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PubMed1989

Transverse and lateral distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus.

The photodimerization of anthracene was used to investigate the transverse and lateral distribution of lipids in the membrane of the Gram-positive bacterium Micrococcus luteus. 9-(2-Anthryl)nonanoic acid (9-AN) is incorporated at a high rate into various membrane lipids of M. luteus. On irradiation of intact bacteria at 360 nm, anthracene-labeled lipids form stable photodimers which can be extracted and separated by thin-layer chromatography. We present here the results of a study on the distribution of two major lipids, phosphatidylglycerol (PG) and dimannosyldiacylglycerol (DMDG), within each leaflet of the membrane lipid bilayer. After metabolic incorporation of a tritiated derivative of 9-AN in M. luteus, the radioactivity associated with the photodimers issued from PG and DMDG was counted. In the bacterial membrane, the ratio of PG-DMDG heterodimer with respect to PG-PG and DMDG-DMDG homodimers is around half of what should be obtained for a homogeneous mixture of the two lipids. In order to find out whether this was due to an asymmetric distribution of the two lipids between the two membrane leaflets or a heterogeneous distribution of the two lipids within the same membrane leaflet, the transverse distribution of PG and DMDG was also investigated. This was carried out by following the kinetics of oxidation of the two lipids by periodic acid in the membrane of M. luteus protoplasts. PG predominated slightly in the outer layer (60%), while DMDG was found to be symmetrically distributed between the two leaflets. By itself, this lipid asymmetry cannot account for the lipid distribution determined from the photodimerization experiments. This indicates that PG and DMDG are not homogeneously distributed in the plane of the bacterial membrane.

Anthracenes↗
PubMed1980

Comparison of the cleavage of pyrimidine dimers by the bacteriophage T4 and Micrococcus luteus UV-specific endonucleases.

A comparison was made of the activity of the UV-specific endonucleases of bacteriophage T4 (T4 endonuclease V) and of Micrococcus luteus on ultravilet light-irradiated DNA substrates of defined sequence. The two enzymes cleave DNA at the site of pyrimidine dimers with the same frequency. The products of the cleavage reaction are the same, suggesting that the scission of DNA by T4 endonuclease V occurs via the combined actin of a pyrimidine dimer specific DNA glycosylase and an apyrimidinic-apurinic (AP) endonuclease as was recently shown for the M. luteus enzyme. The pyrimidine dimer DNA-glycosylase activity of both enzymes is more active on double-stranded DNA than it is on single-stranded DNA.

Base Sequence↗
PubMed1977

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.

DNA Topoisomerases, Type I↗
PubMed1982

Characterization of polynucleotide phosphorylase from Micrococcus luteus and isolation of the 13,000 base poly(A) product of the polymerization reaction.

A new purification procedure for polynucleotide phosphorylase from freeze-dried Micrococcus luteus cells gives approximately 20% yield of nearly homogeneous, primer-independent enzyme which is free of nucleic acid. The physicochemical properties of M. luteus polynucleotide phosphorylase are similar to those previously described for the enzyme from Escherichia coli in terms of Mr, subunit structure, and amino acid composition. The purified enzyme appears to be a trimer composed of three identical subunits (Mr 92,000), but it probably does not exist as such in the cell. Ferguson plot analyses of enzyme in cell extracts indicate that prior to purification the enzyme exists in oligomeric forms characterized by both higher charge and greater Mr. Changes in size and charge of oligomers which occur during purification are probably due to the dissociation of proteins and/or nucleic acids. Dissociation of the oligomers is achieved by dilution and electrophoresis, but reassociation does not occur after concentration. The poly(A) product of the initial polymerization stages migrates as a single band on both nondenaturing and urea-agarose gels. It is 13,000 +/- 2,000 nucleotides long, as measured by electron microscopy, and 8,000 nucleotides long by gel electrophoretic analysis. This poly(A) product remains bound to the enzyme after synthesis, yet can be easily obtained free of protein by proteinase K digestion.

Amino Acid Sequence↗
PubMed1985

Sequence specificity of DNA cleavage by Micrococcus luteus gamma endonuclease.

