Biosynthesis of lasalocid A. Biochemical alteration of polyether antibiotic production

The effect of known precursors, their fluorinated analogs, and biochemical inhibitors on the production of the polyether antibiotic, lasalocid A (1), by resting cells of Streptomyces lasaliensis was determined to study the biochemistry and regulation of antibiotic biosynthesis in vivo.     

Transformation of Streptomyces erythraeus

Streptomyces erythraeus strains were transformed efficiently with six different plasmid DNA vectors by a protocol that uses 0.2 mM Ca2+, 5 mM Mg2+ and 10% DMSO to enhance the stability of protoplasts to storage at -80 degrees C and their transformability at 30 degrees C. The primary thiostrepton-resistant (Thior) transformants for most vectors were unstable even when grown selectively. This instability did not appear to be due to incompatibility with indigenous S. erythraeus plasmids. Conversely, stable transformation was not the result of plasmid or host mutations. Transformation instability or plasmid copy number thus prevented successful shotgun-cloning of DNA that complemented the eryD mutation, which blocks the biosynthesis of erythromycin A, because only plasmid DNA without an insert could be isolated from a Thior Ery+ clone.     

Genetic analysis on bioconversion of aniline to acetaminophen inStreptomyces fradiae

S. fradiae showed the highest acetanilide p-hydroxylation activity in the tested strains. AndS. fradiae was well characterized genetically, especially with respect to tylosin production. Two mutants, which lost hydroxylation, were isolated in 140 regenerated colonies from protoplasts. In restriction enzyme digesion of total DNAs, isolation of giant linear plasmid DNA and determination of antibiotic resistances to chloramphenicol, tylosin, hygromycin B and mitomycin C, any differences among mutants and a wild type strain were not detected. These facts suggest that lesion on 6,000 Kb chromosomal DNA was responsible for the lack of p-hydroxylation activity induced by protoplast formation and regeneration.     

Protoplast fusion and gene recombination in the uncommon Actinomycete Planobispora rosea producing GE2270

An efficient method for protoplast generation for the uncommon actinomycete Planobispora rosea, the producer of the thiazolylpeptide antibiotic GE2270, was developed using a combination of hen egg white lysozyme and Streptomyces globisporus mutanolysin. This method converted more than 70% of vegetative mycelium to protoplasts, which were then regenerated with 50% efficiency in an optimized medium. When P. rosea protoplasts were efficiently fused, recombination between different antibiotic (streptomycin and gentamicin) resistance markers originated sensitive strains (str(s)gen(s)) at frequencies as high as 18% and double resistant fusants (str(r)gen(r)) at frequencies as high as 29%. Double resistant fusants showed GE2270 productivity intermediate between the productivity of the parental strains. Protoplast generation and fusion in P. rosea makes whole genome shuffling feasible as an approach to be used alternately with classical random mutagenesis in industrial strain improvement programs.     

苏氨酸产生菌原生质体的研究

考察苏氨酸产生菌GSR 8-25的原生质体制备及再生的最佳条件,并用紫外线(uv)诱变处理其原生质体,从再生株中筛选苏氨酸高产菌株。经多次连续诱变处理得一再生株25-20,其苏氨酸产量由出发菌株的15mg/ml提高到21.5mg/ml,提高率达43.3％.     

顶头孢霉229与12的分生孢子及原生质体的形成和再生

头孢菌素产生菌顶头孢霉菌株229的沉没培养或斜面培养都可形成分生孢子,并可用普通滤纸将它们与菌丝及节孢子分开,但是它们成活率极低.这种成活的分生孢子的数量与培养基成分有关.菌丝培养基成分对制备顶头孢霉原生质体有显著影响.用一种MM培养基培养的菌丝,不经巯基化合物预处理,酶解(1%纤维素酶)3小时后,可得到大量原生质体.原生质体的再生频率为1.8～4.6%.与分生孢子形成的菌落相比,原生质体再生菌落的产抗生素能力显示出较大的变异性.本文还讨论了山梨醇与Nikkomycin对菌丝生长形态及原生质体形成的影响.     

Genetic transformation ofStreptomyces caespitosus

Genetic transformation ofStreptomyces caespitosus by plasmid pIj 702 was carried out. Optimal conditions for the protoplast preparation ofStreptomyces caespitosus, its regeneration, and its transformation by pIj 702 were evaluated. Addition of 2% glycine to the culture broth was optimal for protoplast yield. Formation and regeneration of protoplasts were most efficient when the mycelium were harvested at between late log and stationary growth phase. The regeneration frequency of the protoplasts was 15% when the protoplasts were regenerated on R2YE agar media containing 0.5M sucrose. Under the best condition for protoplats regeneration, the optimal transformation frequency was achieved with 40% polyethylene glycol (M.W. 4,000) treatment for 2 minutes.     

