Excision repair of gamma-ray-induced alkali-stable DNA lesions with the help of gamma-endonuclease from Micrococcus luteus.

gamma-Endonuclease Y, an enzyme that hydrolyses phosphodiester bonds at alkalistable lesions in gamma-irradiated (N2, tris buffer) DNA, has been partially purified from Micrococcus luteus. The enzyme has a molecular weight of about 19 000, induces single-strand breaks with 3'OH-5'PO4 termini and contains endonuclease activity towards DNA treated with 7-bromomethylbenz(a)anthracene. gamma-Endonuclease Y induces breaks in OsO4-treated poly(dA-dT) and apparently is specific towards gamma-ray-induced base lesions of the t' type. The complete excision repair of gamma-endonuclease Y substrate sites has been performed in vitro by gamma-endonuclease Y, DNA polymerase and ligase.     

Gamma endonuclease of Micrococcus luteus: action on irradiated DNA

Gamma endonuclease is a Mg2+-independent enzyme of Micrococcus luteus that recognizes and cleaves DNA at a variety of altered pyrimidines produced by ionizing radiation. The production of enzyme-recognizable sites (ERS) by ionizing radiation under different irradiation conditions was measured. Ionizing radiation produced the greatest number of ERS when irradiations were performed under anoxic conditions in the presence of the free radical scavenger KI. Since dihydrothymine is a major pyrimidine lesion produced in DNA during anoxic irradiation, the ability of gamma endonuclease to excise this lesion was assessed. Dihydrothymine was released from DNA irradiated under anoxic conditions in a radiation dose-dependent manner, consistent with gamma endonuclease's known DNA glycosylase activity. Gamma endonuclease was also shown to cleave heavily uv-irradiated DNA. When the sequence specificity of gamma-endonuclease cleavage was studied using uv-irradiated DNA, cleavage was seen specifically at cytosines. The identity of this enzyme-recognizable cytosine photoproduct is not known.     

Micrococcus luteus correndonucleases. II. Mechanism of action of two endonucleases specific for DNA containing pyrimidine dimers.

Py pyrimidine dimers Py correndonucleases I and II from Micrococcus luteus act exclusively on thymine-thymine, cytosine-cytosine, and thymine-cytosine cyclobutyl dimers in DNA, catalyzing incision 5' to the damage and generating 3'-hydroxyl and 5'-phosphoryl termini. Both enzymes initiate excision of pyrimidine dimers in vitro by correxonucleases and DNA polymerase I. The respective incised DNAs, however, differ in their ability to act as substrate for phage T4 polynucleotide ligase or bacterial alkaline phosphatase, suggesting that each endonuclease is specific for a conformationally unique site. The possibility that their respective action generates termini which represent different degrees of single strandedness is suggested by the unequal protection by Escherichia coli binding protein from the hydrolytic action of exonuclease VII.     

Death of Micrococcus luteus in Soil

Micrococcus luteus cells died relatively quickly when they were added to natural soil. The results were similar for soil in nature and as soil samples in the laboratory. The cells died more quickly when nutrients were added to the soil. Those cells that survived soil residence exhibited a temporary lengthening of the time required for colonial growth and pigment formation on laboratory media. They had not gained increased survival capability, however. This was evident when they were retested in soil. Good survival of the M. luteus cells was noted when the soil was incubated at lowered temperatures. Some protection to the cells was provided by slow drying of the soil during incubation or by addition of NaCl. Microscopic examination of the soil revealed that the M. luteus cells were being physically destroyed and that two different bacteria were growing in the areas where the cells had lysed. It was suggested that bacterial predators in the soil might be associated with the death of the M. luteus cells.     

DNA-relaxing enzyme from Micrococcus luteus.

