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Optimization of culture conditions and medium composition for the production of micrococcin GO5 by Micrococcus sp. GO5.

To enhance the production of micrococcin GO5, a bacteriocin produced by Micrococcus sp. GO5, cultivation conditions and medium composition were optimized. The optimal initial pH and temperature for bacteriocin production were 7.0-9.0 and 37 degrees C, respectively. Micrococcus sp. GO5 displayed the highest micrococcin GO5 activity when grown in modified MRS medium that contained lactose or sucrose, rather than glucose, as a carbon source. The maximum bacteriocin activity was obtained in modified MRS medium containing 0.5% tryptone and 1.0% yeast extract as nitrogen sources instead of the other nitrogen sources present in MRS medium. Bacteriocin production was greatly affected by the concentration of K(2)HPO(4); strain GO5 produced eight-fold more bacteriocin in medium containing 2.0-2.5% K(2)HPO(4) than in medium containing 0.2% K(2)HPO(4). The optimal concentration of MgSO(4).7H(2)O for bacteriocin production was 0.5%. The production of micrococcin GO5 was increased 32-fold in shake flask culture and 16-fold in a bioreactor using the optimized medium (TY medium), compared with culturing in MRS medium.

Bacteriocins↗

Molecular cloning, overexpression, and purification of Micrococcus luteus K-3-type glutaminase from Aspergillus oryzae RIB40.

We have for the first time found and cloned the cDNA (AoglsA) of Aspergillus oryzae RIB40, which encodes a 49.9-kDa protein sharing 40% homology with the salt-tolerant glutaminase of Micrococcus luteus K-3 (Micrococcus glutaminase). AoglsA was subcloned into a series of expression vectors and expressed in Saccharomyces cerevisiae and Escherichia coli. The gene product, which we named AoGls, showed glutaminase activity and was produced in a cell wall fraction of S. cerevisiae and a soluble protein in E. coli. The highest expression level of 186 U/mg was obtained when the AoglsA was inserted into six bases downstream of the Shine-Dalgarno (SD) sequence of pKK223-3 and expressed in E. coli Rosetta (DE3). AoGls was purified by SuperQ-TOYOPEARL, glutamine affinity chromatography, and Butyl-TOYOPEARL. This is the first report on the overexpression and purification of a M. luteus K-3-type glutaminase cloned from an eucaryote.

Amino Acid Sequence↗

Micrococcus luteus endocarditis: case report and review of the literature.

This paper includes a report on a case of prosthetic valve endocarditis due to Micrococcus luteus and a review of the sixteen cases of endocarditis due to Micrococcus species reported in the literature. The patient was successfully treated with rifampicin combined for two weeks with gentamicin and vancomycin and for another four weeks with teicoplanin. The hospital course was uneventful and no surgery was required.

Aged↗

Characterization of de-emulsification capabilities of a Micrococcus species.

Effect of post harvest washing as well as cell concentration on de-emulsification characteristics of an isolated Micrococcus species has been tested with Tween 60 Span 60 stabilised oil in water (o/w) and L-92 pluronic surfactant stabilised water in oil (w/o) model emulsions (kerosene water). The cells used were 140 h old and grown under submerged conditions at 37 degrees C in a medium containing n-tetradecane (4% v/v) as the carbon source. The harvested bacterial cells when in an unwashed condition (at a cell concentration of 2 mg/ml of emulsion) were found to de-emulsify the o/w system at a much faster rate than the w/o system exhibiting half-life values for the respective system as 10.2 and 127.7 h. Post harvest washing of the cells with any lipid solubilising solvent (n-pentane, n-hexane, kerosene, chloroform-methanol-water (CMW)) yielded a decrease in their de-emulsification power for w/o emulsion. But the decay of o/w emulsion became faster with n-pentane- and kerosene-washed cells as evident from their corresponding half-life values of 3.3 and 4.6 h. Compared to the w/o system, an increase in the concentration of kerosene-washed cell had a direct effect on de-emulsification for the o/w system. For cell concentrations of 2, 3 and 4 mg/ml of the emulsion, the half-life values for the w/o system were 364.8, 442.0 and 454.9 h, respectively. For 2 and 4 mg/ml cell contents, the half-life values for the o/w system were 4.6 and 1.0 h. The decay of both the emulsions was very slow or even incomplete for cell concentrations less than 2 mg/ml. De-emulsifying capacity of the n-tetradecane grown Micrococcus species towards o/w model emulsion improved considerably after washing the cells with n-pentane and kerosene, and use of kerosene-washed cells (4 mg/ml) reduced the half-life to 1 h.

Biodegradation, Environmental↗

Successful immunotherapy with micrococcus, BCG or related polysaccharides on L1210 leukaemia after BCNU chemotherapy.

