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1.
UV-sensitive mutant strain of Haemophilus influenzae Rd MBH3, is 20 times more sensitive to UV irradiation than the wild type strain. The mutation responsible for increased UV sensitivity of the strain was identified as G --> A transition predicting synthesis of truncated UvrAdeltaC44 protein (Balsara & Joshi). Recombinant UvrAdeltaC44 protein was purified for the first time under denaturing conditions. The molecular weight of the recombinant protein was estimated as approximately100 kDa. Recombinant UvrAdeltaC44 protein was found to be less efficient in its ATPase and DNA binding activity as compared to the wild type protein. Recombinant plasmid carrying uvrAdeltaC44 gene could partially complement the UvrA deficiency in E. coli UvrA mutant. 相似文献
2.
A A Kim E S Molchanova B N Il'iashenko 《Molekuliarnaia genetika, mikrobiologiia i virusologiia》1985,(10):25-28
The Escherichia coli K12 mutant having the increased efficiency for plasmid DNA transformation has been shown to possess the different protein composition of the outer membrane of the cellular wall, as compared with that of the wild type strain. Correlation between the level of calcium-dependent plasmid transformation and the portion of infections DNA bound with cytoplasmic membranes is demonstrated for the Escherichia coli cells mutant for outer membrane structure and ability to be transformed by plasmid DNA. 相似文献
3.
4.
Cloning and characterization of the Aeromonas caviae recA gene and construction of an A. caviae recA mutant. 总被引:7,自引:4,他引:3
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A recombinant plasmid carrying the recA gene of Aeromonas caviae was isolated from an A. caviae genomic library by complementation of an Escherichia coli recA mutant. The plasmid restored resistance to both UV irradiation and to the DNA-damaging agent methyl methanesulfonate in the E. coli recA mutant strain. The cloned gene also restored recombination proficiency as measured by the formation of lac+ recombinants from duplicated mutant lacZ genes and by the ability to propagate a strain of phage lambda (red gam) that requires host recombination functions for growth. The approximate location of the recA gene on the cloned DNA fragment was determined by constructing deletions and by the insertion of Tn5, both of which abolished the ability of the recombinant plasmid to complement the E. coli recA strains. A. caviae recA::Tn5 was introduced into A. caviae by P1 transduction. The resulting A. caviae recA mutant strain was considerably more sensitive to UV light than was its parent. Southern hybridization analysis indicated that the A. caviae recA gene has diverged from the recA genes from a variety of gram-negative bacteria, including A. hydrophila and A. sobria. Maxicell labeling experiments revealed that the RecA protein of A. caviae had an Mr of about 39,400. 相似文献
5.
KP1_4563基因是肺炎克雷伯菌NTUH-K2044中假设的蛋白编码基因,与Ⅲ型菌毛的功能有关。本实验首先采用同源重组基因敲除方法构建肺炎克雷伯菌KP1_4563基因缺失的突变株(Kp-△4563),然后PCR扩增KP1_4563基因片段,克隆到质粒p BAD33上,将重组质粒导入Kp-△4563获得回补株(Kpc-△4563)。分别测定野生株、突变株、回补株用普通LB培养基,改良Minka培养基以及含胆汁盐的LB培养基培养时生物膜形成能力,以此来探讨KP1_4563基因以及不同培养基对肺炎克雷伯菌体外生物膜形成的影响。我们成功构建KP1_4563基因缺失的突变株和回补株Kpc-△4563。与野生株相比,突变株Kp-△4563生物膜形成能力减弱,回补株介于野生株和突变株之间。使用改良Minka培养基使菌株菌毛化以及加入胆汁盐可以增加生物膜的形成能力。这些分析表明肺炎克雷伯菌KP1_4563基因能正调控细菌生物膜的形成。体外培养使细菌菌毛化以及加入胆汁盐可以促进生物膜的形成。 相似文献
6.
