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PubMed1989

Bacteriophage-T4 and Micrococcus luteus UV endonucleases are not endonucleases but beta-elimination and sometimes beta delta-elimination catalysts.

Bacteriophage-T4 UV endonuclease nicks the C(3')-O-P bond 3' to AP (apurinic or apyrimidinic) sites by a beta-elimination reaction. The breakage of this bond is sometimes followed by the nicking of the C(5')-O-P bond 5' to the AP site, leaving a 3'-phosphate end; delta-elimination is proposed as a mechanism to explain this second reaction. The AP site formed when this enzyme acts on a pyrimidine dimer in a polynucleotide chain undergoes the same nicking reactions. Micrococcus luteus UV endonuclease also nicks the C(3')-O-P bond 3' to AP sites by a beta-elimination reaction. No subsequent delta-elimination was observed, but this might be due to the presence of 2-mercaptoethanol in the enzyme preparation.

Catalysis↗
PubMed1990

The distribution of electron flow in the branched respiratory chain of Micrococcus luteus.

Endogenous coupled respiration of Micrococcus luteus protoplasts showed a relatively high resistance to low concentrations of KCN, 2-nonyl-4-hydroxyquinoline N-oxide (NQNO) and dicyclohexylcarbodi-imide (DCCD) when the inhibitors were applied individually. In the presence of both KCN and NQNO (or DCCD), O2 uptake was strongly inhibited. The proteolysis of external membrane proteins of protoplasts also induced the high sensitivity of endogenous coupled respiration to low KCN. The effects of NQNO, DCCD and proteolysis were explained by the inhibition of an alternative respiratory system when reducing equivalents passed preferentially down the KCN-sensitive cytochrome oxidase. Uncoupling of the cell membrane increased the electron flow via the cytochrome oxidase-containing respiratory branch. It is suggested that the energy state of cells could control the electron-flow distribution between two branches, and quinones of different levels of reduction could be involved in the mechanism of respiratory branching.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed2002

The rpf gene of Micrococcus luteus encodes an essential secreted growth factor.

Micrococcus luteus secretes a small protein called Rpf, which has autocrine and paracrine signalling functions and is required for the resuscitation of dormant cells. Originally isolated from the supernatant of actively growing cultures, Rpf was also detected on the surface of actively growing bacteria. Most molecules may be sequestered non-productively at the cell surface, as a truncated form of the protein, encompassing only the 'Rpf domain' is fully active. The C-terminal LysM module, which probably mediates binding to the cell envelope, is not required for biological activity. Rpf was essential for growth of M. luteus. Washed cells, inoculated at low density into a minimal medium, could not grow in its absence. Moreover, the incorporation of anti-Rpf antibodies into the culture medium at the time of inoculation also prevented bacterial growth. We were unable to inactivate rpf using a disrupted form of the gene, in which most of the coding sequence was replaced with a selectable thiostrepton resistance marker. Gene disruption was possible in the presence of a second, functional, plasmid-located copy of rpf, but not in the presence of a rpf derivative whose protein product lacked the secretory signal sequence. As far as we are aware, Rpf is the first example of a truly secreted protein that is essential for bacterial growth. If the Rpf-like proteins elaborated by Mycobacterium tuberculosis and other mycobacteria prove similarly essential, interference with their proper functioning may offer novel opportunities for protecting against, and treating, tuberculosis and other mycobacterial disease.

Bacterial Proteins↗
PubMed2003

Structural analysis and genetic variation of the 16S-23S rDNA internal spacer region from Micrococcus luteus strains.

AIMS: To clone and sequence the 16S-23S ribosomal DNA (rDNA) internal spacer region (ISR) from Micrococcus luteus. METHODS AND RESULTS: The primer pair for 16S-23S rDNA ISR amplified a fragment of about 850 bp in length for two strains, JCM3347 and JCM3348 and a fragment of about 790 bp for a strain, ATCC9341. After sequencing the ISRs were identified by the comparison of the ISRs and the flanking regions of ISR. CONCLUSIONS: Although the sequence difference of the ISR occurred at only one position between the two JCM strains, the highly variable length (440 and 370 bp) and sequence similarity (about 40%) were demonstrated between the ISRs of the two JCM strains and a ATCC strain. SIGNIFICANCE AND IMPACT OF THE STUDY: A CCTCCT sequence was first detected at the 3'-end of the 16S rDNA of the three strains. Moreover, highly similar sequence to the 21-bp region containing a putative rRNA processing site was observed in the ISR of the three strains. Interestingly, no intercistronic tRNAs were demonstrated in the ISRs from the three strains.

