Expression of cloned bialaphos resistance gene (bar) in Streptomyces strains

The object of the study was a strain of Streptomyces hygroscopicus producing bialaphos, an antibiotic used as a herbicide, which is promising and ecologically safe. Molecular cloning of a bialaphos resistance gene (bar) was performed in the recipient strain, S. lividans TK64, within the 2.0-kb DNA fragment with the plasmid pIJ699. Introduction of a bar gene into another strain producing bialaphos, i.e. S. viridochromogenus Tu494, led to its higher constitutive resistance to bialaphos. The results confirmed the data on different regulation of bar (S. hygroscopicus) and pat (S. virido-chromogenus) resistance genes.     

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.     

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.     

The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster

Summary We have isolated and studied the organization ofStreptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity toS. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector pIJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.     

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.     

Resistance to macrolides, lincosamides and streptogramin type B antibiotics due to a mutation in an rRNA operon of Streptomyces ambofaciens.

Streptomyces ambofaciens produces spiramycin, a macrolide antibiotic and expresses an inducible resistance to macrolides, lincosamides and streptogramin B antibiotics (MLS). From a mutant of S.ambofaciens exhibiting a constitutive MLS resistance phenotype a resistance determinant was cloned on a low copy number vector (pIJ61) through its expression in Streptomyces lividans. Further characterization has shown that this determinant corresponded to a mutant rRNA operon with a mutation in the 23S rRNA gene. In different organisms, mutations leading to MLS resistance have been located at a position corresponding to the adenine 2058 of Escherichia coli 23S rRNA. In the 23S rRNA from S.ambofaciens a similar position for the mutation has been postulated and DNA sequencing of this region has shown an adenine to guanine transition at a position corresponding to 2058. S.ambofaciens possesses four rRNA operons which we have cloned. In Streptomyces, contrary to other bacteria, a mutation in one among several rRNA operons confers a selectable MLS resistance phenotype. Possible reasons for this difference are discussed.     

Molecular cloning of the nosiheptide resistance gene from Streptomyces actuosus ATCC 25421

An 8.5 kb BamHI DNA fragment conferring resistance to nosiheptide, a peptide antibiotic of the 'thiostrepton group', was cloned from Streptomyces actuosus ATCC 25421 in Streptomyces lividans 1326. Two BamHI fragments of S. actuosus, the 8.5 kb fragment and an additional 3.0 kb fragment, hybridized with a thiostrepton resistance gene probe (pIJ30). The 8.5 kb fragment showed a relatively low degree of homology with the thiostrepton resistance gene. The restriction map of the nosiheptide resistance gene isolated here was significantly different from the map of the thiostrepton resistance gene previously published.     

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。     

Molecular cloning of chlortetracycline resistance gene from chlortetracycline producer Streptomyces aureofaciens

The chlortetracycline (CT) resistance gene ctr was cloned from S. aureofaciens 633, a strain producing the antibiotic. The 6.6-kb DNA Bam HI fragment containing the resistance gene was cloned with the plasmid vector pIJ699. Comparison of the restriction maps of the cloned gene and the oxytetracycline (OT) resistance gene otrA from S. rimosus revealed their similarity which enabled identification of the cloned resistance gene as otrA. Investigation of the resistance determinants in S. aureofaciens 633 made it possible to identify a mtr gene(s). It was demonstrated that introduction of a ctrA gene into S. lividance provided a simultaneous increase in the resistance of the recipient strain to CT and a number of macrolide antibiotics. The CT resistance determinants in S. lividans TK64 showed properties of exogenous induction by CT and the macrolide antibiotics similar to the properties of the mtr gene(s) of S. aureofaciens. Possible adaptation properties of mtr genes are discussed.     

Cloning of grisin resistance gene gsr and the study of its functioning in Streptomyces griseus Kr. strain

S. griseus Kr. is a commercial strain producing grisin, an antibiotic of the streptothricin group used as a feed additive. It was shown earlier that genetic instability of the strain was very high which was evident from a high frequency of nonreverting Grn- Grns mutants. With densitographic analysis of chromosomal DNA electrophoregrams and DNA-DNA hybridization it was revealed that the molecular basis of the genetic instability of the S. griseus strain was deletion of a DNA fragment about 20 kb in size containing a grisin resistance gene. The resistance gene designated as gsr was cloned to S. lividans TK 64 within the plasmid vector pIJ699. The restriction map of a cloned DNA fragment with a gsr gene was constructed and its similarity to that of a nat gene resistant to norseothricin, another streptothricin was observed. Introduction of a gsr gene within the multicopy plasmid pIJ699 into S. griseus 212, a highly productive strain synthesiing the antibiotic, led to an increase in its resistance and productivity. Proceeding from the preliminary data on possible linkage of a gsr gene and grisin biosynthesis genes, it appeared possible to use the cloned gene as a molecular probe in cloning the biosynthesis genes.     

Overexpression of a Streptomyces viridochromogenes gene (glnII) encoding a glutamine synthetase similar to those of eucaryotes confers resistance against the antibiotic phosphinothricyl-alanyl-alanine.

