少。通过室内培养设置四个温度梯度（0,5,10和20℃）和两个湿度梯度（25%和40%）对高寒草甸凋落物分解进行了96 d的培养试验。结果表明,总体上高寒草甸凋落物分解速率随着温度和湿度的增加而加快;高湿度条件下凋落物分解释放CO₂总量的敏感性是低湿度的2.3倍左右;湿度变化对DOC淋溶的温度敏感性影响较小。在25%和40%的湿度培养下,CO₂总排放量的温度敏感性分别是DOC总淋溶量的温度敏感性的10和20倍左右,表明未来气候变化情境下凋落物中的有机质分解更多的是以CO₂形式排放到大气中。因此,未来需要更系统的研究不同气候变化情境下凋落物积累与分解、DOC淋溶和土壤碳库的动态变化,从而更好的理解这些生态系统碳循环过程的变化及其对气候变化产生的反馈作用和机制。     

凋落物多样性及组成对凋落物分解及土壤微生物群落的影响——二氧化碳倍增条件下

受全球变化的影响生物多样性的丧失日益严重,及时了解凋落物物种多样性及其组成如何直接或者通过调节微生物群落来间接影响凋落物分解已经成为生态学领域的热点问题之一。在呼伦贝尔典型草原区,羊草（Leymus chinensis）为原生群落建群种,茵陈蒿（Artemisia capillaris）、麻花头（Serratula centauroides）、二裂委陵菜（Potentilla bifurca）在退化及恢复群落中的多度均较大,本研究以此4种植物的凋落物为研究对象,在两倍于当前大气CO₂浓度（800 μmol/mol）条件下,通过嵌套实验设计来研究凋落物多样性（凋落物组成）对凋落物质量、C、N残余率和残余C/N的影响,并深入分析凋落物初始性质以及土壤革兰氏阳性菌（G⁺）、革兰氏阴性菌（G^-）、细菌（B）、真菌（F）及土壤总微生物磷脂脂肪酸（Phospholipid Fatty Acid,PLFA）含量和F/B对凋落物分解的影响。结果表明：（1）凋落物多样性及组成对凋落物质量、C、N残余率以及残余C/N均具有显著影响;凋落物组成对G⁺ PLFAs含量具有显著影响;（2）冗余分析（Redundancy Analysis,RDA）结果表明凋落物组成对凋落物分解和土壤微生物群落相关指标的影响高于凋落物多样性;（3）结构方程模型（Structural Equation Model,SEM）结果表明凋落物初始木质素含量和初始C/N均对凋落物分解具有显著影响,其中凋落物初始木质素含量起主导作用,其还可通过对土壤真菌PLFAs含量的影响来间接影响凋落物N残余率和残余C/N。所得结果可为大气CO₂浓度升高条件下退化草原生态系统的物质循环特征的预测乃至草原生态系统功能的合理评估提供数据支持。     

黑龙江省帽儿山林区6种主要林分类型凋落物研究

对帽儿山林区水曲柳、落叶松等六种主要林分的年凋落量、凋落物组成及分解动态进行了观测。结果表明：(1) 六种林分的年凋落量分别为水曲柳(5.57 t·hm^-2)、蒙古栎(4. t·hm^-2)、山杨(4.27 t·hm^-2)、落叶松(4.08 t·hm^-2)、红松(5.62 t·hm^-2)、樟子松(5.56 t·hm^-2);(2) 六种林分其叶的年凋落量占年总凋落量的比例明显大于其它组分,是其凋落物中的主要组成部分;(3) 经过近1a时间的分解,测得六种林分凋落叶分解速率依次为：水曲柳>樟子松>落叶松>山杨>蒙古栎>红松;经模拟研究表明,水曲柳凋落叶95%分解需3.5 a;蒙古栎需8.0 a;山杨需6.7 a;落叶松需6.6 a;红松需8.8 a;樟子松需4.4 a。     

