Effect of lincomycin on its own producer, Actinomyces roseolus, in periodic cultivation in a liquid medium

Lincomycin added to the cultivation medium induced a number of changes in the organism producing it during its ontogenesis when grown recurrently on liquid media. It was found that lincomycin inhibited the culture growth and decreased the absolute amount of the antibiotic synthesized while the specific activity of the culture increased. A number of cytomorphological rearrangements relevant to the adaptive protective reactions was found. It is suggested that an increase in the resistance of the culture to the antibiotic produced by it at the late developmental stages is the result of the above protective reactions.     

Streptomycin and lincomycin resistances are selective plastid markers in cultured Nicotiana cells

Summary Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.     

Action of lincomycin, chymotrypsin and their combinations on the course of experimental staphylococcal infection

The culture of Staphylococcus aureus was administered intraperitoneally in a dose of LD30 to albino mice. The animals of the 1st, 2nd and 3rd groups were treated with lincomycin, chymotripsin and combination of lincomycin with chymotripsin respectively. The animals of the 4th group were used as control and were not subjected to the treatment with the drugs. A part of the animals from every group was killed on the 3rd, 7th, 14th, 21st and subsequent days and their organs were investigated microscopically and bacteriologically. It was found that staphylococci was isolated from the control mice during a 50-day period after inoculation. Complete liberation of the organs from the causative agent within 25 days from the beginning of the experiment was registered in the animals treated with lincomycin. Isolation of the staphylococci was over by the 27th day in the animals treated with chymotrypsin. Liberation of the organs from the causative agent by the 17th day was observed in the albino mice treated with the combination of lincomycin with chymotrypsin. The combined use of lincomycin with chymotrypsin proved to be most effective: no death was registered among the albino mice, the levels of the pathogenicity and antibiotic resistance in the pathogenic staphylococci decreased.     

Enterotoxin production by Vibrio cholerae and Vibrio mimicus grown in continuous culture with microbial cell recycle

We have examined the effect of complete cell recycle on the production of cholera toxin (CT) by Vibrio cholerae and CT-like toxin by Vibrio mimicus in continuous culture fermentations. Complete cell recycle was obtained by filtering culture fluids through Amicon hollow fibers with an exclusion limit of 100,000 daltons (H1P100-20) and returning the concentrated cell slurry to the fermentor. A single 1-liter laboratory fermentor system modified with this recycle loop was capable of producing over 20 liters of cell-free culture filtrate per day. Toxin production in this system was compared with yields obtained in traditional continuous cultures and in shake flask cultures. Yields of CT from V. cholerae 569B in the recycle fermentor were highest at the highest dilution rate employed (1.0 vol/vol per h). The use of complete cell recycle dramatically increased yields over those obtained in continuous culture and equaled those obtained in shake flasks. The concentration of CT in the filtrate was slightly less than half of that measured in culture fluids sampled at the same time. Similarly, V. mimicus 61892 grown in the presence of 50 micrograms of lincomycin per ml produced 280 ng of CT per ml in the recycle fermentor, compared with 210 ng/ml in shake flasks under optimal conditions. The sterile filtrate from this fermentation contained 110 ng/ml.     

在林可霉素生物合成过程中铵离子调控作用的酶学研究

在FUS-50L发酵罐内,用林可链霉菌发酵生产林可霉素。研究发现,NH4^＋对林可霉素发酵过程具有显著的调控效应：补入硫酸铵前,发酵液中的NH4^＋浓度由3．0mmol／L消耗至1．0mmol／L以下的控制过程非常关键,这样可能使林可霉素合成酶大量合成,同时,解除了NH4^＋对谷氨酰合成酶（GS）的阻遏效应;20～24h,尽可能提高硫酸铵的平均补入速率,但最高NH4^＋的瞬时浓度应控制在19．0mmol／L以下,一方面可缩短生物量的积累时间,另一方面可避免过高浓度的NH4^＋对GS的抑制作用;24h后逐渐降低NH4^＋的平均补入速率,使主代谢流转入次级代谢;在发酵中期和后期,应将最低NH4^＋浓度控制在3．0～4．0mmol／L的范围内,避免铵离子对GS的阻遏效应,GS比活力与林可霉素的产量呈正相关关系。最终建立了动态的硫酸铵补加工艺。     

Enterotoxin production by Vibrio cholerae and Vibrio mimicus grown in continuous culture with microbial cell recycle.

