链球菌噬菌体裂解酶在大肠杆菌中的表达、纯化及活性检测

噬菌体裂解酶是噬菌体产生的细胞壁水解酶,通过水解宿主菌细胞壁使子代噬菌体释放,在体外能高效且特异性地杀死细菌。本研究旨在克隆和表达链球菌噬菌体裂解酶PlyC,并测定其生物学活性。利用PCR方法扩增PlyC的2条肽链PlyCA和PlyCB,构建表达载体pET-32a(+)-PlyCA和pET-32a(+)-PlyCB,分别转化至大肠杆菌BL21(DE3)中,以0.7 mmol/L IPTG在30 oC诱导7 h实现了高效表达,SDS-PAGE分析表明PlyCA和PlyCB表达量均可达菌体总蛋白的30%以上。采用Ni2+-NTA亲和层析法纯化目的蛋白,其纯度大于95%。用透析复性方法得到目的产物重组链球菌噬菌体裂解酶PlyC,以浊度法和平板计数法检测其体外抗菌效果,扫描电子显微镜观察裂解酶作用前后细菌细胞形态变化。结果表明重组PlyC能特异性裂解化脓性链球菌(A组β-溶血性链球菌),以4μg/mL浓度作用于OD600为0.56的菌液60 min后杀菌率达99.6%,扫描电镜观察结果显示该酶作用于菌体后,链球菌细胞裂解,呈碎片状态。本研究为开发一种新型、高效的链球菌感染疾病治疗药物打下了基础。     

一株产L-天冬氨酸酶大肠埃希氏菌的噬菌体分离和生物学特性

【目的】从大肠埃希氏菌CICC 11021S发酵液中分离一株噬菌体,对其生物学特性进行研究。【方法】采用双层平板法分离噬菌体CICC 80003;利用透射电镜观察噬菌体形态;提取噬菌体基因组,核酸内切酶处理并进行凝胶电泳;分析噬菌体最佳感染复数、一步生长曲线、p H和温度稳定性、宿主谱。考察CICC 80003对CICC 11021S生长和L-天冬氨酸酶活力的影响。【结果】CICC 80003噬菌斑圆形透明,有明显晕环;头部规则,直径约50-60 nm,尾部长约120-130 nm;基因组能被核酸内切酶Bam H I和Mlu I切开;最佳感染复数0.1,潜伏期5 min,裂解期25 min,平均裂解量约86个;最适p H值8.0;90°C温育15 min,噬菌体全部失活;能裂解大肠埃希氏菌和沙门氏菌的部分菌株。发生噬菌体污染时,CICC 11021S无法正常生长,基本检测不到L-天冬氨酸酶活力。【结论】CICC 80003属于长尾噬菌体科ds DNA噬菌体,液体环境中能够彻底裂解大肠埃希氏菌CICC 11021S。     

一株裂解性奶牛乳房炎源大肠杆菌噬菌体的分离鉴定及生物学特性分析

【目的】从新疆石河子地区奶牛粪样中分离裂解性大肠杆菌噬菌体(Escherichia coli phage),对其进行纯化及生物学特性分析。【方法】利用双层平板法从奶牛粪样中分离、纯化噬菌体,将纯化后的噬菌体浓缩液用醋酸双氧铀负染后通过透射电子显微镜观察其形态特征。对该噬菌体进行全基因组测序和遗传进化分析,同时测定噬菌体的宿主谱、最佳感染复数、一步生长曲线、热稳定性及酸碱稳定性。【结果】分离并纯化出一株裂解性噬菌体vB_EcoM_XJ2,噬菌斑圆形不透明,直径0.7 mm–1.2 mm;电镜显示其头部呈正多面体对称,有可伸缩性尾部;核酸类型为双链DNA,基因组大小为75.617 kb,G+C%含量为42.09%;其核酸序列与大肠杆菌噬菌体NJ01和vB_EcoP_SU10相似性高达94%。生物学特性研究显示该噬菌体能裂解多株临床分离的大肠杆菌;能耐受60°C左右高温,在pH 5.0–11.0范围内效价稳定;最佳感染复数为0.1,潜伏期为15 min,暴发期为95 min,裂解量约为10.6 PFU/cell。【结论】vB_EcoM_XJ2是一株在不同温度、不同酸碱性环境中有较强适应能力的裂解性肌尾科大肠杆菌噬菌体。     

Structural proteins of a lipid-containing bacteriophage which replicates in Escherichia coli: phage PR4.