凋落物多样性及组成对凋落物分解和土壤微生物群落的影响

凋落物分解是生态系统营养物质循环的核心过程,而土壤微生物群落在凋落物分解过程中扮演着极其重要且不可替代的角色。随着生物多样性的丧失日益严峻,探讨凋落物多样性及组成对凋落物分解和土壤微生物群落的影响,不仅有助于了解凋落物分解的内在机制,而且可为退化草原生态系统的恢复提供参考。以内蒙古呼伦贝尔草原退化恢复群落中的草本植物为研究对象,依据植物多度、盖度、频度和物种的重要值及其在群落中的恢复程度筛选出排序前4的羊草（Leymus chinensis）、茵陈蒿（Artemisia capillaris）、麻花头（Serratula centauroides）、二裂委陵菜（Potentilla bifurca）的凋落物为实验材料,通过设置3种凋落物多样性水平（1,2,4）,包括11种凋落物组合（单物种凋落物共4种,两物种凋落物混合共6种,四物种凋落物混合共1种）,利用磷脂脂肪酸（PLFA）方法来研究分解60 d后凋落物多样性及组成对凋落物分解和土壤微生物群落的影响。结果表明：（1）凋落物物种多样性仅对C残余率具有显著影响,表现在两物种混合凋落物C残余率显著低于单物种凋落物,而凋落物组成对所观测的4个凋落物分解参数（质量、C、N残余率以及C/N）均具有显著影响;（2）凋落物物种多样性对细菌（B）含量具有显著影响,而凋落物组成对真菌（F）含量具有显著影响,两者对F/B以及微生物总量均无显著影响;（3）冗余分析结果表明凋落物组成与凋落物分解相关指标（凋落物质量、C、N残余率及C/N）和土壤微生物（真菌、细菌含量）的相关关系高于凋落物多样性。（4）进一步建立结构方程模型（Structural Equation Model,SEM）发现,凋落物初始C含量对凋落物质量、C、N残余率及C/N有显著正的直接影响;凋落物木质素含量对凋落物质量、C、N残余率有显著正的直接影响;凋落物初始N含量对N残余率有显著正的直接影响,而对C残余率及C/N有显著负的直接影响;凋落物初始C/N对凋落物质量、N残余率有显著正的直接影响,而对C/N有显著负的直接影响。此外,凋落物初始C、N、木质素含量及C/N均对真菌含量具有显著正影响,并可通过真菌对凋落物质量分解产生显著负的间接影响。以上结果表明该退化恢复区域优势种凋落物分解以初始C、木质素为主导,主要通过土壤真菌影响凋落物的分解进程,这将减缓凋落物的分解速率进而减慢草原生态系统的进程。这些结果为凋落物多样性及组成对自身分解和土壤微生物群落的影响提供了实验依据,也为进一步分析凋落物分解内在机制以及草原生态系统的恢复提供了数据参考。     

模拟氮沉降对两种竹林不同凋落物组分分解过程养分释放的影响

利用原位分解袋法研究了华西雨屏区苦竹(Pleioblastus amarus)和撑绿杂交竹(Bambusa pervariabilis × Dendrocala mopsi)人工林几种凋落物组分在模拟氮沉降下分解过程中养分释放状态,试验周期为2 a。氮沉降水平分别为对照(CK, 0 g · m^-2 · a^-1)、低氮(5 g · m^-2 · a^-1)、中氮(15 g · m^-2 · a^-1)和高氮(30 g · m^-2 · a^-1),每月下旬定量地对各处理施氮(NH₄NO₃)。结果表明,苦竹林和杂交竹林凋落物主要由凋落叶、凋落箨和凋落枝组成,其中凋落叶约占80%;两个竹种凋落物在分解过程中养分元素释放的种间差异主要与初始养分元素含量有关;凋落物养分元素初始含量对元素释放模式和最终净释放率的大小具有重要的决定作用;目前,这两种竹林生态系统土壤氮输入主要以大气氮沉降(8.24 g · m^-2 · a^-1)为主,同时凋落物氮输入(苦竹和杂交竹林分别为1.93,5.07 g · m^-2 · a^-1)也是一个重要途径;模拟氮沉降对苦竹凋落物碳、磷、钾、钙元素和杂交竹凋落物碳、氮、磷、钾、钙、镁元素释放的抑制作用较弱,处理与对照之间元素总释放率差异一般小于10%;氮沉降显著抑制了苦竹林凋落物氮元素释放,减小幅度为19.0%-27.2%,但由于氮沉降增加对土壤肥力的直接改良作用,氮沉降的增加并不会因为凋落物分解速率的降低造成植物生长所需养分供应的减少;从短期来看,在氮沉降继续增加的情况下,该地区这类竹林生态系统的碳吸存能力仍可能会因为N沉降对植物生长的促进作用而增加。     

