产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因（CgGPD）功能分析

【目的】从高产甘油生产菌株产甘油假丝酵母（Candida glycerinogenes）基因组中克隆了NAD+依赖3-磷酸甘油脱氢酶编码基因（CgGPD）,但是该基因及其上游调控序列具体的功能还是未知的。本文研究了CgGPD基因及其上游调控序列的功能。【方法】本文以酿酒酵母（Saccharomyces cerevisiae）及其渗透压敏感型突变株为宿主,构建3种不同的酵母表达载体导入酵母细胞,研究了不同酵母转化子在渗透压胁迫条件下CgGPD基因表达对细胞的耐高渗透压胁迫应答及其细胞的甘油合成能力的影响。【结果】实验结果表明无论是以来源于S. cerevisiae 的TPI启动子还是来源于CgGPD基因的启动子,过量表达CgGPD基因的转化子均能够显著加速葡萄糖消耗速度和提高甘油合成能力,在gpd1/gpd2突变株中表达CgGPD基因能够消除细胞对外界高渗透压的敏感性,同时转化子胞内甘油大量积累。【结论】CgGPD基因在野生型酵母S. cerevisiae W303-1A表达显著提高细胞的甘油合成能力,在gpd/1gpd2突变株中能够互补GPD1基因的功能,CgGPD基因表达受渗透压诱导 调控。     

进化代谢选育高渗透压耐受型产琥珀酸大肠杆菌

在以碳酸钠为酸中和剂的大肠杆菌两阶段发酵产琥珀酸的过程中,由于Na+的积累造成发酵体系中渗透压的提高,严重抑制了琥珀酸的产物浓度。为了增强大肠杆菌对渗透压的耐受性,考察了利用进化代谢方法筛选高渗透压耐受型高产琥珀酸大肠杆菌菌株的可行性。进化代谢系统作为一种菌株突变装置,可以使菌体在连续培养条件下以最大的生长速率生长。以NaCl为渗透压调节剂,通过在连续培养装置中逐步提高NaCl浓度使菌体在高渗透压条件下快速生长,最终得到了一株高渗透压耐受型琥珀酸生产菌株Escherichia coli XB4。以碳酸钠为酸中和剂,在7 L发酵罐中利用Escherichia coli XB4进行两阶段发酵,厌氧培养60 h后,琥珀酸产量达到了69.5 g/L,琥珀酸生产速率达到了1.81 g/(L.h),分别比出发菌株提高了18.6%和20%。     

大肠杆菌Zn2+敏感突变株构建及其功能验证

【背景】Zn²⁺在细胞解毒及许多生理过程中发挥着关键作用,Zn²⁺转运蛋白已逐渐引起人们的重视。在大肠杆菌中,zntA和zitB是2个外排Zn²⁺的关键基因。【目的】构建大肠杆菌Zn²⁺敏感突变株,并对其功能进行验证。【方法】以Escherichia coli DH5α为出发菌株,利用λ Red重组系统,通过携带卡那霉素抗性基因的同源重组片段敲除zntA基因。在单基因敲除菌株基础上,利用携带庆大霉素抗性基因的同源重组片段敲除zitB基因,获得一株敲除了zntA和zitB的双基因敲除菌株KZAB04。通过功能互补实验检测基因敲除菌株及对照菌株对不同浓度Zn²⁺的敏感程度。【结果】基因敲除菌株KZAB04比出发菌株E.coli DH5α具有更高的Zn²⁺敏感性。【结论】大肠杆菌Zn²⁺敏感突变株构建成功。该菌株的构建为zntA和zitB基因功能的研究提供了必要条件,同时也为其他Zn²⁺转运蛋白基因的功能鉴定与分析奠定了基础。     