DNA fragments of defined sequence have been used to determine the sites of cleavage by gamma-endonuclease activity in extracts prepared from Micrococcus luteus. End-labeled DNA restriction fragments of pBR322 DNA that had been irradiated under nitrogen in the presence of potassium iodide or t-butanol were treated with M. luteus gamma endonuclease and analyzed on high resolution, denaturing, polyacrylamide gels. Gamma endonuclease was found to cleave irradiated DNA preferentially at the positions of cytosines and thymines. DNA cleavage occurred immediately to the 3' side of pyrimidines in irradiated DNA and resulted in fragments that terminate in a 5'-phosphoryl group. These studies indicate that both altered cytosines and thymines may be important DNA lesions requiring repair after exposure to gamma radiation.

Base Sequence↗
PubMed1979

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.

Coliphages↗
PubMed1977

Effects of polyamines on the degradation of ribonucleic acids by polynucleotide phosphorylase of Micrococcus luteus.

The effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(C), and poly(U)] by polynucleotide phosphorylase [polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8] from Micrococcus luteus have been studied. Although the breakdown of all the synthetic polynucleotides tested was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C) greater than poly(A) greater than poly(U) at pH 7.5. However, the difference in degree of stimulation among polynucleotides decreased as the pH or monovalent cation concentration was increased. In the presence of heparin, an inhibitor of polynucleotide phosphorylase hydrolysis of polynucleotides, spermidine clearly stimulated the breakdown of poly(C) and poly(A), while the breakdown of poly(U) was stimulated only slightly by the addition of spermidine. Although binding of [14C]spermine to polynucleotide phosphorylase was observed by gel filtration, the amount of spermine bound to the enzyme was much less than that to RNA.

Binding Sites↗
PubMed1982

On the mechanism of de novo polymerization by form I polynucleotide phosphorylase of Micrococcus luteus.

The diastereomers of adenosine 5'-O-(1-thiodiphosphate) (ADP alpha S) have been tested as substrates for the polymerization reaction of primer-independent polynucleotide phosphorylase from Micrococcus luteus. The preferred substrate is ADP alpha S(Sp), which has a similar Km and a greatly reduced Vmax when compared to the natural substrate ADP. The other diastereomer, ADP alpha S(Rp), is preferentially cleaved by a polyphosphate kinase activity (present with the phosphorylase) that may be responsible for the removal of the 5'-beta-phosphate during de novo polymerization, leading to the observed 5'-phospho-poly(A). Inhibitor studies suggest that the kinase and de novo polymerization sites are not coincident. During de novo polymerization of the diastereomeric mixture, ADP alpha S(Rp) is selectively used to form 5' termini, whereas ADP alpha S(Sp) serves to support the chain elongation. Thus there are two stereochemically distinct subsites for initiating polymerization. ADP beta S functions as a substrate for polynucleotide phosphorylase with kinetic properties similar to those of ADP, indicating that removal of the beta-phosphate (a thiophosphate) is not a kinetically important step and probably occurs after polymerization is complete. The average chain length of the polymeric product is considerably smaller for ADP alpha S vs. ADP beta S or ADP, suggesting that the degree of processivity of the polymerization is determined by competition between the rate of polymerization and the rate of dissociation of the growing chain.

Adenosine Diphosphate↗
PubMed1986

Deacylation of penicilloylated penicillin-binding proteins from Micrococcus luteus: kinetic study of thiol-promoted release of the bound penicilloyl moiety.