The experiments aimed at evaluating the optimal parameters in the chemo-immunotherapeutic treatment of the L1210 lymphoid leukaemia grafted to [female BALB/c (H2d) X male DBA/2 (H2d)]F1 hybrid mice, hereafter referred to as CDF1 mice. In vitro irradiation of leukaemic ascites cells by X- or gamma-rays and subsequent inoculation in mice showed that optimum immunogenicity is radiation dose-dependent. Grafting mice with 10(7) leukaemic ascites cells irradiated at optimum dose (80 GyX- or gamma-rays) delays mortality of the animals when challenged later with untreated L1210 cells, but is unable to cure mice. By contrast, specific immunoprophylaxis induced by Micrococcus, complement-triggering polysaccharides or BCG and irradiated leukaemic cells was able to protect mice against grafts of 10(4) L1210 cells. The i.p. route was notably superior to the i.v. route. When mice bearing advanced L1210 tumour were treated by chemotherapy (12 mg/kg of BCNU) on Day 6.5 after grafting 10(4) L1210 cells and subsequently treated by immunotherapy, a very high percentage (up to 90%) of mice with 10(8) leukaemic cells could be cured by repeated 1mg injections of bacterium or polysaccharide, and challenge with irradiated leukaemic cells was unnecessary. Because of the high cure rate obtained, the very regular response pattern and the non-pathogenicity, the bacterium Micrococcus lysodeikticus would seem a promising new candidate for chemo-immunotherapeutic antitumour strategies.

Animals↗

Preparation and properties of a phosphoenzyme from the phosphoglucomutase of Micrococcus lysodeikticus.

1. Phosphoglucomutase from Micrococcus lysodeikticus was incubated with (14)C- and (32)P-labelled glucose 1,6-diphosphate and separated from the cofactor on a Sephadex column. (32)P-labelled phosphate (0.7mol/mol of enzyme) was associated with the enzyme, but no (14)C label was. 2. The (32)P-labelled enzyme exchanged its label with the substrates. When the labelled enzyme was incubated in Tris buffer, pH8.3, at 30 degrees C the proportion of exchangeable label slowly fell indicating a half-life of the phosphoenzyme of about 50h. 3. When HClO(4) was added to the labelled phosphoenzyme all of the label was precipitated with the protein and none was released as P(i). On alkaline hydrolysis P(i) was released at a rate comparable with the rate of hydrolysis of the phosphoenzyme from rabbit muscle. 4. We conclude that the phosphoenzyme from Micrococcus lysodeikticus yields a relatively stable, catalytically active phosphoenzyme when treated with cofactor, and that there is no evidence for the formation of an enzyme-glucose 1,6-diphosphate complex. The properties of the phosphoenzyme, which resemble those of rabbit muscle phosphoglucomutase, suggest that the phosphate may be bound to serine.

Animals↗

Role of the subunits of the energy-transducing adenosine triphosphatase from Micrococcus lysodeikticus membranes studied by proteolytic digestion and immunological approaches.

An energy-transducing adenosine triphosphatase (ATPase, EC 3.6.1.3) that contains an extra polypeptide (delta) as well as three intrinsic subunits (alpha, beta, gamma) was purified from Micrococcus lysodeikticus membranes. The apparent subunit stoichiometry of this soluble ATPase complex is alpha 3 beta 3 gamma delta. The functional role of the subunits was studied by correlating subunit sensitivity to trypsin and effect of antibodies raised against holo-ATPase and its alpha, beta and gamma subunits with changes in ATPase activity and ATPase rebinding to membranes. A form of the ATPase with the subunit proportions 1.67(alpha):3.00(beta:0.17(gamma) was isolated after trypsin treatment of purified ATPase. This form has more than twice the specific activity of native enzyme. Other forms with less relative proportion of alpha subunits and absence of gamma subunit are not active. Of the antisera to subunits, only anti-(beta-subunit) serum shows a slight inhibitory effect on ATPase activity, but its combination with either anti-(alpha-subunit) or anti-(gamma-subunit) serum increases the effect. The results suggest that beta subunit is required for full ATPase activity, although a minor proportion of alpha and perhaps gamma subunit(s) is also required, probably to impart an active conformation to the protein. The additional polypeptide not hitherto described in Micrococcus lysodeikticus ATPase had a molecular weight of 20 000 and was found to be involved in ATPase binding to membranes. This 20 000-dalton component can be equated with the delta subunit of other energy-transducing ATPases and its association with the (alpha, beta, gamma) M. lysodeikticus ATPase complex appears to be dependent on bivalent cations. The present results do not preclude the possibility that the gamma subunit also plays a role in ATPase binding, in which, however, the major subunits do not seem to play a role.

Adenosine Triphosphatases↗

Micrococcus folliculitis in HIV-1 disease.

