棕色固氮菌突变种(UW45、DJ54和DJ35)的缺失FeMoco钼铁蛋白的纯化、特性鉴定及结晶研究

一．棕色固氮菌突变种UW45、缺失nifH（DJ54）和缺失nifE(DJ35)突变种的钼铁蛋白的纯化、特性鉴定及结晶研究 棕色固氮菌突变种UW45的菌体破碎后，所得粗提物经两次DEAE 52柱层析后得到部分纯的nifB- MoFe蛋白和Fe蛋白。再经Sephacryl S-300和 DEAE柱的进一步纯化，便使nifB- MoFe蛋白基本达到SDS凝胶电泳纯。SDS-PAGE结果表明，nifB- MoFe蛋白具有与野生型棕色固氮菌（OP）MoFe蛋白相同的亚基种类和组成。此粗提物可为用NMF抽提的 OP MoFe蛋白的FeMoco激活，所得Fe蛋白具有与OP Fe蛋白相似的互补活性，可使OP MoFe的比活性达到2192 nmol C2H2/min/mg蛋白。FeMoco可使无互补活性的 nifB- MoFe蛋白与nifB- Fe蛋白组成具有可观放氢活性的固氮酶，使FeMoco显出的比活性接近文献报道的还原乙炔的最高值。对nifB- MoFe蛋白的结晶及晶体生长进行了的研究，初步探讨了结晶溶液各组分的种类和浓度、结晶方法和实验操作等与能否出现晶体及晶体的数目、大小、质量、形状和出晶时间等的相互关系。在结晶实验时，一次就得到了国内外尚未报道的该蛋白的短斜四棱柱的棕色晶体。目前所得的最大的晶体的二维边长都为0.1mm。初步结果表明，这种晶体可能就是nifB- MoFe蛋白的晶体。 从棕色固氮菌突变种DJ54中得到了ΔnifH MoFe蛋白;并参与了棕色固氮菌突变种DJ35的ΔnifE MoFe蛋白的分离纯化，所用方法与nifB- MoFe蛋白的分离纯化相似。对这两种突变种蛋白的特性和结晶进行了初步研究。在结晶实验时，也是一次就得到了国内外尚未报道的ΔnifH MoFe蛋白和ΔnifEMoFe蛋白的晶体。 二．新型固氮酶MnFe蛋白和CrFe蛋白的特性与结晶研究 在已有的工作基础上，分离纯化了几批MnFe蛋白和CrFe蛋白，并用部分纯的nifB- Fe蛋白进行活性互补，分别测定了MnFe蛋白和CrFe蛋白的底物还原活性。不断优化MnFe蛋白和CrFe蛋白晶体生长条件，获得了晶质良好的MnFe蛋白和CrFe蛋白的较大晶体。 在2001年的“神舟2号”飞船搭载实验中，MnFe蛋白的出晶率达到100%，所获得的晶体也比地面对照略厚些。继续进行MnFe蛋白和CrFe蛋白的空间计划的地面匹配实验，以满足对蛋白质样品的要求，以保证宇宙飞船“神舟3号”的蛋白质搭载实验获得更好的结果。