原始阔叶红松林下红松、蒙古栎混合凋落叶分解特征及相互作用研究

采用凋落物网袋（litter bag）分解法,模拟红松（Pinus koraiensis）(以下用P表示)和蒙古栎（Quercus mongolica）（以下用M表示）在不同演替阶段可能的组成比例(P、M/P1∶3、M/P1∶1、M/P3∶1、M）进行野外分解实验。分析不同比例的两种凋落叶混合的分解特征、相互作用及机理。结果表明：1.从质量损失率来看,与单种凋落叶分解情况相比,蒙古栎和红松凋落叶混合对凋落物分解具有促进作用,其中蒙古栎和红松（M/P）按1∶3混合分解最快,且随着蒙古栎凋落叶在混合比例中的增加,混合分解速率先减小后增大。2.从C、N元素动态看,C素在各处理的凋落叶主要表现为净释放,而N素在各处理中变现比较复杂,在各处理的红松凋落叶中表现为富集,而在各处理蒙古栎凋落叶中则表现为释放。蒙古栎凋落叶可以促进红松凋落叶C元素释放和N元素的富集,降低红松凋落叶的C/N比,促进红松凋落叶的分解。红松凋落叶能促进蒙古栎凋落物C元素释放,但对蒙古栎凋落叶N元素的释放作用不明显,对蒙古栎凋落叶的C/N比影响也并不一致。     

香樟凋落叶分解过程对两种间作作物生长和光合特性的影响

采用盆栽试验方法,探讨了香樟(Cinnamomum camphora)凋落叶不同土壤添加水平(0 g/盆为CK、25 g/盆、50 g/盆,100 g/盆)对受体作物小白菜(Brassica chinensis)、莴笋(Lactuca sativa)幼苗生长和光合特性的影响。结果显示:(1)香樟凋落叶分解对两种作物的地径、株高、生物量、叶片数和叶面积均有明显的抑制作用,且抑制效应随凋落叶添加量的增加而增强,但随着分解时间的延长其抑制作用逐渐减弱甚至表现为促进作用。(2)香樟凋落叶分解对两种作物的光合色素含量均有明显的抑制效应,并随凋落叶添加量的增加抑制作用增强,且持续时间延长。(3)经凋落叶处理的两种作物叶片净光合速率(P_n)、气孔导度(G_s)、蒸腾速率(T_r)、水分利用率(WUE)总体上均低于CK,而胞间CO₂浓度(C_i)在各凋落叶处理下均高于CK。(4)随着土壤中凋落叶量的增加,两种作物在光饱和以及CO₂饱和状态下的最大净光合速率(P_{n max})、表观量子效率(AQY)、羧化速率(CE)、光呼吸速率(R_p)和暗呼吸速率(R_d)均不断下降,而光补偿点(LCP)、光饱和点(LSP)、CO₂饱和点(CSP)、CO₂补偿点(CCP)因受体作物的不同,表现出不同的变化趋势。研究表明,土壤中香樟凋落叶分解释放的化感物质,能通过降低受体作物的光合色素合成和光合能力,限制其营养生长,最终影响生物量积累;相对于莴笋,小白菜对香樟凋落叶分解产生的化感胁迫效应具有更强的耐受性,可能更适宜在香樟林间种植。     