Organisms with little pathogenic potential in immunocompetent hosts may produce disease in HIV-1 + patients. We describe three HIV-1 + patients in late disease who presented with pruritic papules with central ulceration over the face and arms. In all the patients the eruptions had been present for months, and the patients did not develop sepsis. Biopsy specimens in all the patients showed large Gram-positive cocci, forming tetrads. Colony morphology, catalase positivity and coagulase negativity, and resistance to nitrofurantoin were used to separate micrococci from staphylococci. Micrococcus species are usually considered normal inhabitants of the skin; however, in patients with HIV-1 disease, Micrococcus species can produce localized cutaneous infections.

Adult↗

Micrococcus sedentarius bacteraemia presenting with haemophagocytic syndrome in previously healthy boy.

Haemophagocytic syndromes are the clinical manifestation of an increased macrophagic activity with haemophagocytosis. Infection-associated HS was originally described by Risdall in 1979, in viral disease. Since the initial description HS has also been documented in patients with bacterial, parasitic or fungal infections. We describe a case of Micrococcus sedentarius bacteraemia in a previously healthy 10-y-old boy with haemophagocytic syndrome. Species of micrococci are generally considered as non-pathogenic commensals that colonize the skin, mucosae and oropharynx. We report the first case of Microccoccus sedentarius bacteraemia in an immunocompetent host and first case of HS associated with Micrococcus species.

Bacteremia↗

Structural and topological studies on the lipid-mediated assembly of a membrane-associated lipomannan in Micrococcus luteus.

The biosynthesis of three mannolipids and the presence of a membrane-associated lipomannan in Micrococcus luteus (formerly Micrococcus lysodeikticus) were documented over 30 years ago. Structural and topological studies have been conducted to learn more about the possible role of the mannolipids in the assembly of the lipomannan. The major mannolipid has been purified and characterized as alpha-D-mannosyl-(1 --> 3)-alpha-D-mannosyl-(1 --> 3)-diacylglycerol (Man2-DAG) by negative-ion electrospray-ionization multistage mass spectrometry (ESI-MSn). Analysis of the fragmentation patterns indicates that the sn-1 position is predominantly acylated with a 12-methyltetradecanoyl group and the sn-2 position is acylated with a myristoyl group. The lipomannan is shown to be located on the exterior face of the cytoplasmic membrane, and not exposed on the surface of intact cells, by staining of intact protoplasts with fluorescein isothiocyanate (FITC)-linked concanavalin A (Con A). When cell homogenates of M. luteus are incubated with GDP-[3H]mannose (GDP-Man), [3H]mannosyl units are incorporated into Man1-2-DAG, mannosylphosphorylundecaprenol (Man-P-Undec) and the membrane-associated lipomannan. The addition of amphomycin, an inhibitor of Man-P-Undec synthesis, had no effect on the synthesis of Man1-2-DAG, but blocked the incorporation of [3H]mannose into Man-P-Undec and consequently the lipomannan. These results strongly indicate that GDP-Man is the direct mannosyl donor for the synthesis of Man1-2-DAG, and that the majority of the 50 mannosyl units in the lipomannan are derived from Man-P-Undec. Protease-sensitivity studies with intact and lysed protoplasts indicate that the active sites of the mannosyltransferases catalyzing the formation of Man1-2-DAG and Man-P-Undec are exposed on the inner face, and the Man-P-Undec-mediated reactions occur on the outer surface of the cytoplasmic membrane. Based on all of these results, a topological model is proposed for the lipid-mediated assembly of the membrane-bound lipomannan.

Anti-Bacterial Agents↗

Inactivation of ATP-dependent deoxyribonuclease of Micrococcus luteus by 2,3-butanedione.

ATP-dependent deoxyribonuclease from Micrococcus luteus was purified to near homogeneity by a procedure involving gentle cell lysis, ammonium sulfate fractionation, TEAE-cellulose chromatography, Sephadex G-150 gel filtration and DNA-cellulose chromatography. Treatment of the enzyme with 2,3-butanedione, which binds specifically to arginyl residues, caused rapid loss of enzyme activities and the effect was enhanced by borate ion. The reaction obeyed first order kinetics with respect to the butanedione concentration, indicating that at least one functional arginyl residue is involved in the inactivation reaction. The enzyme was protected from inactivation by the presence of a low concentration of ATP, but not of ADP, AMP or adenosine. These results indicate that ATP-dependent deoxyribonuclease of Micrococcus luteus has functional arginyl residue(s) at an ATP-binding site.

Arginine↗

Enzyme electrophoretograms in the analysis of taxon relatedness of Micrococcus cryophilus, Branhamella catarrhalis and atypical Neisserias.