含铬或锰的新型固氮酶的纯化, 特性和结晶—棕色固氮菌突变种UW3中CrFe蛋白和MnFe蛋白的研究

以含MnSO4或Na2CrO4的无钼无氨的修改Burk’s培养基， 培养不能合成含钼固氮酶体系的棕色固氮菌 (Azotobacter vinelandii Lipmann) 突变种UW3， 发现在一定浓度范围内， MnSO4或Na2CrO4的加入有助于促进其生长。 菌体生长和C2H2还原活性曲线测定结果表明， 这种促进作用很可能是通过Mn或Cr取代Mo， 参与组装固氮酶中心原子簇， 从而影响固氮活性而实现的。 利用阴离子交换 (DEAE-52和Q-Sepharose FF) 和凝胶过滤 (Sephacryl S-200) 柱层析从两种菌体中纯化得到固氮酶组分Ⅰ蛋白 (分别命名为MnFe蛋白和CrFe蛋白)， 并对其进行了特性研究。 厌氧天然聚丙烯酰胺凝胶电泳 (PAGE) 和SDS-PAGE结果显示， 两种蛋白均为两种亚基组成的四聚体。 亚基可以与OP MoFe蛋白抗体发生免疫反应， 分子量分别略小于野生种OP MoFe蛋白的α和β亚基。 CrFe蛋白的C2H2还原活性， Ar下放氢活性和固氮活性分别相当于OP MoFe蛋白的36%， 38%和43%， 而MnFe蛋白活性相当于OP MoFe蛋白的50%左右， 并且两种蛋白与OP MoFe蛋白具有相似的固氮电子利用率。 对两种蛋白金属含量的测定证实其中分别含有Mn和Cr， 但仍存在少量Mo污染。 与OP MoFe蛋白相比，这两种蛋白圆二色谱的摩尔椭圆率 ([θ]) 除在450nm较接近外，在可见光区的其它波长处均显著降低。 与DT还原OP MoFe蛋白相似， CrFe蛋白和MnFe蛋白具有g≈4。3、3。7和2。0的特征EPR信号， 但各处信号强度比例不同。 在对污染Mo可能引起的信号进行校正后，CrFe蛋白的三个信号强度分别相当于DT还原OP MoFe蛋白的20%， 0%和10%， 而MnFe蛋白则分别相当于112%， 49%和65%。 上述结果表明， CrFe蛋白和MnFe蛋白与OP MoFe蛋白金属原子簇的主要差异很可能在于FeMco (M=Cr， Mn或Mo)的M种类， 而P-cluster结构和组成均未见大的差异。 利用气相扩散悬滴法对MnFe蛋白和CrFe蛋白结晶条件进行了筛选和初步优化， 确定了以Tris/Hepes， NaCl， MgCl2和PEG 8000为主要变量的沉淀剂体系， 寻找各组分对于晶体生长的最适浓度。 以此为基础探讨了应用气相扩散坐滴法和液-液扩散法对两种蛋白结晶条件的优化。 在一定条件下， 两种蛋白分别通过液-液扩散法获得了优质大单晶。 对从CrFe蛋白和MnFe蛋白制备物中培养出的蛋白质晶体的SDS-PAGE鉴定显示， 晶体由与OP MoFe蛋白相似的两种亚基组成。 通过 “神舟三号” 飞船搭载实验探讨了空间微重力对于厌氧蛋白质结晶的影响， 结果表明， 微重力有助于避免孪晶形成， 并具有长期培养后获得适于进行X-射线衍射分析的优质大单晶的潜在前景。 结合空间科学使固氮酶结构与功能研究得以发展， 这项工作是有意义并且可行的。

光敏核不育水稻41 kDa和61 kDa蛋白质的发现、N-端序列分析及其雄性不育性状关系的探讨

光敏核不育水稻农垦58S是石明松于1973年在晚粳农垦58的大田中发现的雄性不育突变体，它在长日照下雄性不育可被用于与恢复系杂交生产杂种，而在短日照下雄性可育能用于自交繁殖，它的恢复系来源广泛。基于这些特性，育种学家用光敏核不育水稻建立的二系杂交水稻制种技术有很大的应用潜力。近十几年来，育种学家用农垦58S作基因供体转育了许多新的不育系，研究结果表明育成的粳型不育系均为光敏不育系，但在育成的籼型不育系中，绝大多数丧失光敏核不育特性，变成温敏不育系。目前因不知光敏核不育的分子遗传机制，尚不能解释这些问题。 本文用双向电泳技术分析了农垦58S和农垦58苗期和育性转换光敏感期叶绿体蛋白质的差异，在农垦58S中发现三个蛋白质(Pl，P2和P3)，其中Pl和P2在苗期和光敏感期叶片内均存在，P3仅在光敏感期的叶片中存在，它们不受长日照或短日照处理的影响。农垦58没有这三个蛋白质。 用制备型双向电泳纯化后，得到SDS - PAGE和IEF纯的Pl和P2。经SDS-PAGE和IEF测定，Pl的等电点是6.2，分子量是41 kDa;P2的等电点是5.8，分子量是61 kDa。现称Pl为P41，P2为P61。氨基酸序列分析和同源性检索发现P41与水稻叶绿体ATP合成酶p亚基和酵母转录因子CAD1有同源性，此外，P41的N-端序列中有一个与蛋白激酶催化核心中的多功能motif Y-G-X-G-X- (P/T)-G-V相似的序列;P61的14个氨基酸长的N-端序列与水稻叶绿体ATP合成酶β亚基的一致。P41和P61 N-端前12个氨基酸的序列也完全一致。 PCR扩增和Southern杂交分析没有发现农垦58S和农垦58之间ATP合成酶β亚基基因(atpB)的多态性。Nothern杂交分析表明农垦58S中仅有一种、与农垦58 atpB mRNA分子量相同的atpB转录产物，但它的atpB mRNA丰度明显低于农垦58的。没有检测到突变的atpB和其它形式的atpB转录产物。 分析P41和P61在其它水稻材料中的分布特点发现它们在粳型光敏不育系7001S、5088S、31301S、C407S和1647S，籼型光敏不育系W7415S和W9451S以及温（光）敏不育系培矮64S中存在，而在对照材料三系水稻马协A、珍汕97A、马协B、珍汕97B和明恢63以及常规粳稻C94153中不存在。根据这些不育系的系谱和它们与农垦58S之间基因的等位性研究结果，讨论了P41和P61与光敏核不育性的可能联系。