We have examined the effect of complete cell recycle on the production of cholera toxin (CT) by Vibrio cholerae and CT-like toxin by Vibrio mimicus in continuous culture fermentations. Complete cell recycle was obtained by filtering culture fluids through Amicon hollow fibers with an exclusion limit of 100,000 daltons (H1P100-20) and returning the concentrated cell slurry to the fermentor. A single 1-liter laboratory fermentor system modified with this recycle loop was capable of producing over 20 liters of cell-free culture filtrate per day. Toxin production in this system was compared with yields obtained in traditional continuous cultures and in shake flask cultures. Yields of CT from V. cholerae 569B in the recycle fermentor were highest at the highest dilution rate employed (1.0 vol/vol per h). The use of complete cell recycle dramatically increased yields over those obtained in continuous culture and equaled those obtained in shake flasks. The concentration of CT in the filtrate was slightly less than half of that measured in culture fluids sampled at the same time. Similarly, V. mimicus 61892 grown in the presence of 50 micrograms of lincomycin per ml produced 280 ng of CT per ml in the recycle fermentor, compared with 210 ng/ml in shake flasks under optimal conditions. The sterile filtrate from this fermentation contained 110 ng/ml.     

Eremomycin in the treatment of antibiotic-associated colitis in golden hamsters]

The efficacy of eremomycin, a new glycopeptide antibiotic, was studied on a model of antibiotic-associated colitis in golden hamsters. The colitis was induced by intraperitoneal or intragastric administration of lincomycin. In a dose of 100 mg/kg administered orally once a day for 5 days eremomycin protected the animals from the lincomycin-induced colitis: some animals survived, the others died in later periods. When the animals were infected with a pathogenetic strain of Clostridium difficile followed by exposure to lincomycin the use of eremomycin produced the similar effect.     

林可霉素生物合成培养基的优化

以花生粉和棉籽蛋白粉取代了原培养基中的黄豆饼粉,采用响应面法对林可霉素产生菌的发酵培养基进行了优化.首先通过单因素试验及正交实验确定替代氮源及其浓度,采用Plackett-Burman实验分析各因素的主效应,选出对响应值影响较大的3个因素,即花生粉、K2HPO4和玉米浆.对这些因素做爬坡实验,确定三个重要因素的中心点浓...     

Electrochemiluminescence detection with integrated indium tin oxide electrode on electrophoretic microchip for direct bioanalysis of lincomycin in the urine

In this article, an antibiotic, lincomycin was determined in the urine sample by microchip capillary electrophoresis (CE) with integrated indium tin oxide (ITO) working electrode based on electrochemiluminescence (ECL) detection. This microchip CE-ECL system can be used for the rapid analysis of lincomycin within 40s. Under the optimized conditions, the linear range was obtained from 5 to 100 microM with correlation coefficient of 0.998. The limit of detection (LOD) of 3.1 microM was obtained for lincomycin in the standard solution. We also applied this method to analyzing lincomycin in the urine matrix. The limit of detection of 9.0 microM was obtained. This method can determine lincomycin in the urine sample without pretreatment, which demonstrated that it is a promising method of detection of lincomycin in clinical and pharmaceutical area.     

Photosystem II damage induced by chemically generated singlet oxygen in tobacco leaves

In the present work, we investigated the role of chemically generated singlet oxygen, produced by photodynamic effect of rose bengal, in damaging the PSII complex in tobacco leaves in which protein synthesis-dependent repair was inhibited by infiltration with lincomycin. A 30-min exposure to low-intensity (150 μmol m⁻² s⁻¹) photosynthetically active radiation (PAR) induced singlet oxygen production as detected by quenching of 3-[ N -(β-diethylaminoethyl)- N -dansyl]aminomethyl-2,2,5,5-tetramethyl-2,5-dihydro-1 H -pyrrole fluorescence in leaves infiltrated with both lincomycin and rose bengal. This light treatment caused photoinhibition of PSII, as revealed by the marked loss both of the photochemical yield and the amount of D1 protein in PSII reaction center. When rose bengal was not present in the leaves, these symptoms of photodamage were not induced by the same low-intensity PAR. However, when excitation pressure on PSII was increased to 1500 μmol m⁻² s⁻¹, irreversible photodamage of PSII was also observed, showing that the lincomycin treatment applied in vivo was sufficiently inhibiting protein repair. Our results show that singlet oxygen is able to cause oxidative damage in PSII directly, as suggested earlier and argue against its recently hypothesized role exclusive to inhibiting PSII protein repair ( Nishiyama et al. 2006 ).     

