Characterization of phosphinothricin acetyltransferase and C-terminal enzymatically active fusion proteins

Enhanced expression of the bialaphos resistance (bar) from Streptomyces hygroscopicus, which confers resistance to the herbicides bialaphos and phosphinothricin (PPT), has been obtained in Escherichia coli using a vector system based on translational coupling. The gene product, PPT acetyltransferase, was purified to homogeneity and its enzymatic properties were analyzed. Hybrid gene constructs with gene fragments fused to the 3'-terminus of bar yield fusion proteins having acetyltransferase activity, with a Michaelis constant for the PPT substrate comparable to the unmodified enzyme. The bar gene represents a selectable and assayable reporter gene especially suitable for 3'-terminal gene fusions.     

Genetically engineered herbicide resistance in cyanobacterium Synechococcus sp. by bialaphos resistance gene from Streptomyces hygroscopicus

A novel cyanobacterial vector, pTT201, containing the bar gene encoding resistance to herbicides, bialaphos and phosphinothricin, was constructed. In Synechococcus sp. strain PCC7942-SPc, the bar gene was successfully expressed. Plasmid pTT201 increased a minimum inhibitory concentration for bialaphos 16-fold over Synechococcus sp. strain PCC7942-SPc without pTT201. The combination of the bialaphos as a selective agent and the transformation by bar gene serves as a photostable selection system for Synechococcus.     

Identification and expression of the gene encoding phosphonopyruvate decarboxylase of Streptomyces hygroscopicus

The first step of C-P compound biosynthesis is a C-P bond formation reaction catalyzed by phosphoenolpyruvate phosphomutase, but this reaction favors the cleavage of the C-P bond. This C-P bond forming reaction is driven by the following reaction catalyzed by phosphonopyruvate (PnPy) decarboxylase. We have cloned and sequenced the gene (bcpC) encoding PnPy decarboxylase, a key enzyme of C-P compound biosynthesis, from the bialaphos (BA) producing microorganism Streptomyces hygroscopicus by complementation methods using Streptomyces wedmorensis NP-7, which is a mutant of a fosfomycin producing strain deficient in this step. The location of this gene in the BA biosynthetic gene cluster was determined by using the expression system in Streptomyces lividans. DNA sequencing of this gene revealed a 1203-bp open reading frame encoding a polypeptide of 401 amino acids.     

Expression of the Bar Gene Confers Herbicide Resistance in Transgenic Lettuce

Resistance to bialaphos, a broad-spectrum herbicide, was introduced into Lactuca sativa cv. Evola by Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strains 0310 and 1310, both carrying the bialaphos resistance (bar) and neomycin phosphotransferase (nptII) genes, were used for transformation. Primary transformants were selected on kanamycin sulphate-supplemented shoot regeneration medium. Integration of both transgenes was confirmed by non-radioactive Southern hybridisation. The hypervirulent plasmid ToK47 in A. tumefaciens strain 1310 generated multiple insertions of T-DNA in some transgenic plants; the absence of pToK47 (strain 0310) resulted in single gene inserts in all plants tested. Resistance to glufosinate ammonium was observed in axenic seedlings grown on medium supplemented with the herbicide at 5 mg l–1 and in glasshouse-grown plants sprayed with the compound at 300 mg l–1. Stable expression of the bar gene was observed in R2 generation plants. The kanamycin resistance of R1 seedlings was observed by germinating seeds on medium supplemented with 200 mg l–1 kanamycin sulphate. The presence of NPTII protein and PAT enzyme activity were demonstrated by ELISA and PAT enzyme assay respectively. Transgenes segregated in a Mendelian fashion in some plant lines in the R1 generation; herbicide resistance also segregated in the expected ratio in the R2 generation in most transgenic lines. This study confirmed that an agronomically important transgene can be integrated and stably expressed over several generations in lettuce.     