高温和衰老对光合膜结构与功能影响的研究

本部分研究以菠菜和水稻为材料，比较系统的研究了高温对类囊体膜、PSII颗粒、PSII外周捕光天线LHCII、PSII核心复合物和PSII反应中心等不同层次膜蛋白结构与功能的影响，以探讨高温对光合膜蛋白的伤害机理。其主要结果如下： 1．类囊体膜结构与功能的完整性对于维持PSII的结构与功能在高温胁迫下的稳定性具有重要作用。当类囊体膜的完整性受到破坏，当与PSI有关的一系列保护机制失去作用时，PSII对高温胁迫的敏感性会大大加强。 2．虽然PSI的功能在高温下保持相对稳定，但PSI的结构在高温胁迫下并不稳定。本文的研究发现LHCI对高温非常敏感，在中度高温胁迫下就开始降解，但PSI的核心在高温下比较稳定，所以PSI介导的电子传递活性仍然维持在较高水平。 3．高温胁迫会对PSII的结构和功能产生多重破坏。这个过程首先应该是放氧复合体的失活：其次是反应中心的可逆失活;接下来可能是核心天线CP43和CP47的失活导致捕光天线同反应中心的能量传递受阻;再下来是QA到QR电子传递的受阻、反应中心的不可逆失活、捕光效率下降等过程;最后是大范围色素蛋白的变性和失活，PSII的结构和功能遭到彻底破坏。 4．高湿胁迫下Fo显著升高，Fo的升高的原因可能源于少量捕光天线同反应中心的分离和反应中心的失活。 5．高等植物体的类囊体膜中存在多种Chla和Chlb的光谱吸收形式。这些代表不同的光谱吸收形式的组分在高温胁迫下表现出不同程度的降解，其中C678 和C684组分降解最快。这些不同的光谱吸收形式组分可能以不同的比例存在于每一种色素蛋白复合物中。 6．LHCII的结构与功能对于维持PSII结构与功能的热稳定性具有重要作用，LHCII完全缺失的水稻突变体VG28及分离纯化的PSII核心复介物都人大增强了对高温的敏感性。但一种LHCII减少的水稻突变体249-Mutant,反而增加了PSII的热稳定性，进一步的研究表明，类囊体膜中 LHCII本身含量的多少对PSII热稳定性的影响不足决定性的，关键性因素可能主要取决于 LHCII含量改变而引起的膜脂组成和膜脂不饱和程度的改变，以及由膜脂变化引起的PSII放氧复合体结构与功能的变化。 7．本研究首次发现，高温可以促使分离纯化的LHCII的红区吸收光谱发生显著红移，而680nm处的荧光发射降低，长波长荧光组分大大增强。绿胶电泳表明中度高温胁迫能够诱导LHCII产生寡聚体，这种寡聚体可能在调节能量耗散方面具有重要生理意义：而严重高温胁迫下LHCII倾向于聚合产生大分子的非活性聚集体。 8．分离纯化的反应中心对高温非常敏感，各种色素的结构和功能在轻度高温胁迫下就开始受到破坏和抑制，各种色素变性和降解的顺序由快到慢是：P680>Pheo>Chla>β-Car。反应中心的多肽组分在高温胁迫下显著减少，Dl和D2减少的原因可能归因于高温胁迫导致大分子聚合物的产生，D2的减少显著快于D1的减少。

Anti-HIV-1 property of trichosanthin correlates with its ribosome inactivating activity