Base Sequence↗
PubMed1970

Dinucleotides as products of an exonucleolytic activity association with the Micrococcus luteus DNA polymerase.

The partially purified DNA polymerase from Micrococcus luteus contains a low level of exonucleolytic activity. The enzyme preparation (1200-fold purified) contains approximately 100 times more polymerase than exonuclease activity. Both single- and double-stranded DNA are degraded at the same rate. The predominant products are dinucleoside diphosphates (d-pXpY); mononucleotides and a trace of trinucleotides are also produced. Each of these products is formed at a constant rate throughout the course of the reaction. The nuclease degrades a DNA chain from the 5'-end. The enzyme preparation contains no detectable endonuclease activity.

Carbon Isotopes↗
PubMed1972

Endonucleolytic activity from Micrococcus luteus that acts on -ray-induced damage in plasmid DNA of Escherichia coli minicells.

A partially purified extract from Micrococcus luteus contains endonucleolytic activity toward ultraviolet (UV)-irradiated deoxyribonucleic acid (DNA). We found that the same extract also acts on superhelical, covalently-closed circular lambdadv DNA isolated from gamma-irradiated minicells of E. coli. The introduction of nicks in isolated covalently-closed circular DNA by an endonuclease in the extract results in relaxed circles, and these two circular DNA species are easily distinguishable by their sedimentation properties in alkaline sucrose. The frequency with which the endonuclease-susceptible lesions are produced in superhelical DNA is only marginally enhanced when (60)Co gamma-rays are administered to an aerobic rather than an anoxic minicell suspension. The ratio of endonuclease-sensitive defects to single-strand scissions, induced by gamma-irradiation in air, is about 1:3. The nuclease-sensitive lesions disappear from gamma-irradiated minicells during incubation after radiation presumably as a consequence of excision repair. Since the addition of UV-irradiated calf-thymus DNA depresses the ability of the M. luteus extract to attack not only UV-damaged E. coli DNA (a known substrate for the so-called UV-specific endonuclease that catalyzes the initial single-strand incision adjacent to the structural defect) but, also lambdadv DNA injured by gamma-rays, we conclude that physicochemical alterations induced by both types of radiation are recognized by one and the same endonuclease.

Animals↗
PubMed1972

Role of the penicillin-sensitive transpeptidation reaction in attachment of newly synthesized peptidoglycan to cell walls of Micrococcus luteus.

Cell-wall preparations of Micrococcus luteus (lysodeikticus) catalyze in vitro peptidoglycan synthesis from UDP N-acetyl-D-glucosamine, UDP N-acetylmuramic acid-pentapeptide, and glycine. Newly synthesized peptidoglycan is partially cross-linked by a transpeptidation reaction with concomitant release of C-terminal D-alanine. Penicillin not only strongly inhibits release of D-alanine (98% at 1 mug/ml), but also markedly inhibits incorporation of acetylglucosamine and N-acetylmuramic acid-pentapeptide into the preformed cell-wall peptidoglycan. The simplest explanation for the results is that incorporation of newly synthesized strands of peptidoglycan and their attachment to "older" cell-wall peptidoglycan proceeds mainly by transpeptidation and that transglycosylation is responsible only for part of the elongation of the pre-existing peptidoglycan. Another possibility is that incorporation occurs by transglycosylation, but it cannot continue without concurrent formation of peptide cross-bridges.

Amino Acids↗
PubMed1978

Topographic study of the cell surface of micrococcus radiodurans.

The paracrystalline outer membraneous layer (HPI layer) of Micrococcus radiodurans has been investigated by negative- and positive-staining electron microscopy and subsequent digital image processing. The subunit structure of the major HPI layer protein complex and the lipid-protein distribution in the plane of the membrane have been determined. The HPI layer was found to be highly asymmetric in a transmembrane direction, with the major protein complex only partly penetrating into a lipid-containing backing layer intimately associated with it.