不同水分条件下杨树-玉米复合系统凋落物分解特性

为探讨水分变化对农林复合生态系统凋落物分解特性的影响,以河西走廊杨树（Populus）-玉米（Zea mays）凋落物为研究对象,设置正常水分（9200 m³/hm²,对照）,轻度干旱胁迫（减少15%,7800 m³/hm²）,中度干旱胁迫（减少30%,6400 m³/hm²）3种不同水分处理条件,采用分解袋法研究了不同水分条件下杨树叶和玉米秸秆的质量残留率、分解速率和养分含量变化特征。结果表明：（1）随着干旱胁迫的加剧,两种凋落物的质量残留率均增加,而分解速率降低。经过164 d的分解后,杨树叶和玉米秸秆的质量残留率分别为70.43%-77.49%、63.55%-68.29%。分析表明：水分和时间对各类型凋落物的质量残留率均有极显著的影响（P<0.001）,但二者的交互作用不显著（P>0.05）;干旱胁迫显著降低了玉米秸秆的分解速率,但杨树叶的分解速率却只是在中度干旱胁迫下显著降低（P<0.05）。对于不同类型凋落物而言,分解速率表现为玉米秸秆>杨树叶。（2）两种类型凋落物的氮（N）残留率在分解过程中表现为降低的趋势,但随着干旱程度的加大,N的残留率增加,表明水分抑制了N的释放过程。分解164d后,同一类型凋落物不同水分条件下的N残留率均存在显著差异。对于同一水分条件下不同凋落物而言,玉米秸秆的N残留率最低,而杨树叶最高。总的来说,水分降低对干旱区农林复合系统内凋落物的分解和氮元素含量具有显著的抑制作用。     

模拟氮沉降对华西雨屏区撑绿 杂交竹凋落物分解的影响

从2008年1月至2010年1月,对华西雨屏区撑绿杂交竹(Bambusa pervariabilis × Dendrocala mopsi)人工林进行了模拟氮沉降试验,氮沉降水平分别为对照(CK, 0 g · m^-2 · a^-1)、低氮(5 g · m^-2 · a^-1)、中氮(15 g · hm^-2 · a^-1)和高氮(30 g · m^-2 · a^-1)。利用凋落袋法对杂交竹凋落叶和凋落箨进行原位分解试验,并在每月下旬定量地对各处理施氮(NH₄NO₃)。结果表明,自然状态下杂交竹凋落叶和凋落箨分解95%所需时间分别为2.9,1.5 a;氮沉降显著抑制了凋落叶的分解,在分解后期3个氮沉降处理凋落叶无灰分质量残留率均显著大于对照,氮沉降对凋落箨分解无明显影响;氮沉降显著抑制了凋落叶中木质素和纤维素的分解。杂交竹凋落叶在分解后期质量损失缓慢,处于较稳定状态,氮沉降的增加使得凋落物的残留率稳定在一个更高的水平,表明氮沉降的增加可能会使更多的凋落物残体和稳定有机质留存于杂交竹林土壤中,从而增加杂交竹林土壤碳贮存。     

海拔和郁闭度对祁连山青海云杉林叶凋落物分解的影响

为了探究海拔和郁闭度对青海云杉林叶凋落物分解的影响,本文选择海拔为2850 m,3050 m,3250 m和3450 m四个梯度和高、中、低三个林分郁闭度,采用分解网袋法,研究青海云杉叶凋落物分解速率及分解过程中N、P元素变化。结果表明,质量损失率随时间在波动增大。分解速率先减小后增大,不同海拔下分解速率为K₃₄₅₀ > K₃₀₅₀ > K₃₂₅₀ > K₂₈₅₀,不同郁闭度下分解速率为K_低 > K_中 > K_高,青海云杉叶枯落物分解50%和95%所需时间约为5.3 a和22.7 a。枯落物分解过程中,N、P含量和累积系数在不同海拔和郁闭度下的变化不同,与季节变化有关。研究结果为祁连山森林生态系统地球化学循环奠定基础。     

Interconversions of different forms of vitamin B6 in tobacco plants

There are six different vitamin B₆ (VB₆) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5′-phosphate (PLP), pyridoxamine 5′-phosphate (PMP), and pyridoxine 5′-phosphate (PNP), of which PLP is the active form. Although plants are a major source of VB₆ in the human diet, and VB₆ plays an important role in plants, the mechanisms underlying the interconversions of different VB₆ forms are not well understood. In this study, in vitro tobacco plants were grown on Murashige and Skoog (MS) basal media supplemented with 100 mg/L of PM, PL or PN and the abundance of the different B₆ vitamers in leaf tissue was quantified by high performance liquid chromatography (HPLC). The total amount of VB₆ was about 3.9 μg/g fresh weight of which PL, PM, PN, PLP and PMP accounted for 23%, 14%, 37%, 20% and 6%, respectively. Tobacco plants contained a trace amount of PNP. Supplementation of the culture medium with any of the non-phosphorylated vitamers resulted in an increase in total VB₆ by about 10-fold, but had very little impact on the concentrations of the endogenous phosphorylated vitamers. Administration of either PM or PN increased their endogenous levels more than the levels of any other endogenous B₆ vitamers. PL supplementation increased the levels of plant PN and PM significantly, but not that of PL, suggesting that efficient conversion pathways from PL to PN and PM are present in tobacco. Additionally, maintenance of a stable level of PLP in the plant is not well-correlated to changes in levels of non-phosphorylated forms.     