Trichosanthin (TCS) is a type I ribosome inactivating (RI) protein possessing anti-tumor and antiviral activity, including human immunodeficiency virus (HIV). The mechanism of these actions is not entirely clear, but is generally attributed to its RI property. In order to study the relationship between the anti-HIV-1 activity of TCS and its RI activity, three TCS mutants with different RI activities were constructed by using site-directed mutagenesis. The anti-HIV-1 activities of the three mutants were tested in vitro. Results showed that two TCS mutants, namely TCSM((120-123)), TCSE160A/E189A, with the greatest decrease in RI activity, lost almost all of the anti-HIV activity and cytopathic effect. Another mutant TCSR122G, which exhibited a 160-fold decrease in RI activity, retained some anti-HIV activity. The results from this study suggested that RI activity of TCS may have significant contribution to its anti-HIV-1 property. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Independency of anti-HIV-1 activity from ribosome-inactivating activity of trichosanthin

Trichosanthin (TCS) is a type I ribosome-inactivating (RI) protein possessing multiple biological and pharmacological activities. Its major action is inhibition of human immunodeficiency virus (HIV) replication but the mechanism is still elusive. All evidences showed that this action is related to its RI activity. Previous studies found that TCS mutants with reduced RI activity simultaneously lost some anti-HIV activity. In this study, an exception was demonstrated by two TCS mutants retaining almost all RI activity but were devoid of anti-HIV-1 activity. Five mutants were constructed by using site-directed mutagenesis with either deletion or addition of amino acids to the C-terminal sequence. Results showed that the RI activity of mutants with C-terminal deletion mutants (TCSC2, TCSC4, and TCSC14) decreased by 1.2-3.3-fold with parallel downshifting of its anti-HIV-1 activity (1.4-4.8-fold). Another two mutants, TCSC19aa and TCSKDEL having 19 amino acid extension and a KDEL signal sequence added to the C-terminal sequence, retained all RI activity but subsequently lost most of the anti-HIV-1 activity. These findings suggested that ribosome inactivation alone might not be adequate to explain the anti-HIV action of TCS. (C) 2003 Elsevier Science (USA). All rights reserved.

Site-directed PEGylation of trichosanthin retained its anti-HIV activity with reduced potency in vitro

Trichosanthin (TCS) was the first ribosome inactivating protein found to possess anti-HIV-1 activity. Phase I/II clinical trial of this compound had been done. Antigenicity and short plasma half-life were the major side effects preventing further clinical trial. Modification of TCS is therefore necessary to revive the interest to develop this compound as an anti-HIV agent. Three potential antigenic sites (Ser-7, Lys-173, and Gln-219) were identified by computer modeling. Through site-directed mutagenesis, these three antigenic amino acids were mutated to a cysteine residue resulting in 3 TCS mutants, namely S7C, K173C, and Q219C. These mutants were further coupled to polyethylene glycol with a molecular size of 20 kDa (PEG) via the cysteine residue. This produced another three TCS derivatives, namely PEG(20)k-S7C, PEG(20)k-K173C, and PEG(20)k-Q219C. PEGylation had been widely used recently to decrease immunogenicity by masking the antigenic sites and prolong plasma half-life by expanding the molecular size. The in vitro anti-HIV-1 activity of these mutants and derivatives was tested. Results showed that the anti-HIV-1 activity of S7C, K173C, and Q219C was decreased by about 1.5- to 5.5-fold with slightly lower cytotoxicity. On the other hand, PEGylation produced larger decrease (20- to 30-fold) in anti-HIV activity. Cytotoxicity was, however, weakened only slightly by about 3-fold. The in vitro study showed that the anti-HIV activity of PEGylated TCS was retained with reduced potency. The in vivo activity is expected to have only slightly changed due to other beneficial effects like prolonged half-life. (C) 2004 Elsevier Inc. All rights reserved.

Independent Transport and Sorting of Functionally Distinct Protein Families in Tetrahymena thermophila Dense Core Secretory Granules

Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal beta/gamma-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, Delta GRT1 Delta GRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in Delta GRT1 Delta GRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from Delta GRT1 Delta GRT2 cells appear less adhesive than those from the wild type.