Bacterial Proteins↗
PubMed1978

Micrococcus luteus DNA gyrase: active components and a model for its supercoiling of DNA.

Two active components alpha and beta of micrococcus luteus DNA gyrase, of peptide weights of 115,000 and 97,000, respectively, have been purified. Each individual component exhibits little DNA gyrase activity; the ATP-dependent negative supercoiling of a covalently closed circular DNA duplex is catalyzed by a combination of the two. Covalent closure by Escherichia coli ligase of a circular DNA containing single-chain scissions, when carried out in the presence of a combination of the DNA gyrase components alpha and beta, gives a positively supercoiled DNA upon removal of the bound protein molecules. ATP was not present during the ligase treatment; therefore the positive supercoiling of DNA observed is a result of the binding of gyrase molecules, presumably as multi-subunit oligomers, during the ligation step. This is in contrast to the negative supercoiling of DNA catalyzed by gyrase in the presence of ATP. A model in which negative supercoiling of DNA is achieved by ATP-modulated repetitive wrapping of the DNA around gyrase is described. The model also suggests a plausible mode of action by which translocation of a DNA along its helix axis can be actively driven by an ATPase.

Adenosine Triphosphate↗
PubMed1981

Release of 7-methylguanine residues from alkylated DNA by extracts of Micrococcus luteus and Escherichia coli.

Cell extracts from Micrococcus luteus release both free 3-methyladenine and free 7-methylguanine from alkylated DNA. The glycosylase activity responsible for the liberation of 7-methylguanine is not 3-methyladenine-DNA glycosylase, which, when purified, does not liberate it. Furthermore, the heat inactivation rates of the two enzymatic activities are different. The release of 7-methylguanine by chemical depurination of ethanol-soluble oligonucleotides has been ruled out. A similar activity releasing 7-methylguanine is also found in Escherichia coli.

Animals↗
PubMed1983

Inhibition of Micrococcus luteus DNA topoisomerase I by UV photoproducts.

The activity of Micrococcus luteus DNA topoisomerase I on UV-irradiated supercoiled DNA was studied under either processive or distributive reaction conditions. Changes in DNA structure caused by UV irradiation reduce the rate of DNA relaxation at very low concentration of photoproducts. Under processive conditions the inhibition of the topoisomerase I by photoproducts can be quantitated by measuring the amount of substrate left in the replicative form I band. The mode of action of DNA topoisomerase I was affected by the presence of photoproducts in the DNA substrate, although the ability of the enzyme to form a covalent complex with UV-irradiated supercoiled DNA was not changed. The inhibition of topoisomerase I by UV photoproducts has been compared to the effects of single-stranded DNA and UV-irradiated duplex linear DNA on the enzyme, and the results suggest that the inhibition by photoproducts is caused by changes in the conformation of the supercoil. Our findings indicate the possibility that DNA topoisomerase I plays a role in repair.

DNA, Superhelical↗
PubMed1997

UV endonuclease of Micrococcus luteus, a cyclobutane pyrimidine dimer-DNA glycosylase/abasic lyase: cloning and characterization of the gene.

The gene of Micrococcus luteus UV endonuclease (cyclobutane pyrimidine dimer-DNA glycosylase/ abasic lyase) was cloned and characterized. The cloned gene, whose product had a predicted molecular mass of 17,120 Da, was found to be capable of complementing the Escherichia coli uvrA6 mutation in vivo with respect to resistance to acetonemediated molecular photosensitization, a treatment producing exclusively cyclobutane pyrimidine dimers in DNA. It also generated a nicking activity specific for photosensitization-treated DNA by in vitro transcription/translation. When expressed in E. coli cells, the gene produced a protein structurally identical with UV endonuclease and possessing an activity consistent with cyclobutane pyrimidine dimer-DNA glycosylase/abasic lyase with respect to the effect of inhibitors and the site of the DNA backbone scission. Furthermore, the UV endonuclease-deficient mutant DB7 was shown to regain the enzyme through transformation with the cloned gene. The deduced amino acid sequence of the gene product was at best 27% identical with that of endonuclease V of phage T4, an enzyme strikingly similar to UV endonuclease in molecular and catalytic properties. Despite this marginal overall similarity in amino acid sequence, four of the seven amino acid residues reported to be functionally important in the T4 enzyme were found to be conserved in the M. luteus enzyme. We propose that the gene be called uveA.