Light- and seasonal-induced plasticity in leaf morphology, N partitioning and photosynthetic capacity of two temperate deciduous species

Processes involved in leaf photosynthetic acclimation to light and throughout the growing season were investigated in two hardwood species (Acer saccharum and Betula alleghaniensis), which differed in their level of shade-tolerance. For both species, variation in traits related to (i) leaf morphology (LMA, leaf mass:area ratio), (ii) leaf N content (N_A, leaf nitrogen content on an area basis and N_M, N concentration in leaf dry mass), (iii) leaf N partitioning among photosynthetic functions (P_r, N allocated to Rubisco, and P_b, N allocated to bioenergetics), and (iv) leaf photosynthetic capacity (V_cmax, maximal carboxylation rates, and J_max, maximal light-driven electron flow) were assessed at three different times during the growing season (early, mid- and late summer) and under four contrasting light regimes (40, 17, 6 and 2% of full sunlight). For both species, light-driven variation in most traits was greater than their seasonally driven variation. Furthermore, results showed for both species the pre-eminence of LMA changes in the light-driven acclimation of N_A. Importance of N_M to variation in N_A was restricted to seasonal acclimation, especially for the less shade-tolerant species, B. alleghaniensis. Similarly, for both species, light-driven acclimation of leaf photosynthetic capacities was tightly related to variation in N_A, which was related to LMA changes. However, variation in P_r and P_b better explained seasonally driven variation in V_cmax and J_max, specifically under lower light levels, where N_A was low. Thus, the great variability observed for leaf activity in response to contrasting light environments was related to efficient morphological adjustments, regardless of species level of shade-tolerance. Finally, physiological adjustments were mainly involved in fine-scale changes observed during seasonally driven acclimation of leaves, when LMA was constrained to a slight range of variation.     

Selection of a highly virulent fungal isolate, Metarhizium anisopliae CLO 53, for controlling Hoplia philanthus

The June beetle, Hoplia philanthus Füessly (Coleoptera: Scarabaeidae), has become a widespread and destructive insect pest of lawns, sport turf, pastures, and horticultural crops in Belgium. The virulence of 34 entomopathogenic fungal isolates from the genera Metarhizium, Beauveria, and Paecilomyces to third-instar H. philanthus was tested in bioassays by dipping larvae in 10(7)conidia/ml suspensions. Two isolates of Metarhizium anisopliae (CLO 53 and CLO 54) caused maximally 90% mortality 10 weeks post-inoculation while other isolates only caused mortalities between 10 and 62%. The virulence of M. anisopliae CLO 53 was further tested by exposing H. philanthus larvae to conidial serial concentrations of 10(4)-10(9)conidia/g sandy soil for up to 11 weeks at 15, 20 or 25 degrees C. Mortality was dependant on the fungal concentration, exposure time, and temperature. Eleven weeks after inoculation, the LC50 values for this isolate ranged from 1.3 to 4.0 x 10(6), 1.0 to 3.2 x 10(5), and 2.5 x 10(4) to 10(5)conidia/g soil at 15, 20, and 25 degrees C, respectively. The LT50 values for this isolate ranged from 3.5 to 21.7, 2.4 to 18.7, and 2.9 to 16.1 weeks at concentrations of 10(9) and 10(4)conidia/g soil at 15, 20, and 25 degrees C, respectively. In glasshouse pot experiment with perennial ryegrass (Lolium perenne L.), the isolate CLO 53 caused mortalities of 50 and 88% of H. philanthus larvae 10 weeks after application of 10(4) and 10(6)conidia/cm(2) soil surface, respectively. The present results suggest that the Belgian isolate CLO 53 has excellent potential for biological control of H. philanthus.     