一种快速、精确构建大肠杆菌组氨酸营养缺陷型的方法

将表达Red体内重组蛋白的质粒pKD46转化大肠杆菌：DH5α,用5′端与组氨酸基因同源,3′端与卡那霉素抗性基因同源的引物获得具有卡那霉素抗性基因的PCR产物,然后电击转化DH5α,在λRed重组系统的帮助下,通过卡那霉素抗性基因两侧的组氨酸基因序列在体内与大肠杆菌染色体上的组氨酸基因发生同源重组,置换了DH5α组氨酸操纵元中的hisDCB基因,最后利用卡那霉素抗性基因两端的FRT位点,通过FTP位点专一性重组将卡那霉素抗性基因去除,最终获得了不具抗性的大肠杆菌组氨酸营养缺陷型菌株。为在大肠杆菌及其他菌株中快速、精确的构建营养缺陷型菌株提供了有益的参考。     

大肠杆菌pqqL基因敲除与功能分析

【目的】通过构建大肠杆菌pqqL基因缺陷突变株,研究大肠杆菌pqqL基因的功能。【方法】首先通过PCR扩增得到pqqL基因和kan抗性基因,在体外构建线性打靶片段pqqL-kan-pqqL。然后通过Red同源重组敲除大肠杆菌的pqqL基因,构建大肠杆菌缺失突变体DH5αΔpqqL。在此基础上通过DCIP法检测山梨糖脱氢酶活性来比较大肠杆菌突变株与亲本株中PQQ合成的情况。【结果】成功敲除了大肠杆菌的pqqL基因,DCIP法检测结果显示大肠杆菌pBCP162/DH5αΔpqqL和pMD19T Simple-pqqABCDE/DH5α能够合成PQQ,而大肠杆菌pMD19T Simple-pqqABCDE/DH5αΔpqqL不能合成PQQ。【结论】大肠杆菌pqqL基因和pqqF基因具有同样的功能。     

Hsp提高细菌的逆境生存能力和乙醇产量

目的：用热休克蛋白Hsp（HeatShockProteins）基因重组大肠杆菌,改善细胞生长状况、提高大肠杆菌的逆境耐受性和乙醇产量。方法：将来自Pyrococcus加诬凇的基因Hsp与Lac启动子串联,构建成由Lae启动子调控Hsp表达的操纵子,经该操纵子转化的大肠杆菌分别在高渗透压、酸性条件、高温和高糖的条件下发酵,利用气相色谱检测发酵液中的乙醇含量。结果：含有Hsp基因的工程菌与不含Hsp基因的对照菌相比,在0．4mol／LNaCl的高渗透压下乙醇产量提高1．5倍、在pH4．5的酸性条件下提高1．2倍、在高温高糖的条件下提高5．95倍。结论：热休克蛋白Hsp基因的表达可以提高大肠杆菌在逆境中的代谢能力。     

酿酒酵母渗透敏感新基因的定位及其作用机理的研究

本文通过四分子分析将酿酒酵母中另一控制渗透敏感的基因osm3进行了定位。遗传分析表明,osm3是位于染色体Ⅱ上的1个新基因,与gal1座位相距大约45厘摩。其第二次分裂分离频率为51.01%,它与着丝粒的图距为25.51厘摩,属着丝粒连锁基因。回复突变的研究结果表明,渗透敏感性状很可能是因为osm3座位上发生了错义或无义突变所致。 根据渗透敏感菌株和正常菌株对高渗透压反应试验,证明了高的胞内甘油含量是酵母在高渗条件下生长所必需的。osm3菌株不能耐高渗的主要原因是由于甘油转运失调所致,OSM3基因产物可能与甘油的转运过程有关。本文还就高渗对酿酒酵母发酵力的影响进行了讨论。     