Adenosine Triphosphatases↗
PubMed1995

Purification and cloning of Micrococcus luteus ultraviolet endonuclease, an N-glycosylase/abasic lyase that proceeds via an imino enzyme-DNA intermediate.

Although Micrococcus luteus UV endonuclease has been reported to be an 18-kDa enzyme with possible homology to the 16-kDa endonuclease V from bacteriophage T4 (Gordon, L. K., and Haseltine, W. A. (1980) J. Biol. Chem. 255, 12047-12050; Grafstrom, R. H., Park, L., and Grossman, L. (1982) J. Biol. Chem. 257, 13465-13474), this study describes three independent purification schemes in which M. luteus UV damage-specific or pyrimidine dimer-specific nicking activity was associated with two proteins of apparent molecular masses of 31 and 32 kDa. An 18-kDa contaminant copurified with the doublet through many of the chromatographic steps, but it was determined to be a homolog of Escherichia coli ribosomal protein L6. Edman degradation analyses of the active proteins yielded identical NH2-terminal amino acid sequences. The corresponding gene (pdg, pyrimidine dimer glycosylase) was cloned. The protein bears strong sequence similarities to the E. coli repair proteins endonuclease III and MutY. Nonetheless, traditionally purified M. luteus protein acted exclusively on cis-syn thymine dimers; it was unable to cleave site-specific oligonucleotide substrates containing a trans-syn -I, (6-4), or Dewar thymine dimer, a 5,6-dihydrouracil lesion, or an A:G or A:C mismatch. The UV endonuclease incised cis-syn dimer-containing DNA in a dose-dependent manner and exhibited linear kinetics within that dose range. Enzyme activity was inhibited by the presence of NaCN or NaBH4 with NaBH4 additionally being able to trap a covalent enzyme-substrate product. These last findings confirm that the catalytic mechanism of M. luteus UV endonuclease, like those of other glycosylase/AP lyases, involves an imino intermediate.

Amino Acid Sequence↗
PubMed1996

Characterization of an unusual Rho factor from the high G + C gram-positive bacterium Micrococcus luteus.

A transcription termination factor (Rho) was purified from the Gram-positive bacterium Micrococcus luteus, and the complete gene sequence was determined. The M. luteus Rho polypeptide has 690 residues, which is 271 residues more than its homolog from Escherichia coli. Most of the additional residues compose a highly charged, hydrophilic segment that is inserted in a non-conserved region between two conserved regions of the RNA-binding domain of the known Rho homolog proteins. This segment extends from residues 49 to 311 and includes a stretch of 238 residues that contain no hydrophobic side chains. Biochemical studies indicate that the M. luteus protein is very similar to E. coli Rho in terms of its RNA-dependent NTPase activity and its sensitivity to the Rho-specific inhibitor bicyclomycin. However, the M. luteus protein has a less stringent RNA cofactor specificity. It also acts to terminate RNA transcription with E. coli RNA polymerase on the lambda cro DNA template, but at much earlier termination stop points than those recognized by E. coli Rho. Thus, the M. luteus protein functions as a true Rho factor, but with a different specificity than that of E. coli Rho. We propose that this altered specificity is consistent with its need to function on transcripts that have a high content of G + C residues.

Amino Acid Sequence↗
PubMed1997

Biosynthesis of teichuronic acid in the bacterial cell wall. Purification and characterization of the glucosyltransferase of Micrococcus luteus.

This report describes what is, to our knowledge, the first purification to near homogeneity of an enzyme involved in the biosynthesis of the teichuronic acid of Micrococcus luteus cell walls. The glucosyltransferase of M. luteus, which participates in the biosynthesis of teichuronic acid, was solubilized from cytoplasmic membrane fragments by extraction with buffer solutions containing the detergents Thesit (dodecyl alcohol polyoxyethylene ether; 1 mg/ml) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (0.5 mg/ml). The detergent-solubilized enzyme was purified 150-fold, with a recovery of 13% by adsorbent column chromatography, ion-exchange chromatography, gel filtration, and preparative nondenaturing gradient polyacrylamide gel electrophoresis. On the basis of its mobility on native gradient gel, the glucosyltransferase was estimated to have a molecular mass of 440 kDa. The purified native enzyme was a multisubunit protein consisting of subunits of two sizes; their molecular masses were determined to be 52.5 and 54 kDa, respectively, by observation of the mobility of the protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was approximately 5.