A DNA-relaxing enzyme which catalyzes the conversion of superhelical DNA to a non-superhelical covalently closed form has been purified from Micrococcus luteus to near homogeneity by two chromatographic steps. The enzyme is a single polypeptide chain. As determined by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and gel filtration on Sephadex G 150, the molecular weight is 115,000. The DNA-relaxing activity determined as a function of enzyme concentration follows a sigmoidal curve. The enzyme requires Mg++ for activity. In the presence of 4.5 mM Mg++ addition of 50-250 mM KCl yields incompletely relaxed DNA molecules (intermediates); intermediates are also observed in the absence of KCl, when the reaction is carried out at 0 degree C or at Mg++ concentrations exceeding 10 mM.     

Bacterial Predators of Micrococcus luteus in Soil

Micrococcus luteus cells died relatively rapidly when they were added to natural soil. Microscopic observation showed that the cells were being physically destroyed by bacterial predators in the soil. Two of these predators were responsible for the initial, main attack, and they were isolated. The isolates on laboratory media lysed M. luteus cells in a manner similar to the attacks that occurred in soil. Neither predator was obligate, however, nor were they nutritionally fastidious. One of these bacteria produced mycelium and conidia. Under nutritionally poor conditions it used slender filaments of mycelium to seek out host cells. It had at least some of the characteristics of a Streptoverticillium species. The other bacterium was a short, gram-negative rod that did not easily fit into any of the known groups of gram-negative bacteria. It attached to host cells, but its mechanism of lysing these cells is not known.     

Binding of Dissolved Strontium by Micrococcus luteus

Resting cells of Micrococcus luteus have been shown to remove strontium (Sr) from dilute aqueous solutions of SrCl₂ at pH 7. Loadings of 25 mg of Sr per g of cell dry weight were achieved by cells exposed to a solution containing 50 ppm (mg/liter) of Sr. Sr binding occurred in the absence of nutrients and did not require metabolic activity. Initial binding was quite rapid (<0.5 h), although a slow, spontaneous release of Sr was observed over time. Sr binding was inhibited in the presence of polyvalent cations but not monovalent cations. Ca and Sr were bound preferentially over all other cations tested. Sr-binding activity was localized on the cell envelope and was sensitive to various chemical and physical pretreatments. Bound Sr was displaced by divalent ions or by H⁺. Other monovalent ions were less effective. Bound Sr was also removed by various chelating agents. It was concluded that Sr binding by M. luteus is a reversible equilibrium process. Both ion exchange mediated by acidic cell surface components and intracellular uptake may be involved in this activity.     

一种由藤黄微球菌产生的纤溶酶

血栓栓塞性疾病如心肌梗塞、脑栓塞等是危害人类健康、导致死亡率最高的疾病之一.全球每年约1 700万人死于心脑血管病,我国每年约有300万人死于此类疾病.治疗此类疾病的主要手段之一是溶栓疗法,即注射溶栓剂疏通血管,但传统的溶栓药物如t-PA、尿激酶等有半衰期短、使用量大、价格昂贵、易引发出血症等不良副作用,因此开发新型溶栓类药物,提供廉价高效、副作用小、使用方便的溶栓药物,对栓塞性疾病的治疗具有重要意义.     

Acetylputrescine deacetylase from Micrococcus luteus K-11

An acetylputrescine deacetylase was induced in Micrococcus luteus K-11, and was partially purified and characterized briefly. The enzyme was most active toward acetylputrescine, followed by N⁸-acetylspermidine and acetylcadaverine, but was inactive toward N¹-acetylspermidine and N¹-acetylspermine. The K_m value for acetylputrescine was 0.321 mM. It was almost unaffected by -SH blocking agents but was inhibited by metal ions such as Cu²⁺ and Ni²⁺. Its molecular weight estimated by Sephacryl S-200 column chromatography was 115000.     

Adaptive response of Micrococcus luteus to alkylating chemicals

Wild type M. luteus cells have been adapted by a step-wise treatment with sub-lethal concentrations of MNNG. The adapted cells exhibit 5.7 fold increased resistance to the killing effects of the mutagen and a simultaneous efficient removal of various base modifications present in cellular DNA. A protein extract prepared from adapted cells contains inducible repair functions which can reduce 80-90% of the alkylated DNA content of 06-MeG is effected by a transmethylase and there is no concomitant release of the modified base. However, N-3 MeG is released as a free modified base through the action of a DNA glycosylase. The release of N-3 MeA is unaffected by the induction treatment whereas that of N-7 methylpurine is slightly improved in the adapted cells.     