On the base of plasmid pCV20 (Apr, Tcr mol. weight 5.2 x 10(6) a recombinant plasmid pEH60 (Apr, mol. weight 17.0 x 10(6) with BamHI fragment of phage DNA, containing red+ and gam+ genes was constructed. Selection was found on the ability of phage red- and gam- to propagate in strain E. coli K12 recA-, which was transformed by recombinant plasmid with active red and gam genes. Influence of recombinant plasmid pEH60 on processes of repair and recombination of phage lambda DNA and bacterial DNA was studied. It was shown that red gene in plasmid pEH60 compensates deficiency of redA gene in these processes with phage lambda DNA; in the case of E. coli K12 AB2480 uvr- recA- (pEH60) the processes of multiple reactivation and decombination of phage red- were presented. In the case of bacterial cells, plasmid pEH60 did not compensate deficiency of recA function of bacteria, although it partly compensates deficiency of recBC function. Increase of survival after introduction of plasmid pEH60 in the cell was obtained only for recBC- strain, but not for wild type and recA- strains. 相似文献
7.
《Journal of Fermentation and Bioengineering》1990,69(2):75-78
A new method to stabilize recombinant plasmids extremely well was exploited using Escherichia coli Tna (trpAEI trpR tnaA) and pSC101trpI15-14 (tetracycline resistance, whole trp operon) as a model system. We mutagenized the Tna strain carrying pSC101trpI15-14 and isolated a mutant 6F484 that stably maintained the recombinant plasmid for 100 generations. From 6F484, plasmid-free cells (tetracycline sensitive) were screened for on selective agar plates containing fusaric acid. The host strain FA14 was found to have lost the ability for active transport of tryptophan, in addition to the phenotype of Trp−. Therefore, strain FA14 could not grow normally even in a complete medium. However, when the strain was transformed with the trp operon recombinant plasmid, its growth rate was almost restored to the original level. These results suggest that the recombinant plasmid is indispensable for the normal growth of host cells like FA14. Even if plasmid-free segregants appear during the cultivation, they cannot grow so rapidly and are diluted as a minority in total population. Consequently, owing to the deficiency of both the biosynthesis and uptake of tryptophan in host strain, the trp operun recombinant plasmid can be stably maintained. 相似文献
8.
When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-beta-hydroxybutyrate (PHB). Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source by A. brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments. However, in A. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested. 相似文献
9.
Isolation of a mutant TOL plasmid with increased activity and transmissibility from Pseudomonas putida (arvilla) mt-2. 总被引:8,自引:7,他引:1
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Strains with greater ability to dissimilate m-toluate were obtained from the wild-type Pseudomonas putida (arvilla) mt-2 that harbors the TOL plasmid. Increased growth of a mutant strain on aromatic substrates was coupled with simultaneous increase in the activity of metapyrocatechase, an enzyme coded by the TOL plasmid, without changing its catalytic properties. In the mutant and the wild-type strains, the inducer specificity and the induction kinetics of metapyrocatechase synthesis were the same, and a half-maximal effect of m-toluate on the enzyme synthesis was observed at 0.25 mM. Thus, the increased utilizability seen in a mutant strain appeared to be due to an increased quantity of the enzymes coded by the TOL plasmid. The properties of the mutant strain were dependent upon the mutation on the TOL plasmid but not on the chromosome mutation. Transfer experiments with a strain carrying the mutant TOL (TOL-H) or the wild-type TOL plasmid revealed that the TOL-H transfer was 1,000 times greater than that of the wild type. 相似文献
10.