Anti-Bacterial Agents↗
PubMed1997

Function of the novel subdomain in the RNA binding domain of transcription termination factor Rho from Micrococcus luteus.

Transcription termination factor Rho from Micrococcus luteus, a high G + C Gram-positive bacterium, contains an unusual extra sequence within its RNA binding domain that is rich in Arg, Glu, and Asp residues and deficient in hydrophobic residues. To determine the role of this extra sequence, we compared the biochemical properties of a variant lacking nearly all the extra sequence, des(60-300) Rho, to that of wild-type M. luteus Rho. The two forms had very similar properties except that the des(60-300) Rho was unable to terminate transcription with Escherichia coli RNA polymerase at the promoter proximal sites used by the wild-type Rho on a lambda cro DNA template but could cause termination at more distal sites and did cause termination at proximal sites when ITP replaced GTP in the reaction mixture. The RNA binding properties of the two forms of this Rho with normal and inosine-substituted RNAs were found to correlate fully with their termination properties. These results indicate that the arginine-rich extra sequence is directly involved in the selection of the termination site and support the hypothesis that the sequence is present in M. luteus Rho to facilitate its binding to M. luteus transcripts, which are likely to have a high degree of base-paired secondary structure because of their high proportion of G residues.

Adenosine Triphosphatases↗
PubMed2001

Identification of Significant residues for homoallylic substrate binding of Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase.

The primary structure of cis-prenyltransferase is totally different from those of trans-prenyltransferases (Shimizu, N., Koyama, T., and Ogura, K. (1998) J. Biol. Chem. 272, 19476-19481). To better understand the molecular mechanism of enzymatic cis-prenyl chain elongation, we selected seven charged residues in the conserved Region V and two of Phe-Ser motif in Region III of undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26 for substitutions by site-directed mutagenesis and examined their effects on substrate binding and catalysis. Kinetic studies indicated that replacements of Arg-197 or Arg-203 with Ser, and Glu-216 with Gln resulted in 7-11-fold increases of Km values for isopentenyl diphosphate and 18-1200-fold decreases of kcat values compared with those of the wild-type enzyme. In addition, two mutants with respect to the Phe-Ser motif in Region III, F73A and S74A, showed 16-32-fold larger Km values for isopentenyl diphosphate and 12-16-fold lower kcat values than those of the wild-type. Furthermore, product analysis indicated that three mutants, F73A, S74A, and E216Q, yielded shorter chain prenyl diphosphates as their main products. These facts together with the protein structural analysis recently carried out (Fujihashi, M., Zhang, Y.-W., Higuchi, Y., Li, X.-Y., Koyama, T., and Miki, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4337-4342) indicated that the diphosphate moiety of homoallylic substrate is electrostatically recognized by the three charged amino acids, Arg-197, Arg-203, and Glu-216, in Region V and the Phe-Ser motif in Region III, also indispensable for homoallylic substrate binding as well as catalytic function. It was suggested that the undecaprenyl diphosphate synthase takes a different mode for the binding of isopentenyl diphosphate from that of trans-prenyl chain elongating enzymes.

Alkyl and Aryl Transferases↗
PubMed1975

Affinity chromatography of succinate dehydrogenase from the membranes of Micrococcus lysodeikticus.

Isolated plasma membranes of Micrococcus lysodeikticus were subjected to extraction with n-butanol in a two-phase system. Succinate dehydrogenase obtained in the soluble aqueous phase after high-speed centrifugation was resolved by separation on calcium phosphate gel and affinity chromatography. The affinity ligand used was oxaloacetate and elution from the column was achieved with 0.5 M succinate. In the final product there was an eleven-fold reduction in the 32P-lipid to protein ratio and a fourteen-fold increase in specific activity relative to the high speed supernatant fraction following n-butanol extraction.

Cell Membrane↗