构建胸膜肺炎放线杆菌apxIC基因插入突变株

目的：构建胸膜肺炎放线杆菌（APP）apxIC基因插入突变菌株,以鉴定ApxⅠ毒素的生物学特性。方法：根据apxⅠ核酸序列（U05042）设计1对引物,用于自APP血清10型参考菌株（D13039）基因组DNA中扩增apxIC基因及其上下游约2．8kb的基因片段,经克隆测序后在apxIC基因下游xbI酶切位点处插入约0．9kb的氯霉素（Chl）抗性基因表达盒,构建用于转化的转移载体pUIC-Chl^r,将转移载体DNA经电转化导入APP血清10型参考菌株中进行同源重组,以获得突变菌株。结果：在含有氯霉素的培养基中经筛选获得2株丧失溶血活性的突变菌株（D13039C-Chl^r）;利用PCR和Southern blot对突变菌株鉴定,显示氯霉素抗性基因已被插入细菌基因组中。结论：利用电转化和同源重组技术构建成功APP apxIC基因插入突变菌株,为分析ApxⅠ毒素的生物学特性,进而研制APP基因工程减毒活疫苗奠定了基础。     

应用Red重组工程技术建立asd基因缺失的大肠杆菌DH108菌株

目的：建立一株新遗传表型的大肠杆菌DH10BAasd菌株。方法：应用pKD46介导的重组系统、kan／kil选择反选择系统、双链线性DNA重组技术和重叠引物介导的DNA重组技术,在菌株DH10B体内,对其染色体上的asd基因进行了基因敲除。结果：建立了一株二氨基庚二酸（DAP）营养缺陷型重组大肠杆菌DH10BAasd。结论：为进一步建立以大肠杆菌DH10B为载体的DNA疫苗或基因治疗载体奠定了基础。     

改造thrL前导调控元件获取不同灵敏度苏氨酸感应器

[目的]构建不同灵敏度的苏氨酸感应器,以报告基因的表达表征苏氨酸的变化。[方法]通过克隆苏氨酸操纵子thrL前导调控元件,并与红色荧光蛋白基因rfp偶联构建在高拷贝质粒p LX07上,转化进大肠杆菌DH5α;之后又通过设计引物进行PCR改变thrL前导调控元件上苏氨酸密码子的编码数量;最后进行荧光检测实验确定不同突变菌感应环境中苏氨酸特性的不同。[结果]得到了以红色荧光蛋白基因为报告基因而能感应苏氨酸的大肠杆菌以及分别编码4个和10个苏氨酸密码子的thrL突变菌。三种菌株单位OD下红色荧光蛋白的相对表达量均跨2个浓度梯度而负相关于苏氨酸浓度,同一浓度下正相关于苏氨酸密码子编码数量。[结论]通过构建感应苏氨酸大肠杆菌并改造其thrL前导调控元件获得了负相关于苏氨酸浓度且灵敏度不同的苏氨酸感应器。     

Expression of outer membrane proteins in Escherichia coli growing at acid pH

It is generally accepted for Escherichia coli that (i) the level of OmpC increases with increased osmolarity when cells are growing in neutral and alkaline media, whereas the level of OmpF decreases at high osmolarity, and that (ii) the two-component system composed of OmpR (regulator) and EnvZ (sensor) regulates porin expression. In this study, we found that OmpC was expressed at low osmolarity in medium of pH below 6 and that the expression was repressed when medium osmolarity was increased. In contrast, the expression of ompF at acidic pH was essentially the same as that at alkaline pH. Neither OmpC nor OmpF was detectable in an ompR mutant at both acid and alkaline pH values. However, OmpC and OmpF were well expressed at acid pH in a mutant envZ strain, and their expression was regulated by medium osmolarity. Thus, it appears that E. coli has a different mechanism for porin expression at acid pH. A mutant deficient in ompR grew slower than its parent strain in low-osmolarity medium at acid pH (below 5.5). The same growth diminution was observed when ompC and ompF were deleted, suggesting that both OmpF and OmpC are required for optimal growth under hypoosmosis at acid pH.     