Effects of culture conditions on yield of Shiga-like toxin-IIv from Escherichia coli

The effects of selected culture conditions on production of Shiga-like toxin-II variant by an edema disease strain of Escherichia coli (412) and E. coli TB1 (pCG6) containing the cloned genes for Shiga-like toxin-II variant were examined. Incubation time, culture media, incubation temperature, starting pH of the culture medium, aeration, static culture, anaerobiosis, carbon sources, amino acids, antibiotics, and mitomycin C were investigated. The study showed that Shiga-like toxin-II variant was primarily cell associated and that strain TB1 (pCG6) produced as much as 100 times more toxin than did strain 412. Culture conditions that resulted in the greatest yield of Shiga-like toxin-II variant were incubation at 37 degrees C for 24 h with shaking in syncase broth initially adjusted to pH 8.5. Aerobic culture with shaking resulted in higher yields of Shiga-like toxin-II variant than did static aerobic or anaerobic culture. Addition of various carbon sources or amino acids, or tetracycline, lincomycin, or trimethoprim:sulfadoxine did not increase yields of toxin. The amount of Shiga-like toxin-II variant in supernatant preparations from strain TB1 (pCG6) was significantly increased by addition of mitomycin C to the culture medium.     

Study of the sensitivity of the L-forms of group A Streptococcus to antibiotics in artificial nutrient media and in human cell cultures

Sensitivity of L-forms of group A streptococci to 5 antibiotics such as erythromycin, lincomycin, tetracycline, gentamicin and chloramphenicol was studied in an artificial nutrient medium and cell cultures i.e. human fibroblast diploid cells and transplantable human heart cells (Girardi). In vitro investigation of the antibiotic effect on the streptococcal L-forms revealed their sensitivity to erythromycin (MIC, 0.4 micrograms/ml), lincomycin (MIC, 0.08 microgram/ml) and tetracycline (MIC, 2 micrograms/ml). The streptococcal L-forms were slightly sensitive to gentamicin (MIC, 6 micrograms/ml) and chloramphenicol (MIC, 30 micrograms/ml). Complete inhibition of the growth of the L-forms in the Girardi cells on the 1st day of the experiment after the antibiotics administration in single doses was induced by lincomycin, 5 micrograms/ml, erythromycin, 10 micrograms/ml, and tetracycline, 100 micrograms/ml. In the diploid cells, the respective figures were 50, 100 and 200 micrograms/ml. Chloramphenicol and gentamicin had an inhibitory effect on the growth of the L-forms but produced no sanative effect.     

亚硝基胍诱变选育林肯霉素高产菌株

以林肯链霉菌９４７－８（Ｓｔｒｅｐｔｏｍｙｃｅｓｌｉｎｃｏｌｎｅｎｓｉｓ９４７－８）为出发菌株（产林肯霉素９４０γ／ｍｌ）。采用孢子热处理方法处理出发菌株孢子,得到变异株９４７－８ｓ,产林肯霉素１０８０γ／ｍｌ。对９４７－８ｓ菌株进行ＮＴＧ诱变处理,得变异株９４７－８ｘ,产林肯霉素为１２１８γ／ｍｌ,且生产能力稳定。     

The induction of lincomycin resistance in Lycopersicon peruvianum and Lycopersicon esculentum

Lincomycin-resistant calli were induced from both Lycopersicon esculentum and Lycopersicon peruvianum using N-mitroso-N-methylurea (NMU) mutagenesis. From these calli lincomycin-resistant plants were regenerated. For L. peruvianum it was shown that the resistant plants could be divided in two classes with respect to their resistance to lincomycin and its derivative clindamycin. The first class comprised plants which were resistant to 500 mg/l lincomycin and showed no shoot or root formation in the presence of clindamycin; the second class consisted of plants resistant to 2000 mg/l lincomycin and these plants were able to form shoots and roots on clindamycin containing media. Lincomycin is an inhibitor of peptidyltransferase; chloroplast encoded parts of this enzymatic function are sensitive for this antibiotic. Reciprocal crosses between our lincomycin resistant and wild type L. peruvianum plants indicated a maternal inheritance of the mutation.     

