生物反应器培养转基因鱼腥藻的研究

在反应器中研究了转人TNF-α基因鱼腥藻7120(Anabaena sp．PCC7120,pDC-TNF)的培养。结果表明气升式反应器适合于转基因鱼腥藻的培养。气升式反应器中通气量和光照是主要的影响因素,观察到1L罐中最适通气量为60～75L／h,最适光照强度为1200lx,此时在25℃混养,光照时间／黑暗时间为12h／12h,15d生物量干重大于3g／L,TNF表达水平约占总可溶蛋白的22％,达到了摇瓶培养水平。实验发现添加维生素B1 300μg／L、B12 200μg／L和生物素4μg／L时,生产周期为12d,缩短20％,表达水平相同。培养过程通入含有5％CO2的空气,能促进生长,缩短生产周期,但收获生物量不受影响。从添加维生素和通入CO2的培养结果证明反应器中培养时,光照是限制性因素,当反应器系统一定时,最终生物量有一个最大值,如需进一步提高产量,必须设法改变光照系统。     

Anabaena sp. strain PCC 7120 hetY gene influences heterocyst development

The filamentous cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120 responds to starvation for fixed nitrogen by producing a semiregular pattern of nitrogen-fixing cells called heterocysts. Overexpression of the hetY gene partially suppressed heterocyst formation, resulting in an abnormal heterocyst pattern. Inactivation of hetY increased the time required for heterocyst maturation and caused defects in heterocyst morphology. The 489-bp hetY gene (alr2300), which is adjacent to patS (asl2301), encodes a protein that belongs to a conserved family of bacterial hypothetical proteins that contain an ATP-binding motif.     

FtsZ degradation in the cyanobacterium Anabaena sp. strain PCC 7120

In prokaryotes, cell division is normally achieved by binary fission, and the key player FtsZ is considered essential for the complete process. In cyanobacteria, much remains unknown about several aspects of cell division, including the identity and mechanism of the various components involved in the division process. Here, we report results obtained from a search of the players implicated in cell division, directly associating to FtsZ in the filamentous, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Histidine tag pull-downs were used to address this question. However, the main observation was that FtsZ is a target of proteolysis. Experiments using various cell-free extracts, an unrelated protein, and protein blot analyses further supported the idea that FtsZ is proteolytically cleaved in a specific manner. In addition, we show evidence that both FtsZ termini seem to be equally prone to proteolysis. Taken together, our data suggest the presence of an unknown player in cyanobacterial cell division, opening up the possibility to investigate novel mechanisms to control cell division in Anabaena PCC 7120.     

碳氮源对转基因鱼腥藻Anabaena sp.PCC7120培养的影响

对碳源、氮源种类和用量对转rhTNF-α基因鱼腥藻7120（Anabaena sp.PCC7120）培养的影响进行了研究,发现最适碳源为蔗糖,最适氮源为NaNO3,最佳用量分别为9g/L和2．25g/L,此时生物量远高于自养方式,达2．52g/L,比相同条件下在BG－11培养基培养高71．66％,ATNF－α表达量为16％－22％,生物活性为10^5U/mg。     

Methyl viologen responsive proteome dynamics of Anabaena sp. strain PCC7120

A proteomic approach was employed to elucidate the response of an agriculturally important microbe, Anabaena sp. strain PCC7120, to methyl viologen (MV). Exposure to 2 μM MV caused 50% lethality (LD₅₀) within 6 h and modified the cellular levels of several proteins. About 31 proteins increased in abundance and 24 proteins decreased in abundance, while 55 proteins showed only a minor change in abundance. Of these, 103 proteins were identified by MS. Levels of proteins involved in ROS detoxification and chaperoning activities were enhanced but that of crucial proteins involved in light and dark reactions of photosynthesis declined or constitutive. The abundance of proteins involved in carbon and energy biogenesis were altered. The study elaborated the oxidative stress defense mechanism deployed by Anabaena, identified carbon metabolism and energy biogenesis as possible major targets of MV sensitivity, and suggested potential biotechnological interventions for improved stress tolerance in Anabaena 7120.     