Copper biosorption by chemically treated Micrococcus luteus cells

In order to clarify the binding states of copper in microbial cells, copper biosorption from aqueous systems using the chemically treated Micrococcus luteus IAM 1056 cells (hot water-treated, diluted NaOH-treated, chloroform–methanol-treated, and chloroform–methanol/concentrated KOH-treated cells) was examined. The intact cells of M. luteus adsorbed 527 mol of copper per g cells, and its copper adsorption was very rapid and was affected by the solution pH. The chloroform–methanol/concentrated KOH-treated cells showed higher copper biosorption capacity than the intact and the other chemically treated cells. The electron paramagnetic resonance (EPR) parameters, g and |A |, of Cu(II) ion in microbial cells indicate that Cu(II) ion in the intact and all the chemically treated cells have coordination environments with nitrogen and oxygen as donor atoms, being similar to those of type II proteins. The parameter g also indicated that the coupling between Cu(II) ion and the cell materials in the CHCl₃–MeOH/concentrated KOH-treated cells is rather more stable than those between Cu(II) ion and the cell materials in the other treated cells.     

Further characterization of the polynucleotide phosphorylase of Micrococcus luteus

The purification of polynucleotide phosphorylase from Micrococcus luteus by chromatography on phosphocellulose columns is described. This procedure offers several advantages over previous procedures.     

Degradation of Pyridine by Micrococcus luteus Isolated from Soil

An organism capable of growth on pyridine was isolated from soil by enrichment culture techniques and identified as Micrococcus luteus. The organism oxidized pyridine for energy and released N contained in the pyridine ring as ammonium. The organism could not grow on mono- or disubstituted pyridinecarboxylic acids or hydroxy-, chloro-, amino-, or methylpyridines. Cell extracts of M. luteus could not degrade pyridine, 2-, 3-, or 4-hydroxypyridines or 2,3-dihydroxypyridine, regardless of added cofactors or cell particulate fraction. The organism had a NAD-linked succinate-semialdehyde dehydrogenase which was induced by pyridine. Cell extracts of M. luteus had constitutive amidase activity, and washed cells degraded formate and formamide without a lag. These data are consistent with a previously reported pathway for pyridine metabolism by species of Bacillus, Brevibacterium, and Corynebacterium. Cells of M. luteus were permeable to pyridinecarboxylic acids, monohydroxypyridines, 2,3-dihydroxypyridine, and monoamino- and methylpyridines. The results provide new evidence that the metabolism of pyridine by microorganisms does not require initial hydroxylation of the ring and that permeability barriers do not account for the extremely limited range of substrate isomers used by pyridine degraders.     

藤黄微球菌降解真菌毒素玉米赤霉烯酮的研究

目的研究并优化藤黄微球菌降解真菌毒素玉米赤霉烯酮(ZEN)的因素条件。方法采用HPLC的检测方法对藤黄微球菌降解真菌毒素玉米赤霉烯酮的影响因素(培养基、温度、pH、摇床转速、培养时间和金属离子等)进行优化研究。结果藤黄微球菌在0.05 mol/LMnCl2、初始pH为7.0的LB培养基中,37℃,180 r/min,连续培养120 h,能降解99%的ZEN毒素(初始浓度为2μg/ml)。结论藤黄微球菌降解真菌毒素ZEN的能力与培养基成分、pH和添加的金属离子种类密切相关。     

Riboflavin Production during Growth of Micrococcus luteus on Pyridine

Micrococcus luteus produced 29 μM riboflavin during growth on 6.5 mM pyridine but not during growth on other substrates. On the basis of the results of radiolabelling studies, riboflavin was not directly synthesized from pyridine. Pyridine may interfere with riboflavin biosynthesis or elicit a general stress response in M. luteus.     

