梅岭霉素高产菌株链霉素抗性基因突变株筛选

通过链霉素对梅岭霉素 (Meilingmycin)产生菌南昌链霉菌NS 41 80菌株孢子致死浓度的测定 ,采用诱变剂EMS 4种不同剂量对菌株孢子进行诱变处理 ,然后涂布在含链霉素致死浓度的高氏平板上 ,获得了大量的链霉素抗性基因 (str)突变株。并进一步筛选到梅岭霉素高产菌株 80 5 1 1 2 2 1 ,在摇瓶条件下 ,只产梅岭霉素不产南昌霉素 ,梅岭霉素活性单位达 1 ,52 1 μg/mL,比NS 41 80的摇瓶发酵单位 855μg/mL提高了 77 9% ,该菌株连续传 6代进行摇瓶发酵     

南昌霉素高产菌株的链霉素抗性基因突变诱变筛选研究*

通过链霉素对南昌霉素 (Nanchangmycin)产生菌NS 41 80菌株孢子的致死浓度测定基础上 ,采用诱变剂甲基磺酸乙酯 (EMS)的不同诱变剂量对菌株孢子进行诱变处理 ,诱变处理的孢子涂布在含链霉素 ( 1 0 μg/mL)致死浓度的高氏平板上 ,获得了大量的链霉素抗性基因 (str)突变株。然后从 3,0 0 0株链霉素抗性基因 (str)突变株中通过初筛获得比诱变出发菌株产素能力提高 2 0 %以上的菌株 2 0 2株。再进一步通过摇瓶复筛 ,获得比出发菌株产素能力分别提高 1     

南昌霉素高产菌株的链霉素抗性基因突变诱变筛选研究

通过对链霉素对南昌霉素（Nanchangmycin)产生菌NS－41－80菌株孢子的致死浓度测定基础上,采用诱变剂甲基磺酸乙酯（EMS）的不同诱发剂量对菌株孢子进行诱变处理,诱变处理的孢子涂布在含链霉素（10ug/mL)致死浓度的高氏平板上,获得了大量的链霉素抗性基因（str)突变株。然后从3,000株链霉素抗性基因（str)突变株中通过初筛获得比诱变出发菌株产素能力提高20％以上的菌株202株,再进一步通过摇瓶复筛,获得比出发菌株产素能力分别提高100％,200％,300％高产菌株为48株,7株和1株,分别为复筛菌和初筛菌株的23．76％和1．60％,3．46％和0．23％,0．5％和0．03％,将产素能力提高240％以上5个菌株连同出发菌株连续3批次进行摇瓶发酵结果,5个突变株的产素能力均比出发菌株的产素能力提高57％－96．4％,其中突变株80－5．3－165菌株摇瓶发酵单位达6,000ug/mL以上,3批次摇瓶平均发酵单位达5,855ug/mL,建立了南昌霉素高产菌株的链霉素抗性基因突变诱变快速高效的筛选方法。     

链霉素抗性突变理性筛选新霉素高产菌株

采用化学诱变剂NTG结合链霉素抗性筛选法获得新霉素高产菌株。出发菌株费氏链霉菌(Streptomyces fradiae)FS1109的孢子悬液经不同剂量的化学诱变剂NTG处理后,涂布在含链霉素最小抑制浓度(3μg/m L)的培养基平板上培养,获得大量的链霉素抗性突变株。经影印法初筛和摇瓶发酵复筛,正突变率高于负突变率,获得一株遗传性状稳定的Streptomyces fradiae Str 63菌株,其新霉素生物活性单位比出发菌株提高了50%以上,且C组分较出发菌株的低。     

微波诱变结合抗性突变筛选放线菌素D高产菌株

采用微波结合链霉素抗性筛选法选育放线菌素D的高产菌株。通过考察链霉素对Streptomyces rubiginosohelvolus FIM-N31菌株孢子生长情况的影响确定链霉素致死浓度,出发菌株FIM-N31的孢子经微波辐照处理后,涂布在含链霉素致死浓度（50 μg/mL）的培养基平板上培养,获得了大量的链霉素抗性基因突变株。摇瓶发酵筛选突变株,结果获得一株遗传性状稳定的放线菌素D高产菌Str186,其产放线菌素D的能力比出发菌株提高了8倍以上。     