patS minigenes inhibit heterocyst development of Anabaena sp. strain PCC 7120

The patS gene encodes a small peptide that is required for normal heterocyst pattern formation in the cyanobacterium Anabaena sp. strain PCC 7120. PatS is proposed to control the heterocyst pattern by lateral inhibition. patS minigenes were constructed and expressed by different developmentally regulated promoters to gain further insight into PatS signaling. patS minigenes patS4 to patS8 encode PatS C-terminal 4 (GSGR) to 8 (CDERGSGR) oligopeptides. When expressed by P(petE), P(patS), or P(rbcL) promoters, patS5 to patS8 inhibited heterocyst formation but patS4 did not. In contrast to the full-length patS gene, P(hepA)-patS5 failed to restore a wild-type pattern in a patS null mutant, indicating that PatS-5 cannot function in cell-to-cell signaling if it is expressed in proheterocysts. To establish the location of the PatS receptor, PatS-5 was confined within the cytoplasm as a gfp-patS5 fusion. The green fluorescent protein GFP-PatS-5 fusion protein inhibited heterocyst formation. Similarly, full-length PatS with a C-terminal hexahistidine tag inhibited heterocyst formation. These data indicate that the PatS receptor is located in the cytoplasm, which is consistent with recently published data indicating that HetR is a PatS target. We speculated that overexpression of other Anabaena strain PCC 7120 RGSGR-encoding genes might show heterocyst inhibition activity. In addition to patS and hetN, open reading frame (ORF) all3290 and an unannotated ORF, orf77, encode an RGSGR motif. Overexpression of all3290 and orf77 under the control of the petE promoter inhibited heterocyst formation, indicating that the RGSGR motif can inhibit heterocyst development in a variety of contexts.     

Lipopolysaccharide dependence of cyanophage sensitivity and aerobic nitrogen fixation in Anabaena sp. strain PCC 7120.

Fox- mutants of Anabaena sp. strain PCC 7120 are unable to fix dinitrogen in the presence of oxygen. A fragment of the DNA of Anabaena sp. was cloned by complementation of a spontaneous Fox-, cyanophage-resistant mutant, R56, and characterized. Random insertion of transposon Tn5 delimited the complementing DNA to a 0.6-kb portion of the cloned fragment. Sequencing of this region and flanking DNA showed one complete open reading frame (ORF) similar to the gene rfbP (undecaprenyl-phosphate galactosephosphotransferase) and two partial ORFs similar to genes rfbD (GDP-D-mannose dehydratase) and rfbZ (first mannosyl transferase), all of which are active in the synthesis of the O antigen unit of the lipopolysaccharide (LPS) component of the outer membrane of gram-negative bacteria. In a transposon (Tn5-1087b)-induced, Fox-, cyanophage-resistant mutant, B14, the transposon was found within the same rfbP-like ORF. The three ORFs were insertionally inactivated with the omega cassette (P. Prentki and H. M. Krisch, Gene 29:303-313, 1984) or with Tn5::omega. Only the insertions in the rfbZ- and rfbP-like ORFs led to resistance to cyanophages A-1(L) and A-4(L) and to a Fox- phenotype. Electrophoretic analysis showed that interruption of the rfbZ- and rfbP-like ORFs resulted in a change in or loss of the characteristic pattern of the lengths of the LPS, whereas interruption of the rfbD-like ORF merely changed the distribution of the lengths of the LPS to one with a greater prevalence of low molecular weights. According to electron microscopy, interruption of the rfbP-like ORF may have led to aberrant deposition of the layers of the heterocyst envelope, resulting in increased leakage of oxygen into the heterocyst. The results suggest that modified LPS may prevent cyanophage infection of Anabaena sp. vegetative cells and the formation of a functional heterocyst envelope.     

Novel DNA-binding proteins in the cyanobacterium Anabaena sp. strain PCC 7120

As an approach towards elucidation of the biochemical regulation of the progression of heterocyst differentiation in Anabaena sp. strain PCC 7120, we have identified proteins that bind to a 150-bp sequence upstream from hepC, a gene that plays a role in the synthesis of heterocyst envelope polysaccharide. Such proteins were purified in four steps from extracts of vegetative cells of Anabaena sp. Two of these proteins (Abp1 and Abp2) are encoded by neighboring genes in the Anabaena sp. chromosome. The genes that encode the third (Abp3) and fourth (Abp4) proteins are situated at two other loci in that chromosome. Insertional mutagenesis of abp2 and abp3 blocked expression of hepC and hepA and prevented heterocyst maturation and aerobic fixation of N(2).     

Isolation and complementation of nitrogen fixation mutants of the cyanobacterium Anabaena sp. strain PCC 7120.