The effect of lincomycin on the excretion of diethylstilbestrol and its uterotrophic action in rats.

The enterohepatic circulation of diethylstilbestrol (DES) has been shown to be extensive and to be dependent on enteric β-glucuronidase activity for release of absorbable DES from its nonabsorbable glucuronide excreted in bile. A regimen of the antibiotic lincomycin has been shown in rats to markedly reduce enteric β-glucuronidase activity, intestinal hydrolysis of C¹⁴-DES-glucuronide and absorption of radioactivity. Studies were therefore performed to determine if this lincomycin regimen, by reducing enterohepatic circulation of DES, would alter systemic effects of DES such as uterine weight gain in ovariectomized rats. The lincomycin regimen consisted of 25 mg twice daily by gastric intubation on days 1–4 and 500 mg/l in drinking water on days 1–7. Lincomycin-treated and control rats were injected s.c. with DES (1.6 or 5.0 μg/kg/day) on days 4–6 and sacrificed on day 7 for measurement of uterine weight; the injection on day 4 consisted on monoethyl-l-C¹⁴-DES. Lincomycin did not change the uterotrophic effect of DES. The regimen did, however, reduce the urinary excretion of radioactivity and increase the fecal excretion of glucuronide conjugates, consistent with reduced enterohepatic circulation of DES. In a separate study, bile from rats given DES s.c. was administered into the cecum of lincomycin-treated and control rats; whereas 31.8% of the cecal dose was excreted in the bile of controls, only 1.9% was excreted in lincomycin-treated rats, indicating the marked reduction of DES enterohepatic circulation produced by lincomycin. These findings suggest that the enterohepatic circulation of DES does not significantly contribute to its systemic effects.     

Improvement of lincomycin production by mutant selection and metabolic regulation

Lincomycin is a lincosamide antibiotic produced by Streptomyces lincolnensis. Through mutagenesis by ethylmethansulfonate (EMS) and ultraviolet (UV) irradiation repeatedly, M2 was picked out in plate with glutamine and propylproline orderly. In 50-L stirred bioreactor, the production of lincomycin, fermented by M2, was increased to 8136?u/ml under the optimal condition as compared to original strain S. lincolnensis 07–5 (6634?u/ml). Two-dimensional gel electrophoresis (2-D GE) and mass spectrometry (MS)-shown LmbG, LmbI, and acetohydroxy acid isomeroreductase were remarkably synthesized in M2. The gene lmbG and lmbI are responsible for methylation in the lincomycin biosynthetic cluster, while acetohydroxy acid isomeroreductase contributes to stronger metabolic capability. Finally, we obtained a better strain for industrial production.     

林可霉素生物合成的研究进展

林可霉素是林可链霉菌(Streptomyces lincolnensis)产生的林可酰胺类抗生素,它抑制细菌细胞的蛋白质合成,临床上主要用于治疗革兰氏阳性菌引起的感染性疾病。林可霉素生物合成基因簇已被克隆和测序。近年来,围绕林可酰胺和丙基脯氨酸的生物合成、调控等进行了深入研究,其硫化反应取得了突破性成果,本文综述了林可霉素生物合成的新进展。     

Amplification of lmbB1 gene in Streptomyces lincolnensis improves quantity and quality of lincomycin A fermentation