Two groups of entomopathogenic bacteria, Photorhabdus and Xenorhabdus, share an inhibitory action against phospholipase A2 to induce host immunodepression

Photorhabdus and Xenorhabdus are two genera of entomopathogenic bacteria having a mutualistic relationship with their respective nematode hosts, Heterorhabditis and Steinernema. One of the pathogenic mechanisms of these bacteria includes host immunodepression, which leads to lethal septicemia. It has been known that X. nematophila inhibits phospholipase A2 (PLA2) to induce host immunodepression. Here, we tested the hypothesis of PLA2 inhibition using another bacterial species involved in other genera. P. temperata subsp. temperata is the intestinal symbiont of an entomopathogenic nematode, H. megidis. The bacteria caused potent pathogenicity in a dose-dependent manner against the fifth instar larvae of a test target insect, Spodoptera exigua, as early as 24 h after the intra-hemocoelic injection. In response to the live bacterial injection, hemocyte nodulation (a cellular immune response) and prophenoloxidase (pPO) activation were inhibited, while the injection of heat-killed bacteria significantly induced both immune reactions. The immunodepression induced by the live bacteria was reversed by the addition of arachidonic acid, the catalytic product of phospholipase A2. In contrast, the addition of dexamethasone, a specific PLA2 inhibitor to the heat-killed bacterial treatment, inhibited both immune capacities. In addition to a previously known PLA2 inhibitory action of X. nematophila, the inhibition of P. temperata temperata on PLA2 suggests that bacteria symbiotic to entomopathogenic nematodes share a common pathogenic target to result in an immunodepressive state of the infected insects. To prove this generalized hypothesis, we used other bacterial species (X. bovienni, X. poinarii, and P. luminescens) involved in these two genera. All our experiments clearly showed that these other bacteria also share their inhibitory action against PLA2 to induce host immunodepression.     

The lethal and sub-lethal consequences of entomopathogenic nematode infestation and exposure for adult pine weevils, Hylobius abietis (Coleoptera: Curculionidae)

Entomopathogenic nematodes (EPN) frequently kill their host within 1-2 days, and interest in EPN focuses mainly on their lethality. However, insects may take longer to die, or may fail to die despite being infected, but little is known about the effects of EPN infection on insects, other than death. Here we investigate both lethal and sub-lethal effects of infection by two EPN species, Steinernema carpocapsae and Heterorhabditis downesi, on adults of the large pine weevil, Hylobius abietis. Following 12 h nematode-weevil contact in peat, S. carpocapsae killed a significantly higher proportion of weevils (87-93%) than H. downesi (43-57%) at all concentrations tested. Less than 10% of weevils were dead within 2 days, and weevils continued to die for up to 10 days after exposure (LT₅₀ of 3 days or more). In a separate experiment, live weevils dissected 6 days after a 24 h exposure to nematodes on filter paper harbored encapsulated and dead nematodes, showing that weevils could defend themselves against infection. Some live weevils also harbored live nematodes 6 days after they had been removed from the nematode infested medium. Feeding by weevils was not affected by infection with, or exposure to, either species of EPN. We discuss these results in relation to the use of EPN in biological control against H. abietis.     

Mechanisms of enhanced cellulosic bioethanol fermentation by co-cultivation of Clostridium and Thermoanaerobacter spp

Engineering microbial consortia capable of efficient ethanolic fermentation of cellulose is a strategy for the development of consolidated bioprocessing for bioethanol production. Co-cultures of cellulolytic Clostridium thermocellum with non-cellulolytic Thermoanaerobacter strains (X514 and 39E) significantly improved ethanol production by 194-440%. Strain X514 enhanced ethanolic fermentation much more effectively than strain 39E in co-cultivation, with ethanol production in X514 co-cultures at least 62% higher than that of 39E co-cultures. Comparative genome sequence analysis revealed that the higher ethanolic fermentation efficiency in strain X514 was associated with the presence of a complete vitamin B(12) biosynthesis pathway, which is incomplete in strain 39E. The significance of the vitamin B(12)de novo biosynthesis capacity was further supported by the observation of improved ethanol production in strain 39E by 203% following the addition of exogenous vitamin B(12). The vitamin B(12) biosynthesis pathway provides a valuable biomarker for selecting metabolically robust strains for bioethanol production.     