Approximately 140 mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on media lacking fixed nitrogen (Fix-) were isolated after mutagenesis with diethyl sulfate and penicillin enrichment. A large cosmid library of wild-type Anabaena sp. strain PCC 7120 DNA was constructed in a mini-RK-2 shuttle vector, and seven mutants representing several morphologically abnormal heterocyst phenotypes were complemented. One of these mutants, 216, failed to differentiate heterocysts. All of these mutants except 216 reduced acetylene under anaerobic conditions, indicating that they are not defective in nitrogen fixation per se. Several cosmids were isolated from each complemented mutant and in most cases showed similar restriction patterns. Comparisons of the complementing cosmids from mutant 216 and two other phenotypically distinct mutants by restriction enzyme analysis identified a common region. This region, when present in either a cosmid or a 9.5-kb NheI subclone, is capable of efficiently complementing all three mutants. A 2.4-kb subclone of this region complements mutant 216 only.     

Protein tyrosine phosphorylation in the cyanobacterium Anabaena sp. strain PCC 7120.

Components of a protein tyrosine phosphorylation/dephosphorylation network were identified in the cyanobacterium Anabaena sp. strain PCC 7120. Three phosphotyrosine (P-Tyr) proteins of 27, 36, and 52 kDa were identified through their conspicuous immunoreactions with RC20H monoclonal antibodies specific for P-Tyr. These immunoreactions were outcompeted completely by free P-Tyr (5 mM) but not by phosphoserine or phosphothreonine. The P-Tyr content of the three major P-Tyr proteins and several minor proteins increased with their time of incubation in the presence of Mg-ATP and the protein phosphatase inhibitors sodium orthovanadate and sodium fluoride. Incubation of the same extracts with [gamma-32P]ATP but not [alpha-32P]ATP led to the phosphorylation of five polypeptides with molecular masses of 20, 27, 52, 85, and 100 kDa. Human placental protein tyrosine phosphatase 1B, with absolute specificity for P-Tyr, liberated significant quantities of 32Pi from four of the polypeptides, confirming that a portion of the protein-bound phosphate was present as 32P-Tyr. Alkaline phosphatase and the dual-specificity protein phosphatase IphP from the cyanobacterium Nostoc commune UTEX 584 also dephosphorylated these proteins and did so with greater apparent efficiency. Two of the polypeptides were partially purified, and phosphoamino analysis identified 32P-Tyr, [32P]phosphoserine, and [32P]phosphothreonine. Anabaena sp. strain PCC 7120 cell extracts contained a protein tyrosine phosphatase activity that was abolished in the presence of sodium orthovanadate and inhibited significantly by the sulfhydryl-modifying agents p-hydroxymercuriphenylsulfonic acid and p-hydroxymercuribenzoate as well as by heparin. In Anabaena sp. strain PCC 7120 the presence and/or phosphorylation status of P-Tyr proteins was influenced by incident photon flux density.     

pH值对转基因鱼腥藻培养的影响

研究pH值对转基因鱼腥藻培养的影响,发现培养基初始pH以自然pH8.3为宜,间歇流加稀盐酸控制过程pH值为8.5和9.0,生长较未流加快,每天流加稀盐酸一次控制过程pH为8.5,15d生物量大于3g/L,可溶性蛋白为0.3g/l藻干重,TNF表达量超过22%,生物量比未流加增长12.41%,而TNF表达量未受影响。     

Proteomic analysis of the outer membrane of Anabaena sp. strain PCC 7120

Anabaena is a model to analyze the evolutionary development of plastids, cell differentiation, and the regulation of nitrogen fixation. Thereby, the outer membrane proteome is the place of sensing environmental differences and during plastid development, systems for intracellular communication had to be added to the proteome of this membrane. We present a protocol for the isolation of the outer membrane from Anabaena and the analysis of the proteome using different tools. 55 proteins were identified.     

Cold-stress-altered phosphorylation of EF-Tu in the cyanobacterium Anabaena sp. strain PCC 7120

Growth of prokaryotes at reduced temperature results in the formation of a cold-adapted ribosome through association with de novo synthesized polypeptides. In vitro and in vivo phosphorylation studies combined with affinity purification and mass spectrometry identified that the phosphorylation status of translation elongation factor EF-Tu was altered in response to cold stress in the photosynthetic, Gram-negative cyanobacterium Anabaena sp. strain PCC 7120. In response to a temperature downshift from 30 to 20 degrees C, EF-Tu was rapidly and transiently hyperphosphorylated during the acclimation phase followed by a reduction in phosphorylation below background levels in response to prolonged exposure. EF-Tu was identified as a phosphothreonine protein. Unexpectedly, ribosomal protein S2 was also observed to be a phosphoprotein continuously phosphorylated during cold stress. The phosphorylation status of EF-Tu has previously been associated with translational regulation in other systems, with a reduction in translation elongation occurring in response to phosphorylation. These results provide evidence for a novel mechanism by which translation is initially downregulated in response to cold stress in Anabaena.     