梅岭霉素产生菌抗药性突变标志诱变筛选模型的初步研究

本文通过梅岭霉素 (Meilingmycin)产生菌南昌链霉菌NS 4 1 80菌株孢子对 6种抗生素敏感性测定 ,采用诱变剂EMS四种不同诱变剂量对菌株孢子进行诱变处理 ,诱变处理的孢子涂布在含致死浓度链霉素的高氏平板上。然后从抗药性突变标志菌株中进一步筛选梅岭霉素高产菌株。在 150 0多个抗药性突变株中通过初筛获得了比诱变出发菌株的产素能力提高 50 %以上菌株。通过诱变剂量分别与抗药性突变率和突变株产素产量的变势统计分析表明 ,菌株抗药性突变与产素突变密切相关 ,产素突变的EMS诱变剂量高于抗药性突变诱变剂量 ,在 0 .0 3mol/LEMS剂量作用下 ,菌株致死率为 99.4 3% ,抗药性突变率为 0 .0 4 4 0 % ,建立了梅岭霉素产生菌抗药性突变标志诱变推理性筛选模型。为南昌链霉菌高产菌种选育研究作了有益的尝试 ,并有助于其它链霉菌属的抗生素产生菌育种研究。     

电场诱导棘孢小单胞菌原生质体融合

以小诺霉素（Micromomicin,MCR)产生菌棘孢小单胞菌（Micromonosporaechinospora）为出发菌株,诱变筛选得到一株链霉素抗性菌株,抗性菌株的原生质体分别用紫外线照射和热处理致死,通过单亲致死原生质体融合,以链霉素为选择条件选出融合株,经摇瓶发酵并结合薄层层析扫描（ThinLayerCharamotography,TLC）分析,筛得4株MCR百分含量高于亲株的融合子,MCR百分含量达到90％,效价1076u／ml,分别比亲株提高15％和11％。     

紫外诱变选育恩拉霉素高产菌株

本研究采用紫外诱变育种技术对一株产恩拉霉素抗真菌链霉菌(Streptomyces fungicidicus)F110进行了诱变处理,经链霉素抗性、利福霉素B抗性以及双重抗性筛选,共获得了132株抗生素抗性突变株,其中26株突变菌株的恩拉霉素产量与出发菌株相比均有明显提高。摇瓶发酵条件下,突变株SR93的恩拉霉素产量最高可达2 400μg/m L,与出发菌株相比提高了38%。传代结果表明:该突变株产素水平稳定,因此具备较好的开发及工业应用价值。     

梧宁霉素产生菌链霉素抗性基因突变株的筛选初报

采用紫外线、亚硝酸、亚硝酸与紫外线复合处理对菌株的孢子进行诱变 ,在含有链霉素最小抑制浓度(4μg/mL)的高氏平板筛选 ,分别得到 114株、110株、12 4株链霉素抗性突变株 ,其中梧宁霉素效价高于出发菌株的分别为 9株、9株、18株 ,正突变率分别达到 7.89%、8.18%、14 .5 2 % ,其中突变株HU 74的效价比出发菌株提高了 12 %。     

UV+MW复合诱变结合抗性突变筛选替考拉宁高产菌株

采用微波照射(MW)、紫外照射(UV)和MW+UV处理技术,对替考拉宁产生菌AT-92的孢子进行诱变,诱变处理的孢子悬液涂布在含替考拉宁致死浓度的培养基平板上培养,获得替考拉宁抗性突变株,通过摇瓶发酵对替考拉宁抗性基因突变株进行筛选,获得一株遗传性状稳定的替考拉宁高产菌AT 92-52-37菌株,其产替考拉宁能力比出发菌株的提高5倍。     

Genetic study of plasmid-associated high-frequency mutations to streptomycin resistance in Escherichia coli]

Escherichia coli CTR1(RT1)RHfm1) carrying two H-factors and having unusually high frequency of mutation to high level streptomycin resistance is studied. The high frequency of mutation (about 10(-6) to streptomycin resistance is connected with the presence of R factor RHfm1, controlling the resistance to chloramphenicol and low level streptomacin resistance, but not with RT1, controlling the resistance to tetracycline. Spontaneous or ethidium bromide-induced loss of RHfm1 is accompanied by a decrease of the mutation frequency to 10(-9). RHfm1 is efficiently transmissible to other strains at 28 degrees C. The acquisition of RHfm1 by strains of E. coli K-12 ans S. typhimurium LT2 was followed by a 1000--10000-fold increase of the frequejcy of mutation to streptomycin resistance. Some streptomycin resistant mutants were isolated, and chromosome location of the mutations was demonstrated. The streptomycin resistant mutants were unable to transmit high level of resistance to streptomycin with R factor, but only low level one. The loss of RHfm1 by streptomycin resistant mutants was accompanied by the return to the streptomycin sensitivity of the initial R- strans (E. coli K-12 mutants) or by a decrease of the streptomycin resistance to the level, only 2-fold higher than that of R- wild type (E. coli CTR1 mutant). Thus, the mutantions had practically no effect on streptomycin resistance of R- strains, but could lead to high resistance phenotypes in the presence of RHfm1. The mutant loci in all three studied strains were found to be closely linked to the locus "fus" on the genetic map of E. coli.     