Biochemical properties of penicillin amidohydrolase from Micrococcus luteus.

Some biochemical properties of whole-cell penicillin amidohydrolase from Micrococcus luteus have been studied. This whole-cell enzyme showed its maximal activity at 36 degrees C at pH 7.5. It was found that the activation energy of this enzyme was 8.03 kcal (ca. 33.6 kJ) per mol, and this amidohydrolase showed first-order decay at 36 degrees C. The penicillin amidohydrolase was deactivated rapidly at temperatures above 50 degrees C during storage or preincubation for 24 h. The Michaelis constant, Km, for penicillin G was determined as 2.26 mM, and the substrate inhibition constant, Kis, was 155 mM. The whole-cell penicillin amidohydrolase from M. luteus was capable of hydrolyzing penicillin G, penicillin V, ampicillin, and cephalexin, but not cephalosporin C and cloxacillin. This whole-cell enzyme also had synthetic activity for semisynthetic penicillins or cephalosporins from D-(--)-alpha-phenylglycine methyl ester and 6-alpha-aminopenicillanic acid or 7-amino-3-deacetoxycephalosporanic acid.     

Biochemical properties of penicillin amidohydrolase from Micrococcus luteus.

Some biochemical properties of whole-cell penicillin amidohydrolase from Micrococcus luteus have been studied. This whole-cell enzyme showed its maximal activity at 36 degrees C at pH 7.5. It was found that the activation energy of this enzyme was 8.03 kcal (ca. 33.6 kJ) per mol, and this amidohydrolase showed first-order decay at 36 degrees C. The penicillin amidohydrolase was deactivated rapidly at temperatures above 50 degrees C during storage or preincubation for 24 h. The Michaelis constant, Km, for penicillin G was determined as 2.26 mM, and the substrate inhibition constant, Kis, was 155 mM. The whole-cell penicillin amidohydrolase from M. luteus was capable of hydrolyzing penicillin G, penicillin V, ampicillin, and cephalexin, but not cephalosporin C and cloxacillin. This whole-cell enzyme also had synthetic activity for semisynthetic penicillins or cephalosporins from D-(--)-alpha-phenylglycine methyl ester and 6-alpha-aminopenicillanic acid or 7-amino-3-deacetoxycephalosporanic acid.     

On resuscitation from the dormant state of Micrococcus luteus

It has been found previously that a significant number of Micrococcus luteus cells starved in a prolonged stationary phase (up to 2 months) and then held on the bench at room temperature without agitation for periods of up to a further 2–7 months can be resuscitated in liquid media which contained (statistically) no initially-viable (colony-forming) cells but which were fortified with sterile supernatant from the late logarithmic phase of batch growth. Here it was found that such resuscitation can be done only within a defined time period after taking the first sample from such cultures, necessarily involving agitation of the cells. The duration of this period depends on the age of the starved culture: cells kept on the bench for 3 months possess a 2 month period of resuscitability while cells starved for 6 months can be resuscitated only within 10 days after the beginning of sampling. It is suggested that the input of oxygen to the starved cultures while they are agitated may exert a negative influence on the cells, since cultures stored in anaerobic conditions (under nitrogen) had a more prolonged survival' time. The cells which experienced between 10 and 60 days of starvation on the bench could be resuscitated, although the number of resuscitable cells depended strongly on the concentration of yeast extract in the resuscitation medium. This concentration for cells stored on the bench for more than 2 months was 0.05% while 1-month-old cells displayed a maximum resuscitability in the presence of 0.01% of yeast extract. Application of the fluorescent probe propidium iodide revealed the formation of cells with a damaged permeability barrier if resuscitation was performed by using concentrations of yeast extract of 0.1% and above. Thus the successful resuscitation of bacterial cultures under laboratory conditions may need rather strictly defined parameters if it is to be successfully performed for the majority of cells in a population.