鱼腥蓝细菌PCC7120铁吸收机制

铁离子是鱼腥蓝细菌PCC7120进行呼吸作用、光合作用和固氮作用中相关酶的重要辅基之一,缺铁将严重影响蓝细菌的生存.富氧的生态环境中铁通常以不溶的Fe3+形式存在,不易被细胞吸收利用.低铁条件下,鱼腥蓝细菌PCC7120分泌能螯合铁离子的嗜铁素,通过外膜上相应的转运体将嗜铁素-铁复合物转运到细胞内.综述了近年来在嗜铁素的种类及其生物合成途径、铁吸收系统的组成和功能等方面的最新进展,分析了铁吸收系统的调控机制,为进一步开展鱼腥蓝细菌铁吸收机制的研究提供依据.     

Iron starvation leads to oxidative stress in Anabaena sp. strain PCC 7120

We establish here that iron deficiency causes oxidative stress in the cyanobacterium Anabaena sp. strain PCC 7120. Iron starvation leads to a significant increase in reactive oxygen species, whose effect can be abolished by treatment with the antioxidant tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl). Oxidative stress induced by iron starvation could be a common feature of photosynthetic bacteria.     

Overexpression of pknE blocks heterocyst development in Anabaena sp. strain PCC 7120

The upstream intergenic regions for each of four genes encoding Ser/Thr kinases, all2334, pknE (alr3732), all4668, and all4838, were fused to a gfpmut2 reporter gene to determine their expression during heterocyst development in the cyanobacterium Anabaena (Nostoc) sp. strain PCC 7120. P(pknE)-gfp was upregulated after nitrogen step-down and showed strong expression in differentiating cells. Developmental regulation of pknE required a 118-bp upstream region and was abolished in a hetR mutant. A pknE mutant strain had shorter filaments with slightly higher heterocyst frequency than did the wild type. Overexpression of pknE from its native promoter inhibited heterocyst development in the wild type and in four mutant backgrounds that overproduce heterocysts. Overexpression of pknE from the copper-inducible petE promoter did not completely inhibit heterocyst development but caused a 24-h delay in heterocyst differentiation and cell bleaching 4 to 5 days after nitrogen step-down. Strains overexpressing pknE and containing P(hetR)-gfp or P(patS)-gfp reporters failed to show developmental regulation of the reporters and had undetectable levels of HetR protein. Genetic epistasis experiments suggest that overexpression of pknE blocks HetR activity or downstream regulation.     

Heterocyst-specific expression of patB,a gene required for nitrogen fixation in Anabaena sp. strain PCC 7120

The patB gene product is required for growth and survival of the filamentous cyanobacterium Anabaena sp. strain PCC 7120 in the absence of combined nitrogen. A patB::gfp fusion demonstrated that this gene is expressed exclusively in heterocysts. patB mutants have a normal initial pattern of heterocyst spacing along the filament but differentiate excess heterocysts after several days in the absence of combined nitrogen. Expression of hetR and patS, two critical regulators of the heterocyst development cascade, are normal for patB mutants, indicating that patB acts downstream of them in the differentiation pathway. A patB deletion mutant suffers an almost complete cessation of growth and nitrogen fixation within 24 h of combined nitrogen removal. In contrast, a new PatB mutant that is defective in its N-terminal ferredoxin domain, or a previously described mutant that has a frameshift removing its C-terminal helix-turn-helix domain, grows very slowly and differentiates multiple contiguous heterocysts under nitrogen-deficient conditions.     

环境因子对转基因鱼腥藻培养的影响

摇瓶中对转TNF-α基因鱼腥藻7120（Anabaenasp.PCC7120,pDC-TNF)混合培养条件进行了研究,在含蔗糖9g/L,NaNO32.25g/L的BG-11培养基混合培养时,得到最适培养条件接种量5%,光照强度为1000Lux,光/暗周期（光照时间/黑暗时间）12h/12h,温度25-30℃,自然初始pH值,100mL摇瓶装液量40mL,转基因鱼腥藻15d生物量可达到3g/L以上,可溶性蛋白含量接近30%,TNF表达水平大于22%,与自养相比,生物量增加82.06%,表达水平提高38.79%,证明混合营养型培养是转rhTNF-α基因鱼腥藻7120实现高密度,高表达培养的途径。