The stability of covalent complexes obtained by labelling penicillin-binding proteins 1-6 from Micrococcus luteus with a radioactive derivative of ampicillin has been examined in the presence of thiols. When the incubation medium contained only 1 mM 2-mercaptoethanol, the complexes were almost unaffected for at least 1 h. If 20 mM dithiothreitol was also included in the medium, the amount of bound radioactivity decreased throughout the incubation period. The breakdown of the complexes derived from penicillin-binding proteins 4 and 5 proceeded very slowly, following an apparent first-order kinetics, whereas the kinetics of deacylation of other penicillin-binding proteins exhibited a biphasic pattern with an initial fast phase followed by a slow one, each of which could be approximated by an apparent first-order reaction. This behavior is explained adequately by a two-step mechanism: the penicilloylated penicillin-binding proteins are first deacylated in a reversible exchange with the added thiol, giving rise to an intermediate thioester; once formed, this intermediate is hydrolysed irreversibly. A simple graphical method has been devised to deduce rate constants from the time course of the reaction. Theoretical curves have been constructed, and they fit experimental data satisfactorily. The results point out that added thiols may effectively interfere with the quantitation of penicillin-binding proteins; therefore, the stability of penicilloylated penicillin-binding proteins should be checked carefully when these protecting agents are included in membrane extracts or incubation media.

Ampicillin↗
PubMed1981

Early steps of excision repair of cyclobutane pyrimidine dimers by the Micrococcus luteus endonuclease. A three-step incision model.

The early steps of excision repair of cyclobutane pyrimidine dimers are investigated. It is demonstrated that the apurinic/apyrimidinic endonuclease associated with the Micrococcus luteus uv-specific endonuclease cleaves the phosphodiester bond on the 3' side of the deoxyribose leaving a 3' hydroxy terminus and a 5' phosphoryl terminus. This nick is not a substrate for T4 polynucleotide ligase. The 3' base-free deoxyribose terminus is not a substrate for either the polymerase or the 3' to 5' exonuclease activities of Escherichia coli DNA polymerase I. However, the 3' terminus of the nick is converted to a substrate for DNA polymerization by the action of a 5' apurinic/apyrimidinic endonuclease. A three-step model for the incision step of excision repair of cyclobutane pyrimidine dimers is presented.

Alkaline Phosphatase↗
PubMed1985

Biosynthesis of UDP-N-acetyl-D-glucosaminuronic acid and UDP-N-acetyl-D-mannosaminuronic acid in Micrococcus luteus.

The occurrence and formation of UDP-N-acetyl-D-glucosaminuronic acid (UDP-GlcNAcA) and UDP-N-acetyl-D-mannosaminuronic acid (UDP-ManNAcA) were studied in Micrococcus luteus ATCC 4698. UDP-N-acetylhexosaminuronic acid separated from D-cycloserine-inhibited cells was shown to be a mixture of UDP-GlcNAcA and UDP-ManNAcA in the ratio of 87:13, whereas that obtained from untreated cells was a 96:4 mixture of these two nucleotides. Crude enzyme preparations obtained from the supernatant fraction of cells catalyzed the NAD+-dependent conversion of UDP-GlcNAc into UDP-GlcNAcA and UDP-ManNAcA. Studies on the partial separation and properties of enzymes revealed that UDP-GlcNAcA is synthesized directly from UDP-GlcNAc by the action of UDP-GlcNAc dehydrogenase and that UDP-ManNAcA is synthesized from UDP-GlcNAc through the successive actions of UDP-GlcNAc 2-epimerase and UDP-ManNAc dehydrogenase. However, enzymatic conversion of UDP-GlcNAcA to UDP-ManNAcA was not detected. Ammonium sulfate protects both dehydrogenases from inactivation during storage and incubation. Partially purified UDP-GlcNAc dehydrogenase required dithiothreitol and the particulate fraction for its full activity. The apparent Km values of UDP-GlcNAc dehydrogenase for UDP-GlcNAc and NAD+ were 0.28 and 1.43 mM, respectively. The optimum pH of this enzyme was higher than 9 in Tris-HCl buffer. p-Chloromercuribenzoate at 27 microM as well as 10 mM ethanol almost completely inhibited the UDP-GlcNAc dehydrogenase reaction.

Carbohydrate Dehydrogenases↗
PubMed1978

Purification and characterization of an endonuclease from Micrococcus luteus that acts on depurinated and carcinogen-modified DNA.

