维生素C生产用菌系2980产酸菌基因转移的筛选模型及转座子Tn5诱变

维生素Ｃ的生产目前国内广泛采用由产酸菌（Ｇｌｕｃｏｎｏｂａｃｔｅｒｏｘｙｄａｎｓ）与伴生菌（Ｂａｃｉｌｕｓｍｅｇａｔｅｒｉｕｍ）组成的２９８０菌系，在２９８０中Ｇ．ｏｘｙｄａｎｓ单独生长传代困难，其生长和产酸需要Ｂ．ｍｅｇａｔｅｒｉｕｍ参与。以ＢａｃｉｌｕｓｓｕｂｔｉｌｉｓＫｉ２１３２（ｐＵＢ１１０）作为伴生菌与原２９８０的Ｇ．ｏｘｙｄａｎｓ组合，获得稳定产酸的新菌系。在此基础上建立了适合我国混菌发酵产酸菌外源基因（Ｋａｎｒ）转移的筛选模型。同时报道了以携带有自杀性载体Ｐ１：：Ｔｎ５的大肠杆菌Ｅ．ｃｏｌｉＷ３１１０为供体菌对Ｇ．ｏｘｙｄａｎｓ进行Ｔｎ５诱变的条件和结果。

以转座子Tn5作弗氏中华根瘤菌的可识别生态学标记的研究──Tn5的水平转移及其对R．frediiTn5突变株运动的影响

将一株弗氏中华根瘤菌（Ｒ．ｆｒｅｄｉｉ）ＱＢ１１３０的Ｔｎ５插入突变株ＯＮ－２用于生态学研究，以评估Ｔｎ５在自然环境中的水平转移以及各种水势下Ｔｎ５对突变株ＯＮ－２在土壤中运动的影响．试验表明，在自然潮湿的土壤中，Ｔｎ５本身的水平转移频率很低，且与Ｔｎ５插入相关的突变株卡那霉素抗性表型标记在非选择性平板上连续传４０代后仍然稳定．突变株ＯＮ－２与相对应的野生型菌株ＱＢ１１３０在各种相同水势的土壤中的运动无明显差异（Ｐ＝０．０１），表明Ｔｎ５的插入不影响突变株的运动．因此，Ｔｎ５可作为研究Ｒ．ｆｒｅｄｉｉ基因工程菌大回应用的一个稳定有效的生态学标记．

鱼腥藻PCC7120基因组中一个影响异形胞发育和图式形成的新区域

以Tn5 10 87b诱变鱼腥藻PCC712 0 ,筛选不能利用氮气生长、异形胞图式发生变化的突变株。突变株 # 180 1经缺氮诱导 2 4h后无异形胞形成 ,48h后沿藻丝形成少量成熟异形胞 ,占藻细胞总数比例为 2 .8% ,其异形胞发育速度和形成频率均与野生型有显著区别。以ClaⅠ酶切该突变株总DNA、自环化后以电泳冲法转化大肠杆菌 ,回收带有插入位点两侧DNA片段的转座子Tn5 10 87b。经测序确定转座子位于未知功能基因alr0 0 99第 14 8和 14 9碱基之间。为证明突变性状确由a

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.

alr0117, a two-component histidine kinase gene, is involved in heterocyst development in Anabaena sp PCC 7120

Anabaena sp. PCC; 7120 was mutagenized by transposon Tn5-1087b, generating a mutant whose heterocysts lack the envelope polysaccharide layer. The transposon was located between nucleotides 342 and 343 of alr0117, a 918 bp gene encoding a histidine kinase for a two-component regulatory system. Complementation of the mutant with a DNA fragment containing alr0117 and targeted inactivation of the gene confirmed that alr0117 is involved in heterocyst development. RT-PCR showed that alr0117 was constitutively expressed in the presence or absence of a combined-nitrogen source. hepA and patB, the two genes turned on during wild-type heterocyst development, were no longer activated in an alr0117-null mutant. The two-component signal transduction system involving alr0117 may control the formation of the envelope polysaccharide layer and certain late events essential to the function of heterocysts.

Construction and analysis of a high-CO2-requiring mutant of the cyanobacterium Anabaena sp strain PCC7120

A mutant of Anabaena sp. strain PCC7120 requiring high CO2 was generated using Tn5 mutagenesis. This is the first data for a filamentous cyanobacterium. The mutant was capable of growing at 5% CO2, but incapable of growing at air levels of CO2. Southern hybridization analysis indicated that the Anabaena genome was inserted by the transposon at one site. The apparent photosynthetic affinity of the mutant to external dissolved inorganic carbon (DIC) was about 300 times lower that of the wild type (WT), and the medium alkalization rate as well as the carboxysomal carbonic anhydrase activity of the mutant was also lower than those of the WT. When the mutant was transferred from the culture medium bubbled with 5% CO2 to higher DIC (8.4% CO2) or 1% CO2, it showed similar responses to the WT. However, aberrant carboxysomes were found in the mutant cells through ultrastructural analysis, indicating it was most probably the wrong organization of the carboxysomes that eventually led to the inefficient operation of carboxysomal carbonic anhydrase and the subsequent defectiveness of the mutant in utilizing DIC.