PR4 is a lipid-containing bacteriophage which is able to replicate in Escherichia coli. The virus was labeled with either [14C]leucine and [14C]threonine or H235SO4 and then purified by several rounds of sucrose gradient centrifugation. Autoradiographs showed the virus to be composed of six major protein species with molecular weights (1) 68 000, (2) 47 500, (3) 38 500, (4) 35 000, (5) 20 700, (6) 16 500 daltons. Electropherograms showed protein No. 2 to be the major protein, comprising about 43% of the total weight of viral protein.     

一种新的DyP-type过氧化物酶在大肠杆菌中的重组表达、纯化及鉴定

染料脱色过氧化物酶(DyP-type过氧化物酶)是含有亚铁血红素,能降解各种有毒染料的一类蛋白.为了研究运动发酵单胞菌Zymomonas mobilis ZM4 (ATCC 31821)中一种新的DyP-type过氧化物酶的特点和功能,以Z.mobilis基因组DNA为模板,通过PCR扩增目的基因,克隆到大肠杆菌表达载体pET-21b(+)中.通过ZmDyP与其他DyP-type过氧化物酶的比对,发现它们存在着共同保守氨基酸D149、R239、T254、F256和GXXDG结构基序,说明ZmDyP是Dyp-type过氧化物酶家族的一个新成员.经IPTG诱导大肠杆菌中pET21 b(+)-ZmDyP表达,并将表达的酶进行金属螯合层析纯化.SDS-PAGE分析表明,纯酶分子量为36 kDa,而活性染色显示分子量为108 kDa,表明该酶在活性状态下可能是一个三聚体.光谱扫描显示ZmDyP有一个典型的亚铁血红素吸收峰,说明它是含有亚铁血红素的蛋白.对ZmDyP性质进行了研究,发现以2,2-二氨-双(3-乙基苯并噻唑-6-磺酸)ABTS为底物,ZmDyP表现出更高的转化效率.这些研究结果丰富了DyP-type 过氧化物酶家族信息,并且为ZmDyP的结构功能和反应机制研究奠定了基础.     

Characterization of bacteriophage lambda reverse as an Escherichia coli phage carrying a unique set of host-derived recombination functions

λ reverse (λrev) (Zissler et al., 1971a) is a recombination proficient derivative of a A phage which had lost the phage recombination genes by deletion. In this work, phages with the Rev phenotype have been obtained by a method of selection different from that of Zissler et al. (1971a). Comparison of DNA from two of our isolates and one of Zissler's by electron microscopic heteroduplex mapping shows that all three phages carry substitutions of non-λ DNA which are indistinguishable in extent, location and base sequence. Genetic and biochemical characterization of λrev strongly suggests that the substituted DNA codes for recombination functions different from the λ recombination functions which are deleted. These substituted genes apparently derive from the host chromosome or a prophage, and may be the same as the genes responsible for the SbcA and Rac phenotypes in the host.     

Effects of bacteriophage fd infection on Escherichia coli HB11 envelope: a morphological and biochemical study.

Phage fd-infected host bacteria revealed three characteristic changes in their envelope. (i) The preferred cleavage plane during freeze-fracturing shifted from the inner to the outer membrane (OM). (ii) The total lipids of the OM of the infected cells increased by 25% without major alterations in the relative concentration of phospholipids. We propose that such an increase would to some extent contribute to the change in the freeze-fracture behavior of the OM; however, additional factors will have to play a role in the apparent fracture resistance of the inner membrane. (iii) Ultrathin sectioning and immunolabeling methods revealed that extrusion of fd phages takes place at membrane adhesion sites of the infected cells.     

Genetic and morphological characterization of an Escherichia coli chromosome segregation mutant.