An endonuclease which is active with regard to depurinated, alkylated, arylated, and arylamidated DNA has been purified 500-fold from Micrococcus luteus. In this purification, separation from the pyrimidine-dimer-specific ultraviolet-endonuclease has been achieved. The enzyme has a molecular weight of 30000 on the basis of gel filtration; its activity is not absolutely dependent upon the presence of Mg2+, but 5--30 mM Mg2+ produces a five-fold stimulation. Potassium chloride concentrations of less than 100 mM are optimal, while concentrations exceeding 100 mM inhibit. The enzyme has no effect on native DNA, but introduces single-strand breaks into DNA containing apurinic/apyrimidinic sites produced by heating at an acidic pH. DNA treated with such carcinogens as N-alkyl-N-nitrosoureas, alkyl methanesulfonates, alkyl sulfates, nitrogen mustard, beta-propiolactone, 7-bromomethyl-benz[a]anthracene, N-acetoxy-2-acetylaminofluorene, and 7,12-dimethyl-benz[a]anthracene-5,6-oxide also becomes susceptible to enzymic action. The activity of the enzyme has been detected by making use of the difference in mobility between supercoiled closed-circular DNA of Pseudomonas phage PM2 and its nicked form in agarose gel elctrophoresis. Even depurinated or carcinogen-modified supercoiled PM2 DNA migrated faster than the respective relaxed nicked forms. A comparison of the number of enzyme-catalyzed single-strand breaks with the number of alkali-labile (i.e. apurinic) sites in carcinogen-modified PM2 DNA showed that the enzyme preparation introduced approximately twice as many breaks into the substrates as the number of apurinic sites present. We conclude that the enzyme preparation either recognizes both apurinic sites and DNA bases carrying carcinogenic residues or contains DNA glycosidase activity in addition to the endonuclease activity. Exposure of ultraviolet-irradiated PM2 DNA to the endonuclease preparation showed that pyrimidine dimers were not substrates. The yield of enzyme-catalyzed single-strand breaks found in ultraviolet-irradiated DNA was five times the number of alkali-labile sites present suggesting that minor photoproducts, possibly 5,6-saturated pyrimidine residues, were recognized in addition to apurinic sites.

Carcinogens↗
PubMed1981

Poly(adenosine diphosphoribose) synthesis in ultraviolet-irradiated xeroderma pigmentosum cells reconstituted with Micrococcus luteus UV endonuclease.

Synthesis of DNA and poly(adenosine diphosphoribose) [poly(ADPR)] was examined in permeabilized xeroderma pigmentosum lymphoblasts (XP3BE) before and after UV irradiation and in the presence and absence of Micrococcus luteus UV endonuclease. M. luteus UV endonuclease had no effect on the level of DNA or poly(ADPR) synthesis in control, unirradiated cells. UV irradiation caused a decrease in replicative DNA synthesis without any significant change in poly(ADPR) synthesis. In UV-irradiated cells treated with M. luteus UV endonuclease, DNA synthesis was restored to a level slightly greater than in the unirradiated control cells, and poly(ADPR) synthesis increased by 2- to 4-fold. Time--course studies showed that the UV endonuclease dependent poly(ADPR) synthesis preceded the endonuclease-dependent DNA synthesis. Inhibition of endonuclease-dependent poly(ADPR) synthesis with 3-aminobenzamide, 5-methylnicotinamide, or theophylline produced a partial inhibition of the endonuclease-dependent DNA synthesis. Conversely, inhibition of the endonuclease-dependent DNA synthesis with dideoxythymidine triphosphate, phosphonoacetic acid, or aphidicolin had no effect on the endonuclease-dependent poly(ADPR) synthesis. These studies show that stimulation of poly(ADPR) synthesis in UV-irradiated cells occurs subsequent to the DNA strand breaks created by the specific action of the UV endonuclease on UV-irradiated DNA. The effect of the inhibitors of poly(ADPR) synthesis in UV-irradiated cells indicates that the endonuclease-stimulated DNA synthesis is dependent in part on the prior synthesis of poly(ADPR).

Cell Line↗
PubMed1983

Characterization of succinate dehydrogenase from Micrococcus luteus (lysodeikticus) by electron-spin-resonance spectroscopy.