Phosphinothricyl-alanyl-alanine (PTT), also known as bialaphos, contains phosphinothricin, a potent inhibitor of glutamine synthetase (GS). A 2.75-kilobase NcoI fragment of the Streptomyces viridochromogenes PTT-resistant mutant ES2 cloned on a multicopy vector mediated PTT resistance to S. lividans and to S. viridochromogenes. Nucleotide sequence analysis of the 2.75-kb NcoI fragment revealed the presence of three open reading frames. Open reading frame 3 was termed glnII since significant similarity was found between its deduced amino acid sequence and those from GS of eucaryotes and GSII of members of the family Rhizobiaceae. Subcloning experiments showed that PTT resistance is mediated by overexpression of glnII encoding a 37.3-kilodalton protein of 343 amino acids. A three- to fourfold increase in gamma-glutamyltransferase activity could be observed in S. lividans transformants carrying the glnII gene on a multicopy plasmid. For S. viridochromogenes it was shown that PTT resistance conferred by the 2.75-kb NcoI fragment was dependent on its multicopy state. GS activity encoded by glnII was found to be heat labile. Southern hybridization with seven different Streptomyces strains suggested that they all carry two types of GS genes, glnA and glnII.     

Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance.

The nucleotide sequence of pC194, a small plasmid from Staphylococcus aureus which is capable of replication in Bacillus subtilis, has been determined. The genetic determinant of chloramphenicol (CAM) resistance, which includes the chloramphenicol acetyl transferase (CAT) structural gene, the putative promoter and controlling element of this determinant, have been mapped functionally by subcloning a 1,035-nucleotide fragment which specifies the resistance phenotype using plasmid pBR322 as vector. Expression of CAM resistance is autogenously regulated since the 1,035-nucleotide fragment containing the CAT gene sequence and its promoter cloned into pBR322 expresses resistance inducibly in the Escherichia coli host. A presumed controlling element of CAT expression consists of a 37-nucleotide inverted complementary repeat sequence that is located between the -10 and ribosome-loading sequences of the CAT structural gene. Whereas the composite plasmid containing the minimal CAT determinant cloned in pBR322 could not replicate in B. subtilis, ability to replicate in B. subtilis was seen if the fragment cloned included an extension consisting of an additional 300 nucleotides beyond the 5' end of the single pC194 MspI site associated with replication. This 5' extension contained a 120-nucleotide inverted complementary repeat sequence similar to that found in pE194 TaqI fragment B which contains replication sequences of that plasmid. pC194 was found to contain four opening reading frames theoretically capable of coding for proteins with maximum molecular masses, as follows: A, 27,800 daltons; B, 26,200 daltons; C, 15,000 daltons; and D, 9,600 daltons. Interruption or deletion of either frame A or D does not entail loss of ability to replicate or to express CAM resistance, whereas frame B contains the CAT structural gene and frame C contains sequences associated with plasmid replication.     

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.     

吸水链霉菌Streptomyces hygroscopicus 17997中噬菌体基因转移系统的建立及其应用

由吸水链霉菌Streptomyces hygroscopicus 17997产生的格尔德霉素geldanamycin(GA)属安莎类抗生素,具有良好的抗肿瘤和抗病毒活性。本文应用链霉菌温和噬菌体ΦC31衍生的KC515载体,在吸水链霉菌S．hygroscopicus 17997中建立并优化了S．hygroscopicus 17997的基因转染体系。利用所建立的基因转染体系,以基因阻断技术从S．hygroscopicus 17997基因文库含有多组PKS基因柯斯质粒中,鉴定了与GA PKS生物合成相关基因的柯斯质粒,该工作为GA生物合成基因簇的克隆奠定了基础。     

Effect of a Cloned DNA Fragment from Streptomyces chrysomallusNo. 2 on the Metabolism of Certain Streptomyces Strains

The effects on a cloned DNA fragment carrying an actinomycin resistance determinant on physiological processes in strains of streptomycetes with various potencies in producing this antibiotic, their inactive mutants, and the model strain ofStreptomyces lividans66 were studied. This fragment was shown to modulate bacterial resistance to actinomycin and biosynthesis of antibiotics.     

Effect of cloned DNA fragment from Streptomyces chrysomallus No.2 on metabolism in Streptomyces strains

The effects on cloned DNA fragment carrying an actinomycin resistance determinant on physiological processes in streptomyces strains with various potencies in producing this antibiotic, their inactive mutants, and model strain of Streptomyces lividans 66 were studied. This fragment was shown to modulate bacterial resistance to actinomycin and biosynthesis of antibiotics.     

Cloning of a lincosamide resistance determinant from Streptomyces caelestis, the producer of celesticetin, and characterization of the resistance mechanism.

Self-resistance has been investigated in Streptomyces caelestis (producer of the lincosamide antibiotic celesticetin), from which a lincosamide resistance determinant (clr) has been isolated on a 1-kilobase DNA fragment and cloned in Streptomyces lividans. The clr product is a specific methylase which produces a single residue of N6-monomethyladenine in 23S rRNA at position 2058, thereby rendering the 50S ribosmal subunit resistant to the action of lincosamides.     

Analysis of the nourseothricin-resistance gene (nat) of Streptomyces noursei

A gene (nat) conferring resistance to the streptothricin antibiotic nourseothricin (Nc) was cloned from the producer Streptomyces noursei into Streptomyces lividans on the vector pIJ702 to form pNAT1. The nat gene was localized on a 1-kb SalI-MboI fragment, which also carries the nat promoter. Divergent promoter activity from the nat promoter region was identified on the cloned fragment using promoter probe plasmids pIJ486 and pIJ487. The nat gene is not expressed from its own promoter in Escherichia coli as shown by its failure to promote cat expression in promoter-less plasmid pBB100 and by the expression of NcR in only one orientation, when cloned in pUC19. In S. lividans 7A, harbouring plasmid pNAT1, an Nc-acetylating activity (NAT) was associated with the cloned resistance gene. The substrate specificity of NAT correlated well with the substrate range of the acetyltransferase in S. noursei and Tn1825-determined streptothricin resistance in Gram-negative bacteria. Moreover, an extract of S. lividans carrying pNAT1 showed specific serological cross-reactivity with an extract of E. coli carrying Tn1825.