Nucleotide sequence of the Serratia marcescens SR50 chromosomal ampC β-lactamase gene

A novel type of osmoregulatory mutant of Escherichia coli K-12 exhibiting constitutive expression of the ompC gene was isolated and characterized at the molecular level In this particular mutant ( cec ; c onstitutive e xpression of Omp C ). an insertion sequence (IS-1) was found to be located at right upstream of the regulatory sequence for the ompC promoter. We demonstrate that the IS1 insertion observed in the cec mutant does not provide the ompC gene with an artificial promoter, but rather perturbs normal regulation of the ompC promoter, which is mediated by the regulatory gene, ompR .     

Escherichia coli mutants with an altered sensitivity to cecropin D.

Cecropins are a family of small, basic antibacterial polypeptides which can be isolated from pupae of immunized Lepidoptera. They are active against both gram-negative and gram-positive bacteria. We studied a mutant of Escherichia coli, strain SB1004, which is more sensitive to cecropin D than is the parental strain. The mutant was selected as resistant to a host range mutant of a Serratia marcescens phage. When the protein composition of the outer membrane was examined, strain SB1004 and some other phage-resistant mutants were found to be deficient in the OmpC protein. It was concluded that the OmpC protein is the receptor of the phage. Strain SB1004 was found to differ from other ompC mutants in being especially sensitive to hydrophobic antibiotics and to cecropin D. Furthermore, strain SB1004 has a tendency for spontaneous autolysis. A genetic analysis showed the mutations in strain SB1004 and a suppressor mutant to map in the ompC region. The activity of cecropin D against different strains of E. coli was specifically enhanced when divalent cations were absent. No such effect was found with cecropins A and B, which are less hydrophobic than the D form.     

OmpC and OmpF are required for growth under hyperosmotic stress above pH 8 in Escherichia coli

AIMS: To investigate the requirement of outer membrane porins for osmotic adaptation at alkaline pH in Escherichia coli. METHODS AND RESULTS: Escherichia coli mutants deficient in ompC, ompF and both genes were constructed and the growth of these mutants was observed at alkaline pH. The growth rate of the mutant deficient in both ompC and ompF was slower than that of the wild type and mutants deficient in one of these genes under hyperosmotic stress at pHs above 8.0. The decreased rate was recovered when a cloned ompC was introduced to the mutant, but the growth recovery with a cloned ompF was partial. Such growth diminution was not observed at pHs below 8.0. CONCLUSION: OmpC and OmpF were shown to participate in hyperosmotic adaptation at alkaline pH in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report to demonstrate that OmpC and OmpF are required for hyperosmotic adaptation at pHs above 8.0, but not below 8.0.     

大肠杆菌ptsG和ptsM突变体的构建及特性研究

目的：敲除大肠杆菌DH5α中与葡萄糖磷酸化转运相关的ptsG、ptsM基因,考察缺陷株生长特性及其可能的应用。方法：PCR扩增靶基因,构建两翼带有靶基因序列并嵌合抗药基因标记的线性片段,利用Red同源重组技术敲除靶基因。结果：成功敲除了大肠杆菌DH5α的ptsG和ptsM基因;在含有葡萄糖的LB培养基中,DH5αΔptsG最高菌密度是亲本的2.8倍,添加吡咯喹啉醌或导入其生物合成基因后能够产酸;DH5αΔptsM最高菌密度是亲本的4/10,有明显的产酸现象。结论：DH5αΔptsG可用于大肠杆菌高密度发酵和吡咯喹啉醌生物合成基因缺陷株筛选。     

The porin OmpC of Salmonella typhimurium mediates adherence to macrophages.

Macrophages recognize, adhere to, and phagocytose Salmonella typhimurium. The major outer membrane protein OmpC is a candidate ligand for macrophage recognition. To confirm this we used transposon mutagenesis to develop an ompC-deficient mutant in a known virulent strain of S. typhimurium; mutant and wild type were compared in macrophage adherence and association assays. Radiolabeled wild type S. typhimurium bound to macrophages at five-fold higher levels than did the ompC mutant. In association assays, macrophages in monolayers bound and internalized three-fold more wild type than mutant, while macrophages in suspension bound and internalized 40-fold more wild type than mutant. The ompC gene of our test strain of S. typhimurium contains several discrete differences compared with the ompC genes of Salmonella typhi and Escherichia coli. The deduced OmpC amino acid sequence of S. typhimurium shares 77 and 98% identity with OmpC amino acid sequence of E. coli and S. typhi, respectively. Evidence from this study supports a role for the OmpC protein in initial recognition by macrophages and distinguishes regions of this protein that potentially participate in host-cell recognition of bacteria by phagocytic cells.     