盐藻肌动蛋白基因启动子驱动的bar基因表达作为核转化筛选标记

运用基因组步行方法克隆盐藻肌动蛋白基因5′上游调控序列,发现相对于ATG上游-573和-424bp的位置上分别有75bp长的两个重复序列。没有典型的TATA盒,但有两个TATA样结构、一个CCAAT结构和一个与GCTC(G／C)AAGGC一致的序列。以700bp的盐藻肌动蛋白基因启动子区序列驱动bar基因的表达作为转化盐藻的筛选标记。转化的藻细胞暗光恢复24h后,在含0．5μg／mL除草剂的培养基中常规培养生长1周,然后将细胞平铺于含0．5μg／mL除草剂的固体培养基上继续筛选培养。约20d后从固体培养板上挑选出5个藻落并作了进一步培养和分析。结果显示,5个转化藻中携带bar嵌合基因的整合位点均位于核基因组内。Southern blotting分析表明,仅有一个转化藻整合单拷贝的bar基因,而另外4个转化藻株则包含多个拷贝bar基因片段,提示盐藻核基因转化主要是外源基因的随机整合,外源基因在转化盐藻中的整合拷贝数并不影响其除草剂抗性。RT-PCR方法证明了bar基因在转化藻中的转录。5个转化藻在含除草剂的液体培养基中维持生长了至少7个月,表明核基因转化的稳定性。     

Streptomyces hygroscopicus has two glutamine synthetase genes.

Streptomyces hygroscopicus, which produces the glutamine synthetase inhibitor phosphinothricin, possesses at least two genes (glnA and glnB) encoding distinct glutamine synthetase isoforms (GSI and GSII). The glnB gene was cloned from S. hygroscopicus DNA by complementation in an Escherichia coli glutamine auxotrophic mutant (glnA). glnB was subcloned in Streptomyces plasmids by insertion into pIJ486 (pMSG3) and pIJ702 (pMSG5). Both constructions conferred resistance to the tripeptide form of phosphinothricin (bialaphos) and were able to complement a glutamine auxotrophic marker in S. coelicolor. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of S. lividans(pMSG5) revealed a highly overexpressed 40-kilodalton protein. When GS was purified from this strain, it was indistinguishable in apparent molecular mass from the 40-kilodalton protein. The nucleic acid sequence of the cloned region contained an open reading frame which encoded a protein whose size, amino acid composition, and N-terminal sequence corresponded to those of the purified GS. glnB had a high G + C content and codon usage typical of streptomycete genes. A comparison of its predicted amino acid sequence with the protein data bases revealed that it encoded a GSII-type enzyme which had previously been found only in various eucaryotes (47 to 50% identity) and nodulating bacteria such as Bradyrhizobium spp. (42% identity). glnB had only 13 to 18% identity with eubacterial GSI enzymes. Southern blot hybridization experiments showed that sequences similar to glnB were present in all of the five other Streptomyces species tested, as well as Frankia species. These results do not support the previous suggestion that GSII-type enzymes found in members of the family Rhizobiaceae represent a unique example of interkingdom gene transfer associated with symbiosis in the nodule. Instead they imply that the presence of more than one gene encoding GS may be more common among soil microorganisms than previously appreciated.     

Transgenic cotton resistant to herbicide bialaphos

Resistance to bialaphos, a non-selective herbicide, was intro duced into cotton through genetic engineering. A gene encoding phosphinothric in acetyltransferase (bar) from Streptomyces hygroscopicus was inserted into elite varieties of cotton through particle bombardment. Based on the marker gene, -glucuronidase (gus) expression, a total of 18 Pima (Gossypium barbadense), 45 DP50 (G. hirsutum L.), 20 Coker 312 (G. hirsutum) and 2 El Dorado (G. hirsutum) transgenic plants were recovered. Integration of the bar gene into cotton genomic DNA was confirmed by Southern blot analysis and gene expression was confirmed by northern blot and enzyme assays. Herbicide (Basta®) tolerance up to 15 000 ppm was demonstrated in greenhouse trials. The newly introduced herbicide tolerance trait is inherited in a Mendelian fashion in the progenies of germline transformants. This study demonstrates the potential for particle bombardment to introduce commerically important genes directly into elite varieties of cotton. This mode of gene transfer can expedite the introduction of transgenic cotton products into world markets     

Longevity of 5-azacytidine-mediated gene expression and re-establishment of silencing in transgenic rice

Epigenetic silencing of a bialaphos resistance (bar) gene in R1 progeny of a transgenic rice line was found to be meiotically stable since selfed (R2) progeny were also susceptible and the bar locus highly methylated. A high proportion of R2 seedlings germinated in the presence of 5-azacytidine (AzaC) were herbicide-resistant and also contained at least one unmethylated copy of the bar gene, further establishing the relationship between silencing and methylation. Restored bar gene expression was typically maintained for 20–50 days, but eventual methylation and silencing of the bar locus underscores the ability of the recipient genome to recognize and inactivate intrusive DNA.     