Elongation Factor ELL (Eleven-Nineteen Lysine-rich Leukemia) Acts as a Transcription Factor for Direct Thrombospondin-1 Regulation

The eleven-nineteen lysine-rich leukemia (ELL) gene undergoes translocation and fuses in-frame to the multiple lineage leukemia gene in a substantial proportion of patients suffering from acute forms of leukemia. Studies show that ELL indirectly modulates transcription by serving as a regulator for transcriptional elongation as well as for p53, U19/Eaf2, and steroid receptor activities. Our in vitro and in vivo data demonstrate that ELL could also serve as a transcriptional factor to directly induce transcription of the thrombospondin-1 (TSP-1) gene. Experiments using ELL deletion mutants established that full-length ELL is required for the TSP-1 up-regulation and that the trans-activation domain likely resides in the carboxyl terminus. Moreover, the DNA binding domain may localize to the first 45 amino acids of ELL. Not surprisingly, multiple lineage leukemia-ELL, which lacks these amino acids, did not induce expression from the TSP-1 promoter. In addition, the ELL core-response element appears to localize in the -1426 to -1418 region of the TSP-1 promoter. Finally, studies using zebrafish confirmed that ELL regulates TSP-1 mRNA expression in vivo, and ELL could inhibit zebrafish vasculogenesis, at least in part, through up-regulating TSP-1. Given the importance of TSP-1 as an anti-angiogenic protein, our findings may have important ramifications for better understanding cancer.

A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae

Isolation of high neutral lipid-containing microalgae is key to the commercial success of microalgae-based biofuel production. The Nile red fluorescence method has been successfully applied to the determination of lipids in certain microalgae, but has been unsuccessful in many others, particularly those with thick, rigid cell walls that prevent the penetration of the fluorescence dye. The conventional "one sample at a time" method was also time-consuming. In this study, the solvent dimethyl sulfoxide (DMSO) was introduced to microalgal samples as the stain carrier at an elevated temperature. The cellular neutral lipids were determined and quantified using a 96-well plate on a fluorescence spectrophotometer with an excitation wavelength of 530 nm and an emission wavelength of 575 run. An optimized procedure yielded a high correlation coefficient (R-2 = 0.998) with the lipid standard triolein and repeated measurements of replicates. Application of the improved method to several green algal strains gave very reproducible results with relative standard errors of 8.5%, 3.9% and 8.6%, 4.5% for repeatability and reproducibility at two concentration levels (2.0 mu g/mL and 20 mu g/mL), respectively. Moreover, the detection and quantification limits of the improved Nile red staining method were 0.8 mu g/mL and 2.0 mu g/mL for the neutral lipid standard triolein, respectively. The modified method and a conventional gravimetric determination method provided similar results on replicate samples. The 96-well plate-based Nile red method can be used as a high throughput technique for rapid screening of a broader spectrum of naturally-occurring and genetically-modified algal strains and mutants for high neutral lipid/oil production. (C) 2009 Published by Elsevier B.V.

Enhanced protection against oxidative stress in an astaxanthin-overproduction Haematococcus mutant (Chlorophyceae)

Many unicellular green algae can become yellow or red in various natural habitats due to mass accumulation of a secondary carotenoid, such as lutein, or astaxanthin. The accumulation of secondary carotenoids is generally thought to be a survival strategy of the algae under photo-oxidative stress or other adverse environmental conditions. The physiological role of the carotenoids in stress response is less well understood at the subcellular or molecular level. In this study, a stable astaxanthin overproduction mutant (MT 2877) was isolated by chemical mutagenesis of a wild type (WT) of the green microalga Haematococcus pluvialis Flotow NIES-144. MT 2877 was identical to the WT with respect to morphology, pigment composition, and growth kinetics during the early vegetative stage of the life cycle. However, it had the ability to synthesize and accumulate about twice the astaxanthin content of the WT under high light, or under high light in the presence of excess amounts of ferrous sulphate and sodium acetate. Under stress, the mutant exhibited higher photosynthetic activities than the WT, based on considerably higher chlorophyll fluorescence induction, chlorophyll autofluorescence intensities, and oxygen evolution rates. Cell mortality caused by stress was reduced by half in the mutant culture compared with the WT. Enhanced protection of the mutant against stress is attributed to its accelerated carotenogenesis and accumulation of astaxanthin. Our results suggest that MT 2877, or other astaxanthin overproduction Haematococcus mutants, may offer dual benefits, as compared with the wild type, by increasing cellular astaxanthin content while reducing cell mortality during stress-induced carotenogenesis.