Effects of the resistance plasmid R124 on the level of the OmpF outer membrane protein and on the response of Escherichia coli to environmental agents

R ossouw F.T. & R owbury R.J.1984. Effects of the resistance plasmid R124 on the level of the OmpF outer membrane protein and on the response of Escherichia coli to environmental agents. Journal of Applied Bacteriology , 56 , 63–79.
The introduction of the F-like resistance plasmid R124 into an ompC mutant of Escherichia coli K12 conferred altered sensitivity to a wide range of inhibitory agents. Sensitivity to ampicillin, chloramphenicol, ethionine, copper ions, deoxycholate, two fatty acids and colicins L and M was decreased by the plasmid. In contrast the plasmid-bearing ompC derivatives were more sensitive than the plasmid-free ompC mutant to erythromycin, cetyltrimethylammonium bromide and phenol. Introduction of R124 into the ompC strain also decreased the level of the OmpF protein and some (but not all) of the changed sensitivities listed above clearly resulted from this outer membrane protein deficiency. The presence in the ompC mutant of R124 (rather than the more efficient introduction of the plasmid into variants of the ompC strain) led to at least most of the changes described above because those tested were accentuated by the presence of a copy mutant of R124 and reversed by plasmid curing.     

Temperature-sensitive mutants of Escherichia coli B which can grow in high-osmotic medium at the nonpermissive temperature

Two temperature sensitive (TS) mutants (C4 tos and D2 tos) were isolated after mutagenesis of E. coli B/SM by N-methyl-N'-nitro-N-nitrosoguanidine (NMNG), which can grow even at 42 C in high-osmotic medium supplemented by addition of sucrose, NaCl or other compounds. Neither of the mutants lyzed when transferred to low-osmotic medium after growning at the nonpermissive temperature in high osmotic medium. One of these mutants, C4 tos, grew at 42 C in a long filamentous form. When bacteria growing exponentially at 30 C were shifted to 42 C, they continued to grow at a reduced rate even in low-osmotic medium. This strain could also grow or start to grow in low-osmotic medium when supplied with a factor or factors secreted from growing bacteria of another strain. This mutant strain could grow in low-osmotic medium at 42 C when it was cultured anaerobically. The other mutant strain obtained (D2 tos) displayed normal morphology even when grown at 42 C. When it was shifted from 30 to 42 C in low-osmotic medium, increase of mass, measured as optical density, continued for a while, but viability, measured as the number of colony-formers, stopped increasing and then decreased rapidly.     

Effects of the resistance plasmid R124 on the level of the OmpF outer membrane protein and on the response of Escherichia coli to environmental agents

The introduction of the F-like resistance plasmid R124 into an ompC mutant of Escherichia coli K12 conferred altered sensitivity to a wide range of inhibitory agents. Sensitivity to ampicillin, chloramphenicol, ethionine, copper ions, deoxycholate, two fatty acids and colicins L and M was decreased by the plasmid. In contrast the plasmid-bearing ompC derivatives were more sensitive than the plasmid-free ompC mutant to erythromycin, cetyltrimethylammonium bromide and phenol. Introduction of R124 into the ompC strain also decreased the level of the OmpF protein and some (but not all) of the changed sensitivities listed above clearly resulted from this outer membrane protein deficiency. The presence in the ompC mutant of R124 (rather than the more efficient introduction of the plasmid into variants of the ompC strain) led to at least most of the changes described above because those tested were accentuated by the presence of a copy mutant of R124 and reversed by plasmid curing.