【目的】马奶酒样乳杆菌ZW3含有一段长度为14.4 kb的胞外多糖合成基因簇,包含17个与胞外多糖合成相关的基因(WANG_1283?WANG_1299),主要分析17个基因在马奶酒样乳杆菌ZW3生长过程中不同时间段的表达量,探究其中一个表达量发生变化的基因对乳酸菌产胞外多糖的影响。【方法】通过半定量RT-PCR实验,对基因簇上各基因的表达量进行分析;通过构建含有表达量变化基因的重组乳酸乳球菌,比较重组菌与野生菌的产胞外多糖差异。【结果】经分析,WANG_1284、WANG_1286、WANG_1287、WANG_1288、WANG_1290、WANG_1291、WANG_1292、WANG_1294、WANG_1296、WANG_1297、WANG_1298、WANG_1299这12个基因在菌体生长的50 h和60 h (产糖量上升阶段)表达量最高,推测这些基因在多糖聚合过程中起作用。从这12个基因中选出一个表达量发生明显变化的基因WANG_1291做进一步研究。将WANG_1291插入乳酸菌表达载体pMG36e中,构建了重组表达载体pMG36e-1291。将构建的重组表达载体转化到乳酸乳球菌WH-C1中,得到重组菌株。测定重组菌与野生菌生长特性,发现重组菌与野生菌之间的生长速度存在一定差异。然后利用苯酚-硫酸法测得重组乳酸乳球菌的胞外多糖产量是野生菌的2.1倍,胞外多糖产量有了明显的提高。【结论】确定WANG_1291基因是调控马奶酒样乳杆菌ZW3产胞外多糖的关键基因之一。 相似文献
11.
Mutants of Diplococcus pneumoniae that Lack Deoxyribonucleases and Other Activities Possibly Pertinent to Genetic Transformation 总被引:58,自引:36,他引:22
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Sanford Lacks 《Journal of bacteriology》1970,101(2):373-383
Mutants of Diplococcus pneumoniae that lacked the two major deoxyribonucleases of the cell—one an endonuclease, the other an exonuclease preferentially active on native deoxyribonucleic acid (DNA)—were obtained. The development of a method for detecting mutant colonies, based on the binding of methyl green to DNA, facilitated isolation of the mutants. Neither enzyme was essential for growth of the cells, for repair of ultraviolet damage, or for any phase of DNA-mediated transformation. Residual deoxyribonuclease activity in the double mutant corresponded to an exonuclease, approximately one-fifth as active as the major exonuclease, that attacked native and denatured DNA equally well. This activity appeared to be associated with the DNA-polymerase enzyme. A mutant that apparently lacked a cell wall lytic enzyme was also fully transformable. A mutant strain that was four times more sensitive to ultraviolet light than the wild type also transformed normally. Recipient cells of this strain were deficient in the repair of ultraviolet-irradiated transforming DNA. Mutants were found which, unlike the wild type, integrated donor markers only with high efficiency, thereby indicating that a particular cellular component that is susceptible to loss by mutation, such as an enzyme, is responsible for low integration efficiency. 相似文献
12.
S V Molotov D E Duzhi? V N Danilevich V V Sukhodolets 《Molekuliarnaia genetika, mikrobiologiia i virusologiia》1991,(4):3-7
The ability of the industrial strains of Streptococcus lactis to synthesize the enzyme beta-galactosidase was studied. Five strains among sixteen were found to produce high levels of the enzyme. The beta-galactosidase gene in the most active strain Streptococcus lactis 111 was shown to be located on the 50 kb conjugative plasmid. The plasmid was transferred by conjugation into Streptococcus thermophilus cells and subsequently the gene for beta-galactosidase was studied in transconjugants. The beta-galactosidase gene from Streptococcus lactis 111 was subcloned in Escherichia coli cells on the plasmid pBR322. The gene was localized on the 4.8 kb BgIII fragment of DNA. Following the restriction of DNA by the Sau3A the gene was subcloned on the birepliconed plasmid vector pCB20 capable of replication in the Gram-negative as well as Gram-positive microorganisms. The recombinant derivatives of pCB20 were isolated that carry the beta-galactosidase gene on the DNA fragments of different size. 相似文献
13.
Involvement of the Reserve Material Poly-β-Hydroxybutyrate in Azospirillum brasilense Stress Endurance and Root Colonization
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When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-β-hydroxybutyrate (PHB). Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source by A. brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments. However, in A. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested. 相似文献
14.