第二固氮酶系的研究

从四川攀枝花钒铁矿周围的土壤中分离出一株具有较高乙炔还原活性的固氮菌，经鉴定为棕色固氮菌(Azotobacter vinelandii)。在Asby's选择性培养基上产生黄绿色萤光色素，还原乙炔，当培养基中加入lUMN_aVO_3·2H_2O时生长旺盛，产生深绿色萤光，具有较高的乙炔还原作用，实验表明，该菌株含有第二固氮酶，用Tn5对该菌进行接合诱变，获得了大量突变株，但进一步筛选nifH-突变株遇到了困难，可能是由于接合诱变效率太低的原因。用含有克氏肺炎杆菌nifH基因的质粒PPC1201对该菌进行定点整合诱变，得到了29株抗氨苄菁老素的菌株。进一步乙炔还原活性测定表明，其中可能含有14株第一固氮酶nifH基因突变的菌株，10株在第二固氮酶nifH突变的菌株，2株可能在第三固氮酶的nifH-like基因或在三个固氮酶结构基因之处的区域突变的菌株，3株可能在第一、第二固氮酶所共有的基因区域发生突变的菌株。

慢生大豆根瘤菌(Bradyrhizobiumjaponicum)聚羟丁酸合成酶基因(phbC)的克隆和序列测定

以苜蓿根瘤菌Rm10 2 1的 phaC基因突变体菌株Rm1114 4 (phaC ::Tn5 - 2 33)为受体菌 ,通过功能互补 ,成功地从构建的Bradyrhizobium japonicum USDA110基因文库中 ,筛查到能与Rm1114 4互补 ,使之恢复在以乙酰乙酸为唯一碳源的M9培养基 (M9-AA)平板上 5d形成明显可见菌落 ,以及在MOPS平板上形成粘液型菌落的表型的重组粘粒 pDC2 ;经证实 ,该粘粒带有 phbC基因 .完成了该基因的全序列测定并已在GenBank登记 ,登记号为AY0 775 80 .B .japonicumphbC基因由 180 3碱基对组成 ,GC含量 6 1.8% ,AT含量 38.2 % ;编码 6 0 0个氨基酸 ,Mr=6 6 .95× 10 3 .图 3表 3参 13

慢生型大豆根瘤菌(Bradyrhizobiumjaponicum)聚羟丁酸合成酶基因(phbC)突变体的构建及其特性

通过转座子Tn5诱变和同源重组 ,构建了BradyrhizobiumjaponicumUSDA110聚羟丁酸合成酶基因 (phbC)突变体 .序列测定确定了转座子插入的精确位置 ,所获得的 4个转座子诱变的质粒其Tn5插在 phbC基因内两个相距仅 9bp的位点 .被Southern和PCR证实的突变体菌株仍能产生相当于野生型菌株 12 .97%～ 2 5 .10 %的PHB ,并且在突变体和野生型菌株总DNA杂交图上都呈现出一条约 5kb的阳性带 ,推测在B .japonicum基因组中存在不止一个聚羟丁酸合成酶基因 .图 3表 4参 17

苜蓿根瘤菌聚羟丁酸代谢突变体的竞争生长和结瘤能力以及外源生物素的影响

对苜蓿根瘤菌(Sinorhizobium meliloti)聚羟丁酸(PHB)代谢突变体与野生型菌株之间,以及不同突变体之间的竞争生长和竞争结瘤能力在不同培养条件下进行了测定,并研究了外源生物素对各突变体竞争生长和竞争结瘤能力的影响。结果表明:①PAbC 突变体菌株与野生型菌株共培养,不论培养基中添加、不添加外源生物素,pAbC 突变体均表现出生长竞争能力的严重缺陷;竞争结瘤实验也显示,该突变体同野生型菌株竞争结瘤能力大幅下降;说明 PHB 合成能力的缺陷影响了菌株的竞争生长和竞争结瘤能力。②bdhA 突变体与野生型菌株共培养,在不添加外源生物素的情况下,bdhA 突变体同野生菌株竞争生长的能力有明显缺陷,但在添加外源生物素的情况下,其竞争生长能力有明显提高;bdhA::Tn5突变体与 phbC::Tn5-233突变体共培养,如培养基中不添加外源生物素,二者间的竞争生长能力无大的差异;但若添加外源生物素,则 bdhA 突变体的竞争生长能力明显高于 phbC 突变体;表明外源生物素对 bdhA 突变体的竞争生长能力有重要作用。