Low-temperature electron spin resonance spectroscopy has been used to study the biophysical properties of succinate dehydrogenase from the gram-positive bacterium Micrococcus luteus. The paramagnetic redox centres of the enzyme were identified in a succinate-dehydrogenase--antigen complex, which had been purified with the aid of monospecific serum from membranes solubilized with Triton X-100. The centres were characterized in further detail using the membrane-bound and Triton-solubilized forms of the enzyme. These studies distinguished two types of iron-sulphur centres, viz. a [4Fe-4S]3+ cluster displaying a narrow signal at g = 2.01 in the oxidized state (conventionally termed centre S-3) and a [2Fe-2S )0 cluster with an axial signal at g = 2.03 and 1.93 in the reduced state (conventionally termed centre S-1). Centre S-3 had a mid-point redox potential of +10 mV, a comparatively low value for this type of cluster. The behaviour of the g = 1.93 signal of centre S-1 was a complex function of the redox potential, microwave power and temperature of measurement. When measured at low power (i.e. non-saturating conditions), the intensities observed for the g = 1.93 signal poised at various critical potentials in the redox titration were similar. However, the corresponding intensities differed markedly at high power, where conditions were saturating. It is proposed that under saturating conditions the spin-lattice relaxation of the [2Fe-2S] cluster S-1 (mid-point potential +70 mV) is enhanced by centre S-3 between the potential range +10-+70 mV and by an ESR-silent centre, termed centre S-2, with a mid-point potential of -295 mV.

Chemical Precipitation↗
PubMed1980

Synthesis of DNA by Micrococcus luteus is resistant to 6-(p-hydroxyphenylazo)uracil.

The DNA synthesis inhibitor 6-(p-hydroxyphenylazo)uracil (HPUra), at concentrations ranging from 0.2 to 2000 micro M, had essentially no effect on DNA synthesis in Micrococcus luteus strain ML-1. Seven other M. luteus strains were unaffected by 200 micro M HPUra. In vitro DNA synthesis in toluene-treated M. luteus strain ML-1 ws resistant to both HPUra and reduced HPUra.

Adenosine Triphosphate↗
PubMed1984

Staphylococcus aureus and Micrococcus luteus peptidoglycan transglycosylases that are not penicillin-binding proteins.

Major peptidoglycan transglycosylase activities, which synthesize uncross-linked peptidoglycan from lipid-linked precursors, were solubilized from the membranes of Staphylococcus aureus and Micrococcus luteus and were partially purified. The transglycosylase activities were separated from penicillin-binding proteins by solubilization and by purification steps. Therefore, we concluded that these activities were not activities of the penicillin-binding proteins, which are the presumptive peptidoglycan transpeptidases in these gram-positive cocci. Unlike Escherichia coli, in which the network structure of peptidoglycan is synthesized by multiple two-headed penicillin-binding proteins with both transpeptidase and transglycosylase activities, these gram-positive cocci have cell wall peptidoglycan which seems to be synthesized by penicillin-binding protein transpeptidases and a separate transglycosylase.

Bacterial Proteins↗
PubMed1979

Preparation and antibacterial activity upon Micrococcus luteus of derivatives of iturin A, mycosubtilin and bacillomycin L, antibiotics from Bacillus subtilis.

Methylated and acetylated derivatives of iturin A and mycosubtilin and methylated derivatives of bacillomycin L were prepared and their antibacterial activity on Micrococcus luteus was compared with the activity of the original substance. the results obtained show the importance of polar groups for the antibiotic activity of the substances of iturin group.

Anti-Bacterial Agents↗
PubMed1978

Formation of spirodilactone of 4-(2'-carboxyphenyl)-4,4-dihydroxybutyrate from 2-succinylbenzoate in cell-free extracts of Micrococcus luteus.

The enzyme mediated ATP- and, to a lesser extent, CoASH-dependent synthesis of the spirodilactone of 4-(2'-carboxyphenyl)-4,4-dihydroxybutyrate from 2-succinylbenzoate has been demonstrated in membrane-free extracts of Micrococcus luteus and Escherichia coli. The suggestion is made that the spirodilactone is the product of an aberrant reaction involving a compound that is normally an intermediate in the conversion of 2-succinylbenzoate to 1,4-dihydroxy-2-naphthoate.

Adenosine Triphosphate↗