The temperature-sensitive nucleoid segregation mutant of Escherichia coli, PAT32, formerly described as a parA mutant, has been shown to carry a mutation near 66 min on the genetic map. Fine mapping with phages from the collection of Kohara et al. is consistent with its being a parC allele. Observation by fluorescence microscopy revealed the formation, at a nonpermissive temperature, of filaments containing one or two large nucleoids and of normal-size anucleate cells. There was also a significant loss of viability.     

Isolation and characterization of motile Escherichia coli mutants resistant to bacteriophage chi.

Four mutants of Escherichia coli that are resistant to the flagellotropic phage chi, but are motile, were isolated. When they were observed in liquid culture bylight microscopy, one mutant exhibited circular movement and another tumbled at high frequency on the surface of a glass slide. The remaining two mutants moved normally. None of these mutants adsorbed the wild-type strain of chi. P1 transduction revealed that the mutation sites of these four mutants were more than 97% contransducible with a site in hag, the structural gene for flagellin. When flagellins of these mutants were chromatographed on a diethylaminoethyl-cellulose column, two eluted slower and one eluted slightly faster than the flagellin of the parental strain. The other flagellin eluted at the same position as that of the parent. Host range mutants of phage chi, which could infect these bacterial mutants, were isolated.     

Genetic and morphological characterization of ftsB and nrdB mutants of Escherichia coli.

The ftsB gene of Escherichia coli is believed to be involved in cell division. In this report, we show that plasmids containing the nrdB gene could complement the ftsB mutation, suggesting that ftsB is an allele of nrdB. We compared changes in the cell shape of isogenic nrdA, nrdB, ftsB, and pbpB strains at permissive and restrictive temperatures. Although in rich medium all strains produced filaments at the restrictive temperature, in minimal medium only a 50 to 100% increase in mean cell mass occurred in the nrdA, nrdB, and ftsB strains. The typical pbpB cell division mutant also formed long filaments at low growth rates. Visualization of nucleoid structure by fluorescence microscopy demonstrated that nucleoid segregation was affected by nrdA, nrdB, and ftsB mutations at the restrictive temperature. Measurements of beta-galactosidase activity in lambda p(sfiA::lac) lysogenic nrdA, nrdB, and ftsB mutants in rich medium at the restrictive temperature showed that filamentation in the nrdA mutant was caused by sfiA (sulA) induction, while filamentation in nrdB and ftsB mutants was sfiA independent, suggesting an SOS-independent inhibition of cell division.     

一株强裂解性大肠杆菌T1样噬菌体新成员的分离与鉴定

【目的】自然界中噬菌体种类繁多,其裂菌功能在针对细菌耐药方面具有潜在应用价值。不同噬菌体也呈现出显著的基因多样性及宿主特异性。从上海某猪场仔猪肠内容物样品中分离、纯化大肠杆菌的裂解性噬菌体,分析其生物学特性和病毒学特征,为探索应用噬菌体治疗细菌性感染提供研究材料。【方法】采用双层琼脂平板法分离、纯化噬菌体,观察噬菌斑特征,通过电镜观察噬菌体形态特征,测定其裂菌谱、最佳感染复数、一步生长曲线和生物学特性,进行噬菌体全基因组测序和遗传进化分析。【结果】分离、纯化获得一株能高效裂解大肠杆菌K-12菌株的噬菌体,命名为v B_Eco S_SH2(SH2),噬菌斑呈圆形、大而透明、边缘整齐。电镜观察SH2的头部呈二十面体立体对称,尾部较长。噬菌体的潜伏期为10 min,暴发期为60 min,裂解量高达121 PFU/感染细胞,其最佳感染复数为0.1。基因组测序和比对结果表明,SH2的核酸类型为ds DNA,基因组全长为49 088 bp,G+C%含量为45%,Gen Bank登录号为KY985004,结合电镜观察及BLASTp分析,确定其属于有尾噬菌体目长尾噬菌体科成员。同源性及进化分析表明,该噬菌体为大肠杆菌T1样噬菌体的新成员。【结论】分离鉴定了一株裂解效率极高的大肠杆菌T1样噬菌体,并确认其为T1样噬菌体新成员,为研究大肠杆菌噬菌体及其抗菌应用提供了新的实验材料。     

Cloning and biological characterization of the immunity region of Escherichia coli phage Mu.