Global changes in gene expression related to antibiotic synthesis in Streptomyces hygroscopicus

Two-dimensional gel electrophoresis was used to follow changes in gene expression associated with antibiotic (bialaphos) biosynthesis in Streptomyces hygroscopicus. Cultures were pulse-labelled with [35S]-methionine before, during, and after the switch from primary to secondary metabolism in order to compare kinetic profiles of bialaphos (antibiotic) production (bap) genes during this metabolic transition. Separation of gene products on two-dimensional gels revealed that 27 were dependent on brpA for optimal expression and were activated as the culture approached stationary phase. Genes which encoded 10 brpA-dependent proteins were mapped to a 10 kb SstI fragment of the 35 kb bap gene cluster by expressing them in Streptomyces lividans using the thiostrepton-inducible tipA promoter. N-terminal amino acid sequences of two brpA-dependent proteins, obtained by direct microsequencing of protein spots excised from two-dimensional gels, identified them as gene products mapping to the same region and involved in secondary metabolic conversions of the bap pathway. The kinetics of synthesis of 16 brpA-dependent gene products were characterized using QUEST computer software. Cluster analysis performed on the kinetics of synthesis of 346 of the most highly expressed gene products of HP5-29, including 16 brpA-dependent ones, identified 75 families having distinct patterns of expression. Many brpA-dependent proteins were clustered together; 10 were found in one kinetic family. These kinetic families also included brpA-independent gene products perhaps subject to similar regulatory mechanisms and thus possibly involved in bialaphos biosynthesis. The activation/derepression of bap expression took place as cultures approached stationary phase and was temporally related to synthesis of ppGpp.     

Molecular cloning of tylosin-producing Streptomyces fradiae B-45 genes determining increased inducible resistance to tylosin in Streptomyces lividans TK64

DNA of S. fradiae B-45 partially cleaved by Sau3A restrictase was cloned in S. lividans TK64 in the plasmid vector pIJ702. Three recombinant plasmids pVG251, pVG262, and pVG253 with tlr1, tlr2 and tlr3 genes were isolated from the transformed clones of S. lividans TK64 with higher inducible resistance to tylosin as compared to the plasmid-free strain. DNA-DNA blot hybridization was performed between the total DNA cleaved by several restrictases from S. fradiae B-45 and some other strains and the DNA probes containing the tlr genes. It was shown that tlr1 and tlr3 genes were unique in S. fradiae B-45. Sequences homologous to tlr2 gene were present both in DNA of S. fradiae B-45 in 7 copies and in strains of S. antibiotics and S. hygroscopicus producing respectively oleandomycin and turimycin.     

Production of fertile transgenic wheat plants by laser micropuncture.

A modified, non-damaging, protocol for the production of fertile transgenic wheat (Triticum aestivum L. cultivar Giza 164) plants by laser micropuncture was developed. The new homemade setup secures the transformation of as many as 60 immature embryo-derived calli (10000 cells each) in less than one hour using a UV excimer laser with two dimensional translation stages, a suitable computer program and a proper optical system. Five-day-old calli were irradiated by a focused laser microbeam to puncture momentarily made self-healing holes ( approximately 0.5 microm) in the cell wall and membrane to allow uptake of the exogenous DNA. The plant expression vector pAB6 containing bar gene as a selectable marker for the herbicide bialaphos resistance and GUS (uidA) gene as a reporter gene was used for transformation. No selection pressure was conducted during the four-week callus induction period. Induced calli were transferred to a modified MS medium with 1 mg l(-1) bialaphos for regeneration, followed by selection on 2 mg l(-1) bialaphos for rooting. Three regenerated putative transgenic events were evaluated for the integration and stable expression of both genes and results indicated that this modified procedure of laser-mediated transformation can be successfully used in transforming wheat.     

Resistance of the strain Streptomyces hygroscopicus 155 to autogenous and other antibiotics]

Streptomyces hygroscopicus 155, a strain producing pandavir (nigericin) and azalomycin, was studied with respect to resistance to its own and some other antibiotics i.e. tetracycline, gentamicin, streptomycin, erythromycin, tylosin, thiostreptone, benzylpenicillin, monensin and salinomycin. An inactive variant of the culture was used in the control.     