Microbial transformation of aniline to acetaminophen

In order to obtain acetaminophen, a popular analgesic-antipyretic, through microbial p-hydroxylation and N-acetylation of aniline, various fungi and bacteria were screened. Among them,Streptomyces species were chosen for strain improvement by the use of interspecific protoplast fusion technique. Two interspecific fused strains were developed betweenS. rimosus (N-cetylation function) andS. aureofaciens (p-hydroxylation function) and also betweenS. lividans andS. globisporus. For efficient protoplast fusion and cell wall regeneration, various conditions were examined. In a typical experiment of mixedS. rimosus (pro⁻ his⁻) andS. aureofaciens (ilv⁻) protoplasts with 40% (w/v) polyethylene glycol 3350 (PEG) for 3 min gave 8.3×10⁻⁷ of fusion frequency. Treatment of mixedS. lividans (pant⁻) andS. globisporus (leu⁻) protoplasts with 50% (w/v) PEG for 3 min at 30°C gave 1.2×10⁻⁶ of frequency. Among the fused strains, up to 40–50% increase in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation of acetanilide, plasmid curing was attempted. We found that cells treated with acriflavine (at the frequency of 100%) and cells regenerated from protoplsts ofS. aureofaciens (2% frequency) lost their p-hydroxylation function.     

Virulence factors of salmonella ser. enteritidis strains isolated in Italy from food-borne outbreaks

Twelve Salmonella ser. Enteritidis strains phage type 4 isolated in Italy from different food-borne outbreaks were characterized for the expression of different virulence traits, for antibiotic resistance, and for plasmid DNA profile. All the twelve S. Enteritidis strains were able to invade and multiply within HeLa cell monolayers, even if at a lower efficiency if compared to an invasive Shigella flexneri strain. The strains were not hemolytic and produced only a moderate-level cytotoxic effect on HeLa cell monolayers. Moreover, all the strains examined produced mannose-sensitive hemagglutination with chicken erythrocytes but were not able to adhere to tissue culture cells. The strains did not produce the hydroxamate-type siderophore aerobactin or the specific ferric-aerobactin receptor. The S. Enteritidis strains were resistant only to spectinomycin, and eleven strains harbored a 38 MDa non-conjugative plasmid, while one strain harbored a 64 MDa conjugative plasmid which carried a colicinogenic activity-encoding locus. The uniformity of antibiotic resistance pattern, of the plasmid DNA content, and of the virulence factors produced indicated that the S. Enteritidis clinical isolates examined are clonally-related.     

Spectrofluorometric determination of the antibiotic lasalocid in blood.

A spectrofluorometric assay was developed for the determination of the antibiotic lasalocid in dog blood, based on the intrinsic fluorescence of the compound in ethyl acetate. The assay can measure "total" levels of drug and any metabolites present. The specificity of the assay was verified by TLC separation of the dog blood extract, which indicated the presence of only intact drug. The overall recovery (+/- SD) of lasalocid was 62.0 +/- 3.6% in the concentration range of 1.0-10 mug of compound/ml of dog blood. The sensitivity of the assay is 0.5 mug/ml. The assay was applied to the determination of blood levels of lasalocid in the dog following the intravenous administration of a 5-mg/kg dose.     

Activation of tyrosine 3-mono-oxygenase in pheochromocytoma cells by lasalocid.

Tyrosine 3-mono-oxygenase (TH) activity was measured in extracts of cells prepared from a transplantable rat phenochromocytoma. Incubation of the cells with the ionophore lasalocid (X-537A) results in an increase in TH activity. The activation of TH by lasalocid is associated with an increase in the V_max of the enzyme, but is not accompanied by a change in the apparent K_m of the enzyme for its pteridine cofactor or for tyrosine. In these respects, the activation of TH by lasalocid resembles that produced by incubation of the cells in media containing 56 mM K⁺. This effect of lasalocid is not dependent upon the presence of extracellular Ca²⁺. It is proposed that the activation of TH by lasalocid may be mediated by the ionophore-induced release of Ca²⁺ from some intracellular store.     

Enhanced sinefungin production by medium improvement, mutagenesis and protoplast regeneration of Streptomyces incarnatus NRRL 8089

Increased production of sinefungin, a very potent antifungal and antiparasitic nucleoside antibiotic was achieved by medium and strain improvement. When soybean-meal, dextrin and yeast extract were added as carbon and nitrogen sources to the fermentation medium, instead of corn steep liquor, soya-oil and glucose; the antibiotic yield increased from 40 micrograms/ml to 126 micrograms/ml with low biomass production. Strain improvement was attempted by two methods. The mean antibiotic yield of the variants after multistep mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine and ethyleneimine was 466 micrograms/ml. Protoplasts of the parental strain were prepared by lysozyme digestion from mycelia grown in a medium containing 0.7% glycine. The mean activity of the regenerated protoplasts was 664 micrograms/ml. Thus, the overall sinefungin production could be increased 16-fold.     