Thermodynamic and kinetic characterisation of individual haems in multicentre cytochromes c3

The characterisation of individual centres in multihaem proteins is difficult due to the similarities in the redox and spectroscopic properties of the centres. NMR has been used successfully to distinguish redox centres and allow the determination of the microscopic thermodynamic parameters in several multihaem cytochromes c₃ isolated from different sulphate-reducing bacteria. In this article we show that it is also possible to discriminate the kinetic properties of individual centres in multihaem proteins, if the complete microscopic thermodynamic characterisation is available and the system displays fast intramolecular equilibration in the time scale of the kinetic experiment. The deconvolution of the kinetic traces using a model of thermodynamic control provides a reference rate constant for each haem that does not depend on driving force and can be related to structural factors. The thermodynamic characterisation of three tetrahaem cytochromes and their kinetics of reduction by sodium dithionite are reported in this paper. Thermodynamic and kinetic data were fitted simultaneously to a model to obtain microscopic reduction potentials, haem-haem and haem-proton interacting potentials, and reference rate constants for the haems. The kinetic information obtained for these cytochromes and recently published data for other multihaem cytochromes is discussed with respect to the structural factors that determine the reference rates. The accessibility for the reducing agent seems to play an important role in controlling the kinetic rates, although is clearly not the only factor.     

Tuning of functional heme reduction potentials in Shewanella fumarate reductases

The fumarate reductases from S. frigidimarina NCIMB400 and S. oneidensis MR-1 are soluble and monomeric enzymes located in the periplasm of these bacteria. These proteins display two redox active domains, one containing four c-type hemes and another containing FAD at the catalytic site. This arrangement of single-electron redox co-factors leading to multiple-electron active sites is widespread in respiratory enzymes. To investigate the properties that allow a chain of single-electron co-factors to sustain the activity of a multi-electron catalytic site, redox titrations followed by NMR and visible spectroscopies were applied to determine the microscopic thermodynamic parameters of the hemes. The results show that the redox behaviour of these fumarate reductases is similar and dominated by a strong interaction between hemes II and III. This interaction facilitates a sequential transfer of two electrons from the heme domain to FAD via heme IV.     

Structure/function studies on a S-adenosyl-L-methionine-dependent uroporphyrinogen III C methyltransferase (SUMT), a key regulatory enzyme of tetrapyrrole biosynthesis

The crystallographic structure of the Pseudomonas denitrificans S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase (SUMT), which is encoded by the cobA gene, has been solved by molecular replacement to 2.7A resolution. SUMT is a branchpoint enzyme that plays a key role in the biosynthesis of modified tetrapyrroles by controlling flux to compounds such as vitamin B(12) and sirohaem, and catalysing the transformation of uroporphyrinogen III into precorrin-2. The overall topology of the enzyme is similar to that of the SUMT module of sirohaem synthase (CysG) and the cobalt-precorrin-4 methyltransferase CbiF and, as with the latter structures, SUMT has the product S-adenosyl-L-homocysteine bound in the crystal. The roles of a number of residues within the SUMT structure are discussed with respect to their conservation either across the broader family of cobalamin biosynthetic methyltransferases or within the sub-group of SUMT members. The D47N, L49A, F106A, T130A, Y183A and M184A variants of SUMT were generated by mutagenesis of the cobA gene, and tested for SAM binding and enzymatic activity. Of these variants, only D47N and L49A bound the co-substrate S-adenosyl-L-methionine. Consequently, all the mutants were severely restricted in their capacity to synthesise precorrin-2, although both the D47N and L49A variants produced significant quantities of precorrin-1, the monomethylated derivative of uroporphyrinogen III. The activity of these variants is interpreted with respect to the structure of the enzyme.     

Detection of metallo-β-lactamase genes in clinical specimens by a commercial multiplex PCR system

hyplex®-MBL ID Multiplex PCR-ELISA, a novel method for identifying metallo-β-lactamase genes directly in clinical specimens, was evaluated using a consecutive collection of 326 samples from three hospitals in Greece characterized by high prevalence of VIM producers. The method exhibited high sensitivity (98.0%) and specificity (98.6%) and was proven reliable in detecting bla_VIM genes in blood, urine, pus, and sputum samples that, as confirmed by conventional methods, contained various VIM-producing species. Future multicenter studies should be considered for the thorough evaluation of this method and its potential diagnostic utility.