吸水链霉菌17997格尔德霉素部分生物合成基因簇的克隆和分析

吸水链霉菌17997(Streptomyceshy groscopicus17997)是我所从中国云南土壤中分离到的格尔德霉素(geldanamycin,GDM)产生菌,GDM具有良好的抗肿瘤和抗病毒活性,但其肝毒性和水溶性差的缺点限制了其在临床上的应用。为了实现对GDM结构的生物学改造,首先要获得GDM的生物合成基因。根据GDM后修饰基因——氨甲酰基转移酶基因(gdmN)的保守序列筛选S.hygroscopicus17997的柯斯质粒基因组文库,共获得6个阳性克隆,选择CT-4阳性柯斯质粒进行亚克隆和测序,又通过PCR延伸的方法获得了与CT4连锁的将近5kb的外源序列,共获得28.356kb的外源DNA序列,其中包含了13个可能阅读框架,通过同源比较证实该序列与S.hygroscopicusNRRL3602中的GDM生物合成基因有很高的同源性。为进一步研究GDM生物合成基因的功能,并通过组合生物学的方法改造GDM的结构奠定了基础。     

Green, herbicide-resistant plants by particle inflow gun-mediated gene transfer to diploid bahiagrass (Paspalum notatum)

We have established an efficient particle-bombardment transformation protocol for the diploid non-apomictic genotype of the warm season forage crop Paspalum notatum (bahiagrass). A vector containing a herbicide resistance gene (bar) together with the GUS reporter gene was used in transformation experiments. The bar gene confers resistance to the herbicide bialaphos. An improved culture system, highly regenerative callus, dense in compact polyembryogenic clusters, was produced on medium with a high CuSO4 content at elevated temperature. Target tissue (360 calli) produced under these conditions yielded 52 rooted plants on herbicide-containing medium, and 22 of these plants were PCR-positive. DNA gel blot analysis revealed a copy number of 1-5 for the GUS gene in different independent transformants. There was no correlation between copy number and GUS activity. While conventional cultures yielded exclusively albino plants on herbicide-containing medium, improved culture conditions for the target tissue resulted in the recovery of 100% green transgenic plants. All green herbicide-resistant regenerants were morphological normal and fertile.     

酪蛋白水解物对雷帕霉素产生菌Streptomyces hygroscopicus ATCC29253 接合转移效率的影响

吸水链霉菌(Streptomyces hygroscopicus)ATCC29253能够产生非常丰富的次级代谢产物,除了雷帕霉素外,还可以分泌尼日利亚菌素、洋橄榄叶素、六烯类抗生素等多种具有生物活性的物质,具有重要的研究价值和应用前景。【目的】而建立高效的遗传操作系统是研究该链霉菌相关代谢产物合成机理和构建基因工程菌株的基础。【方法】我们测试了不同培养基及供体菌对吸水链霉菌及其它链霉菌接合转移效率的影响。【结果】我们发现酪蛋白水解物和镁离子能显著提高S.hygroscopicus ATCC29253接合转移效率。通过随机组合试验,筛选出最佳的MgCl2和酪蛋白水解物浓度组合,使得S.hygroscopicus ATCC29253接合转移效率达到1.5×10-4。同时我们还发现酪蛋白水解物可以明显改变S.lividans、S.albus、S.avermitilis的接合转移效率。【结论】本研究首次发现酪蛋白水解物不光对S.hygroscopicus ATCC29253接合转移有作用,对其它常用的链霉菌如S.lividans、S.albus、S.avermitilis等的接合转移效率都有显著的影响。     

Identification and characterization of hmr19 gene encoding a multidrug resistance efflux protein from Streptomyces hygroscopicus subsp. yingchengensis strain 10-22

[(The over-usage of antibiotics may result in the develop-)]ment of extensive antibiotic resistance in microorganisms[1]. Export of toxic compounds as means of resistancehas been well documented in pathogenic bacteria as wellas antibiotic-producing microorganisms [2,3]. Drugresistance efflux proteins comprise the primary efflux[(system, namely the )58.3(A)106.2(TP-binding cassette \(ABC\) family)][(transporters ener)10.9(gized by )68.1(A)104.5(T)0.8(P)113.8(,)-0.1( and the secondary active)…