The construction of three hybrid plasmids containing different parts of the left or immunity and end of phage Mu DNA is described. The recombinant plasmids pKN05 and pKN54 carry the HindIII.C and PstI.C fragments of Mu DNA, respectively. Neither of these plasmids expresses the killing function. Moreover, they do not allow plating of superinfecting Mu phages. Plasmid pKN62 harbors the fragment located in between the left PstI and EcoRI cleavage sites on Mu DNA, allows plating of superinfecting Mu phages, but does not express the killing function. These data suggest that the gene coding for the killing function is either positively regulated by a product from the EcoRI.C fragment, or the killing function requires a second product not coded for by pKN62. Mu Vir A- or Mu Vir B- phages are able to grow on bacteria harboring the recombinant plasmid pKN001 which carries the left and EcoRI-C fragment of Mu DNA. This indicates that the superinfecting phages can induce the corresponding gene functions from pKN001. No such induction could be detected in cells harboring the hybrid plasmids pKN05, pKN54 or pKN62.     

Isolation and characterization of a lambda transducing bacteriophage carrying the cysE gene of Escherichia coli K-12

A specialized lambda transducing phage carrying the cysE and gpsA genes of E. coli K-12 has been isolated. The transducing phage has been separated from the helper phage on equilibrium gradients and has been shown to be defective. Evidence is presented that the phage kil gene is not expressed.     

Cnu, a novel oriC-binding protein of Escherichia coli

We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.     

Isolation and characterization of novel cold-sensitive dnaA mutants of Escherichia coli

We developed an efficient method for isolation of novel dnaA mutations based on PCR mutagenesis in the presence of manganese ion and shuffling of dnaA-carrying plasmids in a dnaA deletion host bacterium. Using this system, we obtained 30 cold-sensitive mutants from 4000 clones carrying plasmids with a mutagenized dnaA gene. All 27 cold-sensitive mutants analyzed were defective in DNA replication; none had a DnaAcos (over-initiation) phenotype. Nucleotide sequencing revealed that novel 15 alleles (mutations in 14 amino acid residues) are responsible for the cold-sensitive phenotype and are all located in the carboxy-terminal half of the DnaA protein.     

Identification and characterization of novel low-temperature-inducible promoters of Escherichia coli.

Escherichia coli promoters that are more active at low temperature (15 to 20 degrees C) than at 37 degrees C were identified by using the transposon Tn5-lac to generate promoter fusions expressing beta-galactosidase (beta-Gal). Tn5-lac insertions that resulted in low-temperature-regulated beta-Gal expression were isolated by selecting kanamycin-resistant mutants capable of growth on lactose minimal medium at 15 degrees C but which grew poorly at 37 degrees C on this medium. Seven independent mutants were selected for further studies. In one such strain, designated WQ11, a temperature shift from 37 degrees C to either 20 or 15 degrees C resulted in a 15- to 24-fold induction of beta-Gal expression. Extended growth at 20 or 15 degrees C resulted in 36- to 42-fold-higher beta-Gal expression over that of cells grown at 37 degrees C. Treatment of WQ11 with streptomycin, reported to induce a response similar to heat shock, failed to induce beta-Gal expression. In contrast, treatment with either chloramphenicol or tetracycline, which mimics a cold shock response, resulted in a fourfold induction of beta-Gal expression in strain WQ11. Hfr genetic mapping studies complemented by physical mapping indicated that in at least three mutants (WQ3, WQ6, and WQ11), Tn5-lac insertions mapped at unique sites where no known cold shock genes have been reported. The Tn5-lac insertions of these mutants mapped to 81, 12, and 34 min on the E. coli chromosome, respectively. The cold-inducible promoters from two of the mutants (WQ3 and WQ11) were cloned and sequenced, and their temperature regulation was examined. Comparison of the nucleotide sequences of these two promoters with the regulatory elements of other known cold shock genes identified the sequence CCAAT as a putative conserved motif.