目的:构建炭疽芽胞杆菌假想S-层蛋白SLP缺失突变体,以进行后续SLP的功能研究,为炭疽芽孢杆菌重要基因功能的研究建立技术平台。方法:利用PCR技术,分别扩增得到目的基因的上游同源臂(slp-F)和下游同源臂(slp-R),将抗性基因(S)和上、下游同源臂先后连入穿梭质粒pKSV7,构建打靶载体pKSV7-FSR,经去甲基化后,电转化入炭疽芽胞杆菌A16R,通过同源重组敲除slp基因,并通过DNA测序和Western blot实验验证;对野生株和突变株37℃时的生长曲线及生化反应进行比较研究。结果:分别从DNA水平和蛋白质水平证实slp基因被成功敲除;突变株对数期生长较快,衰退较慢,与野生株的生化反应差异不明显。结论:获得了炭疽芽胞杆菌假想S-层蛋白SLP缺失突变体。 相似文献
15.
Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex that is composed of the products of the UL5, UL52, and UL8 genes. A subcomplex consisting of the UL5 and UL52 proteins retains all the enzymatic activities exhibited by the holoenzyme in vitro. The UL52 protein contains a putative zinc finger at its C terminus which is highly conserved among both prokaryotic and eukaryotic primases. We constructed a mutation in which two highly conserved cysteine residues in the zinc finger motif were replaced with alanine residues. A UL52 expression plasmid containing the mutation in the zinc finger region is unable to support the growth of a UL52 mutant virus in a transient complementation assay. Wild type and mutant UL5.UL52 subcomplexes were purified from insect cells infected with recombinant baculoviruses. Surprisingly, the mutant protein was severely affected in all biochemical activities tested; no helicase or primase activities could be detected, and the mutant protein retains only about 9% of wild type levels of single-stranded DNA-dependent ATPase activity. Gel mobility shift assays showed that DNA binding is severely affected as well; the mutant subcomplex only retains approximately 8% of wild type levels of binding to a forked substrate. On the other hand, the mutant protein retains its ability to interact with UL5 as indicated by copurification and with UL8 as indicated by a supershifted band in the gel mobility shift assay. In addition, the ability of individual subunits to bind single-stranded DNA was examined by photo cross-linking. In the wild type UL5.UL52 subcomplex, both subunits are able to bind an 18-mer of oligo(dT). The mutant subcomplex was severely compromised in the ability of both UL5 and UL52 to bind the oligonucleotide; total cross-linking was only 2% of wild type levels. These results are consistent with the proposal that the putative zinc binding motif of UL52 is required not only for binding of the UL52 subunit to DNA and for primase activity but also for optimal binding of UL5 to DNA and for the subsequent ATPase and helicase activities. 相似文献
16.
Yukihiro Kitade Shohei Okino Wataru Gunji Kazumi Hiraga Masako Suda Nobuaki Suzuki Masayuki Inui Hideaki Yukawa 《Applied microbiology and biotechnology》2013,97(18):8219-8226
Expression plasmids that facilitate production of bio-based products are susceptible to toxic effects that frequently affect plasmid structural stability in recombinant microbial cells. In order to enhance plasmid stability in recombinant Corynebacterium glutamicum, an expression plasmid containing genes of the Clostridium acetobutylicum butyryl-CoA synthesis operon with high structural instability within wild-type C. glutamicum was employed. From a total of 133 mutants exhibiting disruptions in 265 suspect genes, only cgR_0322-deficient mutant was able to maintain the expression plasmid intact. The mutant exhibited normal growth under standard laboratory conditions but its transformation efficiency was about one order of magnitude lower than that of wild-type strain. The cgR_0322 gene encodes an endonuclease that is active against single- as well as double-stranded DNA substrates in the presence of Mg2+. The cgR_0322-deficient strain should therefore facilitate the development of more robust C. glutamicum strains to be used as microbial production hosts. 相似文献
17.
The lpd gene product functions as the L protein in the Escherichia coli glycine cleavage enzyme system. 总被引:5,自引:5,他引:0
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The lpd-encoded lipoamide dehydrogenase, common to the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes, also functions as the lipoamide dehydrogenase (L protein) in the Escherichia coli glycine cleavage (GCV) enzyme complex. Inducible GCV enzyme activity was not detected in an lpd deletion mutant; lpd+ transductants had normal levels of inducible GCV enzyme activity. A serA lpd double mutant was unable to utilize glycine as a serine source and lacked detectable GCV enzyme activity, the phenotype of a serA gcv mutant. Transformation of the double mutant with a plasmid encoding a functional lpd gene restored the ability of the mutant to use glycine as a serine source and restored inducible GCV enzyme activity to normal levels. The presence of acetate and succinate in the growth medium of a strain wild type for lpd and gcv resulted in a 50% reduction in inducible GCV enzyme activity. Enzyme levels were restored to normal under these growth conditions when the strain was transformed with a plasmid encoding a functional lpd gene. 相似文献
18.