农用抗生素TS99高产菌株的选育

在明确链霉素对农抗TS99产生菌Streptomyces fungicidicus YH04孢子的致死浓度为1.2μg/mL的基础上,以链霉素致死浓度为选择压力,采用不同剂量的紫外线照射对菌株孢子进行诱变处理,获得了大量的链霉素抗性基因突变株,进而从中筛选到发酵效价较出发菌株提高60%以上,且遗传稳定性良好的高产菌株Streptomyces fungicidicus YH9407.     

Novel approach for improving the productivity of antibiotic-producing strains by inducing combined resistant mutations

We developed a novel approach for improving the production of antibiotic from Streptomyces coelicolor A3(2) by inducing combined drug-resistant mutations. Mutants with enhanced (1.6- to 3-fold-higher) actinorhodin production were detected at a high frequency (5 to 10%) among isolates resistant to streptomycin (Str(r)), gentamicin (Gen(r)), or rifampin (Rif(r)), which developed spontaneously on agar plates which contained one of the three drugs. Construction of double mutants (str gen and str rif) by introducing gentamicin or rifampin resistance into an str mutant resulted in further increased (1.7- to 2.5-fold-higher) actinorhodin productivity. Likewise, triple mutants (str gen rif) thus constructed were found to have an even greater ability for producing the antibiotic, eventually generating a mutant able to produce 48 times more actinorhodin than the wild-type strain. Analysis of str mutants revealed that a point mutation occurred within the rpsL gene, which encodes the ribosomal protein S12. rif mutants were found to have a point mutation in the rpoB gene, which encodes the beta-subunit of RNA polymerase. Mutation points in gen mutants still remain unknown. These single, double, and triple mutants displayed in hierarchical order a remarkable increase in the production of ActII-ORF4, a pathway-specific regulatory protein, as determined by Western blotting analysis. This reflects the same hierarchical order observed for the increase in actinorhodin production. The superior ability of the triple mutants was demonstrated by physiological analyses under various cultural conditions. We conclude that by inducing combined drug-resistant mutations we can continuously increase the production of antibiotic in a stepwise manner. This new breeding approach could be especially effective for initially improving the production of antibiotics from wild-type strains.     

Compensatory adaptation to the deleterious effect of antibiotic resistance in Salmonella typhimurium

Most chromosomal mutations that cause antibiotic resistance impose fitness costs on the bacteria. This biological cost can often be reduced by compensatory mutations. In Salmonella typhimurium, the nucleotide substitution AAA42 --> AAC in the rpsL gene confers resistance to streptomycin. The resulting amino acid substitution (K42N) in ribosomal protein S12 causes an increased rate of ribosomal proofreading and, as a result, the rate of protein synthesis, bacterial growth and virulence are decreased. Eighty-one independent lineages of the low-fitness, K42N mutant were evolved in the absence of antibiotic to ameliorate the costs. From the rate of fixation of compensated mutants and their fitness, the rate of compensatory mutations was estimated to be > or = 10-7 per cell per generation. The size of the population bottleneck during evolution affected fitness of the adapted mutants: a larger bottleneck resulted in higher average fitness. Only four of the evolved lineages contained streptomycin-sensitive revertants. The remaining 77 lineages contained mutants that were still fully streptomycin resistant, had retained the original resistance mutation and also acquired compensatory mutations. Most of the compensatory mutations, resulting in at least 35 different amino acid substitutions, were novel single-nucleotide substitutions in the rpsD, rpsE, rpsL or rplS genes encoding the ribosomal proteins S4, S5, S12 and L19 respectively. Our results show that the deleterious effects of a resistance mutation can be compensated by an unexpected variety of mutations.     

Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2).

A strain of Streptomyces lividans, TK24, was found to produce a pigmented antibiotic, actinorhodin, although S. lividans normally does not produce this antibiotic. Genetic analyses revealed that a streptomycin-resistant mutation str-6 in strain TK24 is responsible for induction of antibiotic synthesis. DNA sequencing showed that str-6 is a point mutation in the rpsL gene encoding ribosomal protein S12, changing Lys-88 to Glu. Gene replacement experiments with the Lys88-->Glu str allele demonstrated unambiguously that the str mutation is alone responsible for the activation of actinorhodin production observed. In contrast, the strA1 mutation, a genetic marker frequently used for crosses, did not restore actinorhodin production and was found to result in an amino acid alteration of Lys-43 to Asn. Induction of actinorhodin production was also detected in strain TK21, which does not harbor the str-6 mutation, when cells were incubated with sufficient streptomycin or tetracycline to reduce the cell's growth rate, and 40 and 3% of streptomycin- or tetracycline-resistant mutants, respectively, derived from strain TK21 produced actinorhodin. Streptomycin-resistant mutations also blocked the inhibitory effects of relA and brgA mutations on antibiotic production, aerial mycelium formation or both. These str mutations changed Lys-88 to Glu or Arg and Arg-86 to His in ribosomal protein S12. The decrease in streptomycin production in relC mutants in Streptomyces griseus could also be abolished completely by introducing streptomycin-resistant mutations, although the impairment in antibiotic production due to bldA (in Streptomyces coelicolor) or afs mutations (in S. griseus) was not eliminated. These results indicate that the onset and extent of secondary metabolism in Streptomyces spp. is significantly controlled by the translational machinery.     