巨大芽孢杆菌原生质的产生和紫外线诱变

本文报道了巨大芽孢杆菌原生质体的制备和再生。并用紫外线对原生质体进行了诱变处理,从中筛选出几株产酸率高于亲代的菌株。本文还研究了芽孢形成和古龙酸产量的关系;所有的高产菌株或能形成耐热的芽孢,或不耐热的内孢子,而所有低产菌株均不能形成芽孢或内孢子。可能巨大芽孢杆菌在形成芽孢的过程中,产生出某些物质,促进了2-酮基古龙酸的生成。     

A bacterial test in liquid culture for the detection of mutagenic activity of antibacterial compounds: studies with cephalosporine HR 756

A detailed procedure for the short term evaluation of mutagenic activity in the Salmonella assay in liquid cultures is given for Cephalosporine HR 756. The essential steps of the test are: a) determination of absence of interference of the pKM101 plasmid present in the bacterial tester strains, b) determination of the doses of the antibiotic required to obtain a limited killing, c) introduction of appropriate controls with known mutagenic compounds active in the presence and in the absence of metabolic activation, d) segregation of mutants. Following this procedure we have determined the absence of any mutagenic activity in the liquid assay of Cephalosporine HR 756.     

Phenotypic changes associated with loss of expression of tylosin biosynthesis and resistance genes in Streptomyces fradiae

Two mutants of the tylosin-producing Streptomyces fradiae defective in the biosynthesis of the macrolide antibiotic tylosin were isolated from colonies derived from regenerated protoplasts. Both strains were unable to carry out any of at least seven tylosin biosynthetic steps and were sensitive to tylosin. One strain, JS82, was also more sensitive to chloramphenicol (Cm), mitomycin C (Mc), hygromycin B (Hm) and kanamycin (Km) than its parent strain. The other strain, JS87, was also more sensitive to Cm than wild type but expressed normal levels of resistance to Mc and Hm. Both strains expressed genetic instabilities associated with auxotrophy or expression of antibiotic resistance. Since the genetic instabilities were not due to defective error-free or error-prone DNA repair, they appear to be due to genetic rearrangements associated with the deletion or amplification of sequences linked to and perhaps encompassing tylosin biosynthesis genes.     

Giant linear plasmids in Streptomyces: a treasure trove of antibiotic biosynthetic clusters

Many giant linear plasmids have been isolated from Streptomyces by using pulsed-field gel electrophoresis and some of them were found to carry an antibiotic biosynthetic cluster(s); SCP1 carries biosynthetic genes for methylenomycin, pSLA2-L for lankacidin and lankamycin, and pKSL for lasalocid and echinomycin. Accumulated data suggest that giant linear plasmids have played critical roles in genome evolution and horizontal transfer of secondary metabolism. In this review, I summarize typical examples of giant linear plasmids whose involvement in antibiotic production has been studied in some detail, emphasizing their finding processes and interaction with the host chromosomes. A hypothesis on horizontal transfer of secondary metabolism involving giant linear plasmids is proposed at the end.     

The distribution of DNA sequences hybridizing with antibiotic production and resistance gene probes within type strains and wild isolates of Streptomyces species.

Total DNA preparations from 74 antibiotic-producing type strains and 102 natural Streptomyces isolates were examined by dot blots for homology to 6 antibiotic production and resistance genes. Pattern diversity of hybridizations decreased as stringency increased from 65% to 85%. There were 146 unique profiles at 65% stringency with 13 repeated patterns, whilst there were only 14 unique and 11 repeated profiles at 85% stringency. Most of the strains which hybridized at 85% reacted with one or two probes although a few strains showed multiple homologies. This data was used to cluster strains and the groups defined were examined for phenotypic antibiotic resistance. Producers of certain classes of antibiotics clustered to specific groups and some gene homologies were more common amongst strains which produced similar antibiotics.     

The possible involvement of a plasmid(s) in actinomycin synthesis by Streptomyces parvulus and Streptomyces antibioticus.

The treatment of Streptomyces parvulus and Streptomyces antibioticus with acriflavine or novobiocin resulted in the loss of ability to produce actinomycin. The concomitant loss of ability to form aerial mycelium and the incidence of auxotrophic progeny (S. parvulus) were both low relative to the loss of the antibiotic-producing property. Protoplast fusion induced by polyethylene glycol 4000, using suitable auxotrophic strains of S. parvulus, resulted in high recombination frequencies to prototrophy (9.6 approximately 15%). When fusion was carried out between auxotrophic act+ and act- strains, respectively, there was a high frequency (84 approximately 95%) of the actinomycin synthesizing character among the prototrophic recombinants examined. No actinomycin-producing recombinant was detected in similar experiments between auxotrophic non-producing strains.     

紫外线和氯化锂对核糖甙链霉菌原生质体诱变作用的探讨

核糖霉素产生菌核糖甙链霉菌（Streptomyces ribosidificus)原生质体经紫外线和氯化锂复合处理得到2株新的菌株RF2－25和RF3－173,产生抗生素核糖霉素的能力分别比出发株RF－5提高了17％和15．3％,且已供工业化正常生产。该方法简单易行,效果很好。     

纳他霉素产生菌基因组重排育种

Streptomyces gilvosporeus SG-1原生质体经紫外线诱变并筛选链霉素抗性菌株，获得纳他霉素高产菌株。在上述高产突变株中选择4株作为亲本进行基因组重排育种，筛选得到了高产重排菌株，其中一株重排菌株S．gilvosporeus GS-74的纳他霉素产量为3574mg／L。为产量最高的亲本菌株的153％，比原始出发菌株SG-1提高1.17倍。