PCR扩增了蓝细菌集胞藻6803(Synechocystis sp.PCC6803)的agp基因(编码ADP-葡萄糖焦磷酸羧化酶),进一步以pUC118为载体将其克隆到大肠杆菌中,构建了pUCA质粒。通过DNA体外重组,以红霉素抗性基因部分取代agp基因片段,构建了既含agp基因上游及下游序列、又携带选择性标记-红霉素抗性的pUCAE质粒。该质粒转化野生型集胞藻6803细胞,获得了能在含红霉素的培养基上正常生长的agp基因缺失突变株。对该突变株基因组DNA进行PCR扩增,验邝了其基因结构的正确性。突变株细胞生长速度较野生型细胞快,胞内的叶绿素含量比野生型细胞高,表明该突变株具有较高的光合效率。在突变株中未检测到糖原的存在,进一步从生理水平上验证了突变株构建的正确性。 相似文献
19.
Temperature-dependent and amber copy mutants of plasmid R1drd-19 in Escherichia coli. 总被引:9,自引:0,他引:9
The isolation of conditional mutants with an altered copy number of the R plasmid R1drd-19 is described. Temperature-dependent as well as amber-suppressible mutants were found. These mutant plasmids have been named pKN301 and pKN303, respectively. Both types of mutations reside on the R plasmid. No difference in molecular weight could be detected by neutral sucrose gradient centrifugation for any of the mutant plasmids when compared with the wild-type plasmid. The number of copies of the plasmids was determined by measurement of the specific activity of the R plasmid-mediated β-lactamase and by measurement of covalently closed circular (CCC) DNA in alkaline sucrose gradients and dye-CsCl density gradients. Below 34 °C the temperature-dependent mutant, pKN301, had the same copy number as the wild type, while this was four times that of the wild type above 37 °C. The amber mutant pKN303 had a copy number indistinguishable from that of the wild-type plasmid in a strain containing a strong amber suppressor and a copy number about five times that of the wild-type plasmid in a strain lacking an amber suppressor. In a strain containing a temperature-sensitive amber suppressor, the amber mutant's copy number increased with the decrease in amber suppressor activity. Thus, the existence of the temperature-dependent and the amber-suppressible R-plasmid copy mutants indicates that the system that controls the replication of plasmid R1drd-19 contains an element with a negative function and that this element is a protein. 相似文献
20.
P Ghosh C Meyer E Remy D Peterson J Preiss 《Archives of biochemistry and biophysics》1992,296(1):122-128
The Escherichia coli B mutant strain CL1136 accumulates glycogen at a 3.4- to 4-fold greater rate than the parent E. coli B strain and contains an ADPglucose synthetase with altered kinetic and allosteric properties. The enzyme from CL1136 is less dependent on the allosteric activator, fructose 1,6-bisphosphate, for activity and less sensitive to inhibition by AMP than the parent strain enzyme. The structural gene, glgC, for the allosteric mutant enzyme was selected by colony hybridization and cloned into the bacterial plasmid pBR322 by insertion of the chromosomal DNA at the PstI site. One recombinant plasmid, designated pKG3, was isolated from the genomic library of CL1136 containing glgC. The cloned ADPglucose synthetase from the mutant CL1136 was expressed and characterized with respect to kinetic and allosteric properties and found to be identical to the enzyme purified from the CL1136 strain. The mutant glgC was then subcloned into pUC118/119 for dideoxy sequencing of both strands. The mutant glgC sequence was found to differ from the wild-type at the deduced amino acid residue 67 where a single point mutation resulted in a change from arginine to cysteine. 相似文献