Development of high-level streptomycin resistance affected by a plasmid in lactic streptococci.

Some lactose-negative (Lac-) mutants of Streptococcus lactis C2 and ML3 exhibited development of very high level streptomycin resistance after incubation with subinhibitory concentrations of the drug for 18 to 22 h. These drug-resistant mutants showed no loss of resistance even after 6 months of subculturing in broth without any drug. The parental Lac+ strains did not show mutation to high-level streptomycin resistance. The Lac+ characteristic of the parental strain was conjugally transferred to Lac- derivatives of C2 and ML3, showing the ability to mutate to high-level resistance. When transconjugants were analyzed for this characteristic, they showed both mutable and nonmutable Lac+ types. The results suggested that genetic information for mutation to high-level streptomycin resistance in lactic streptococci resides on the chromosome, and its expression is affected by a plasmid. The plasmid profiles of strains C2, ML3, C2 Lac-, ML3 Lac-, and two kinds of transconjugants confirmed the presence of a plasmid of approximately 5.5 megadaltons in strains showing no mutation to high-level streptomycin resistance, while strains missing such a plasmid exhibited high-level streptomycin resistance after incubation with subinhibitory concentrations of the drug.     

Development of high-level streptomycin resistance affected by a plasmid in lactic streptococci

Some lactose-negative (Lac-) mutants of Streptococcus lactis C2 and ML3 exhibited development of very high level streptomycin resistance after incubation with subinhibitory concentrations of the drug for 18 to 22 h. These drug-resistant mutants showed no loss of resistance even after 6 months of subculturing in broth without any drug. The parental Lac+ strains did not show mutation to high-level streptomycin resistance. The Lac+ characteristic of the parental strain was conjugally transferred to Lac- derivatives of C2 and ML3, showing the ability to mutate to high-level resistance. When transconjugants were analyzed for this characteristic, they showed both mutable and nonmutable Lac+ types. The results suggested that genetic information for mutation to high-level streptomycin resistance in lactic streptococci resides on the chromosome, and its expression is affected by a plasmid. The plasmid profiles of strains C2, ML3, C2 Lac-, ML3 Lac-, and two kinds of transconjugants confirmed the presence of a plasmid of approximately 5.5 megadaltons in strains showing no mutation to high-level streptomycin resistance, while strains missing such a plasmid exhibited high-level streptomycin resistance after incubation with subinhibitory concentrations of the drug.     

A clinical isolate of transposon Tn5 expressing streptomycin resistance in Escherichia coli.

The central region of transposon Tn5 carries three antibiotic resistance markers: neo, ble, and str. The str gene codes for a phosphotransferase that inactivates streptomycin. This activity is phenotypically expressed in several gram-negative bacteria but not in Escherichia coli. We identified a Tn5 variant in E. coli clinical isolates that express streptomycin resistance. This transposon carries a 6-base-pair deletion within the str gene, near the 3' end. The same kind of mutation had been previously obtained experimentally from Tn5.     

[3H] dihydrostreptomycin accumulation and binding to ribosomes in Rhizobium mutants with different levels of streptomycin resistance.

Rhizobium trifolii B1, a symbiotic nitrogen fixer, is sensitive to streptomycin (10 microgram/ml) and spontaneously produces spheroplast-like forms during cultivation. Streptomycin-resistant mutants selected with high doses of antibiotic (1,000 microgram/ml) showed pleiotropic changes, including loss of spheroplast formation and infectivity to plants, whereas mutants selected with low doses of streptomycin (10 to 100 microgram/ml) retained properties of parent strain B1 (I. Zelazna-Kowalska, Acta Microbiol. Pol., in press). The present studies revealed that strain B1 and its mutant with a high level of streptomycin resistance, B1 strH, accumulated the antibiotic at similar rates. Mutant B1 strL, with a low level of streptomycin resistance (up to 100 microgram/ml), accumulated the antibiotic at a lower rate. Ribosomes isolated from strains B1 and B2 strL bound [3H]dihydrostreptomycin, whereas those from strain B1 strH did not. These observations indicate that, in R. trifolii B1, mutation to a high level of streptomycin resistance affects ribosomal structure, whereas low-level resistance involves a change in membrane permeability.