Abstract

As a lincosamide antibiotic, lincomycin is still important for treating diseases caused by Gram-positive bacteria. Manufacturing of lincomycin needs efforts to, e.g. maximize desirable species and minimizing unwanted fermentation byproducts. Analysis of the lincomycin biosynthetic gene cluster of Streptomyces lincolnensis, lmbB1, was shown to catalyze the conversion of L-dopa but not of L-tyrosine and then further generated the precursor of lincomycin A. Based on the principle of directed breeding, a strain termed as S. lincolnensis 24-2, was obtained in this work. By overexpressing the lmbB1 gene, this strain produces efficacious lincomycin A and suppresses melanin generation, whereas contains unwanted lincomycin B. The good fermentation performance of the mutant-lmbB1 (M-lmbB1) was also confirmed in a 15?L-scale bioreactor, which increased the lincomycin A production by 37.6% compared with control of 6435?u/mL and reduced the accumulation of melanin by 29.9% and lincomycin B by 73.4%. This work demonstrated that the amplification of lmbB1 gene mutation and metabolic engineering could promote lincomycin biosynthesis and might be helpful for reducing the production of other industrially unnecessary byproduct.     

Lincomycin at Subinhibitory Concentrations Potentiates Secondary Metabolite Production by Streptomyces spp.

Antibiotics have either bactericidal or bacteriostatic activity. However, they also induce considerable gene expression in bacteria when used at subinhibitory concentrations (below the MIC). We found that lincomycin, which inhibits protein synthesis by binding to the ribosomes of Gram-positive bacteria, was effective for inducing the expression of genes involved in secondary metabolism in Streptomyces strains when added to medium at subinhibitory concentrations. In Streptomyces coelicolor A3(2), lincomycin at 1/10 of its MIC markedly increased the expression of the pathway-specific regulatory gene actII-ORF4 in the blue-pigmented antibiotic actinorhodin (ACT) biosynthetic gene cluster, which resulted in ACT overproduction. Intriguingly, S. lividans 1326 grown in the presence of lincomycin at a subinhibitory concentration (1/12 or 1/3 of its MIC) produced abundant antibacterial compounds that were not detected in cells grown in lincomycin-free medium. Bioassay and mass spectrometry analysis revealed that some antibacterial compounds were novel congeners of calcium-dependent antibiotics. Our results indicate that lincomycin at subinhibitory concentrations potentiates the production of secondary metabolites in Streptomyces strains and suggest that activating these strains by utilizing the dose-response effects of lincomycin could be used to effectively induce the production of cryptic secondary metabolites. In addition to these findings, we also report that lincomycin used at concentrations for markedly increased ACT production resulted in alteration of the cytoplasmic protein (F_oF₁ ATP synthase α and β subunits, etc.) profile and increased intracellular ATP levels. A fundamental mechanism for these unique phenomena is also discussed.     

Interspecific protoplast fusion to rescue a cytoplasmic lincomycin resistance mutation into fertile Nicotiana plumbaginifolia plants

Summary A lincomycin-resistant cell line, LR105, was isolated in a mutagenized (0.1 mM N-ethyl-N-nitrosourea) callus culture initiated from a haploid Nicotiana sylvestris plant. The regenerated plants had an abnormal morphology and did not set viable seeds.Transfer of lincomycin resistance was attempted from the original N. sylvestris nuclear background into Nicotiana plumbaginifolia by protoplast fusion, since it was expected that resistance would be cytoplasmically coded. LR105 protoplasts were irradiated with a lethal dose (120 J kg^-1; ⁶⁰ Co source), fused with sensitive N. plumbaginifolia protoplasts and the colonies grown from the fused population were screened for lincomycin resistance. Expression of resistance was expected only if the cytoplasm of the irradiated cells had mixed with nonirradiated cytoplasm, and was reactivated as a result of cell fusion (Menczel et al. 1982).Plants were regenerated in 44 resistant clones. Plants in 41 clones had a N. plumbaginifolia nuclear genome. In three clones somatic hybrids were obtained. The resistant N. plumbaginifolia cybrid plants were fertile, unlike the original LR105 plants. Lincomycin resistance was inherited maternally in the eight clones in which crosses were made. In these clones the introduction of N. sylvestris chloroplasts into a N. plumbaginifolia nuclear background was confirmed by the SmaI restriction endonuclease pattern of the chloroplast DNA. The involvement of chloroplast DNA in determining lincomycin resistance is therefore implied.