Bifunctional enzyme FBPase/SBPase is essential for photoautotrophic growth in cyanobacterium Synechocystis sp PCC 6803

From a random insertion mutant library of Synechocystis sp. PCC 6803, a mutant defective in photoautotrophic growth was obtained. The interrupted gene was identified to be slr2094 (rbpl), which encodes the fructose-1,6-biphosphatase (FBPase)/sedoheptulose-1,7-biphosphatase (SBPase) bifunctional enzyme (F-I). Two other independently constructed slr2094 mutants showed an identical phenotype. The FBPase activity was found to be virtually lacking in an slr2094 mutant, which was sensitive to light under mixotrophic growth conditions. These results indicate that slr2094 is the only active FBPase-encoding gene in this cyanobacterium. Inactivation of photosystem II by interrupting psbB in slr2094 mutant alleviated the sensitiveness to light. This report provides the direct genetic evidence for the essential role of F-I in the photosynthesis of Synechocystis sp. PCC 6803. (c) 2007 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

alpha-tocopherol is essential for acquired chill-light tolerance in the cyanobacterium Synechocystis sp strain PCC 6803

Unlike Escherichia coli, the cyanobacterium Synechocystis sp. strain PCC 6803 is insensitive to chill (5 degrees C) in the dark but rapidly losses viability when exposed to chill in the light (100 mu mol photons m(-2) s(-1)). Preconditioning at a low temperature (15 degrees C) greatly enhances the chill-light tolerance of Synechocystis sp. strain PCC 6803. This phenomenon is called acquired chill-light tolerance (ACLT). Preconditioned wild-type cells maintained a substantially higher level of alpha-tocopherol after exposure to chill-light stress. Mutants unable to synthesize alpha-tocopherol, such as slr1736, slr1737, slr0089, and slr0090 mutants, almost completely lost ACLT. When exposed to chill without light, these mutants showed no or a slight difference from the wild type. When complemented, the slr0089 mutant regained its ACLT. Copper-regulated expression of slr0090 from P-petE controlled the level of et-tocopherol and ACLT. We conclude that alpha-tocopherol is essential for ACLT of Synechocystis sp. strain PCC 6803. The role of a-tocopherol in ACLT may be based largely on a nonantioxidant activity that is not possessed by other tocopherols or pathway intermediates.

A gene cluster that regulates both heterocyst differentiation and pattern formation in Anabaena sp strain PCC 7120

Wild-type Anabaena sp. strain PCC 7120, a filamentous nitrogen-fixing cyanobacterium, produces single heterocysts at semi-regular intervals. asr0100 (patU5) and alr0101 (patU3) are homologous to the 5' and 3' portions of patU of Nostoc punctiforme. alr0099 (hetZ) overlaps the 5' end of patU5. hetZ, patU5 and patU3 were all upregulated, or expressed specifically, in proheterocysts and heterocysts. Mutants of hetZ showed delayed or no heterocyst differentiation. In contrast, a patU3 mutation produced a multiple contiguous heterocyst (Mch) phenotype and restored the formation of otherwise lost intercalary heterocysts in a patA background. Decreasing the expression of patU3 greatly increased the frequency of heterocysts in a mini-patS strain. Two promoter regions and two principal, corresponding transcripts were detected in the hetZ-patU5-patU3 region. Transcription of hetZ was upregulated in a hetZ mutant and downregulated in a patU3 mutant. When mutants hetZ::C.K2 and hetZ::Tn5-1087b were nitrogen-deprived, P-hetC-gfp was very weakly expressed, and in hetZ::Tn5-1087b, P-hetR-gfp was relatively strongly expressed in cells that had neither a regular pattern nor altered morphology. We conclude that the hetZ-patU5-patU3 cluster plays an important role in co-ordination of heterocyst differentiation and pattern formation. The presence of homologous clusters in filamentous genera without heterocysts is suggestive of a more general role.

Predicted glycosyl transferase genes located outside the HEP island are required for formation of heterocyst envelope polysaccharide in Anabaena sp strain PCC 7120

During maturation, heterocysts form an envelope layer of polysaccharide, called heterocyst envelope polysaccharide (HEP), whose synthesis depends on a cluster of genes, the HEP island, and on an additional, distant gene, hepB, or a gene immediately downstream from hepB. We show that HEP formation depends upon the predicted glycosyl transferase genes all4160 at a third locus and alr3699, which is adjacent to hepB and is cotranscribed with it. Mutations in the histidine kinase genes hepN and hepK appear to silence the promoter of hepB and incompletely down-regulate all4160.