Extracts were prepared from Micrococcus cryophilus, several strains of Branhamella catarrhalis and Neisseria spp. Esterases, NADP-dependent isocitrate dehydrogenase and malate dehydrogenase activities were assayed after electrophoresis of extracts of polyacrylamide gels. Except for Neisseria perflava and N. sicca which resolved activity bands for the acetate-esterase only, the remaining bacteria exhibited species-specific esterase patterns also for the propionate and butyrate substrates. The multiple esterase patterns from B. catarrhalis ATCC25238 were qualitatively and quantitatively different from those of B. catarrhalis ATCC23246. This finding and other evidence supports a taxonomic shift of the latter to a species level of that genus. The atypical neisserias N. caviae and N. ovis appeared to exhibit an intrageneric specificity in their esterase patterns with those from B. catarrhalis but not to the other Neisseria spp. tested. The malate dehydrogenase patterns from the atypical neisserias and B. catarrhalis ATCC23246 were qualitatively similar; however, the patterns of isocitrate dehydrogenase activity were variable for these species. Micrococcus cryophilus was distinct in its esterase and dehydrogenase bands, strongly suggesting its taxon unrelatedness to the genus Branhammella or the atypical neisserias. Of the enzymes assayed, esterase proved to be the most reliable for taxonomic identifications.

Acetates↗

Membrane-bound F1 ATPase from Micrococcus Sp. ATCC 398E. Purificationa and characterization ny affinity chromatography.

A chemically reactive ATP analogue, 6-[(3-carboxy-4-nitrophenyl)thio]-9-beta-D-ribofuranosylpurine 5'-triphosphate (Nbs6ITP) has been synthesized. It has the ability to form stable thioether bonds between the 6-position of the purine ring and aliphatic mercapto groups. The nucleotide moiety of the reagent has been covalently bound to agarose, via iminobispropylamine and N-acetyl-homocysteine as space with the purpose of producing an affinity chromatography material. The affinity matrix binds solubilized F1 ATPase from a crude extract of Micrococcus sp. membranes. Afterwards the enzyme can be selectively eluted from the column at a defined ATP concentration. This method is superior to the conventional purification with respect to speed and convenince of the preparation. The affinity chromatography leads in a one-step process to the same purity to enzyme, substituting several steps of the conventional method. In addition, the affinity matrix was used for binding studies. Although the presence of Mg2+ ions is a prerequisite for the hydrolysis of nucleoside 5'-triphosphates, evidence is presented indicating that the binding of the nucleoside triphosphates to highly purified F1 ATPase from Micrococcus sp. appears not to be influenced by Mg2+ ion concentrations so far examined.

Adenosine Triphosphatases↗

Antimicrobial susceptibilities of Stomatococcus mucilaginosus and of Micrococcus spp.

The in vitro susceptibilities of 63 isolates of Stomatococcus mucilaginosus and of 188 isolates of Micrococcus spp. to 18 antimicrobial agents were determined by the agar dilution method. Many beta-lactams, imipenem, rifampin, and the glycopeptides were shown to be active in vitro against Stomatococcus and Micrococcus isolates, whereas the activities of antibiotics such as some aminoglycosides, erythromycin, and fosfomycin against an important number of these microorganisms are limited.

Anti-Bacterial Agents↗

Preliminary studies on the characterization and distribution of Staphylococcus and Micrococcus species on animal skin.

A total of 221 strains of staphylococci and 98 strains of micrococci isolated from the skins of Eastern gray squirrels, Southern flying squirrels, raccoons, opossums, squirrel monkeys, swine, sheep, horses, cattle, and dogs were characterized in a preliminary attempt to resolve their natural relationships and distribution in nature. Staphylococci demonstrating the widest host range included Staphylococcus xylosus and unnamed Staphylococcus sp. 3. Unnamed Staphylococcus sp. 2 was isolated only from sheep, Staphylococcus sp. 4 only from opossums, Staphylococcus sp. 5 only from squirrel monkeys, and Staphylococcus sp. 6 only from swine. The predominant species isolated from human skin, including S. epidermidis, S. hominis, S. haemolyticus, and S. capitis, were either not isolated or only rarely isolated from animal skin. Micrococcus varians was the predominant Micrococcus species isolated from animal skin. M. luteus was only occasionally isolated. M. lylae, M. sedentarius, M. roseus, M. kristinae, and M. nishinomiyaensis, species occasionally isolated from human skin, were not isolated from animal skin.

Animals↗

Micrococcus luteus teichuronic acids activate human and murine monocytic cells in a CD14- and toll-like receptor 4-dependent manner.

Teichuronic acid (TUA), a component of the cell walls of the gram-positive organism Micrococcus luteus (formerly Micrococcus lysodeikticus), induced inflammatory cytokines in C3H/HeN mice but not in lipopolysaccharide (LPS)-resistant C3H/HeJ mice that have a defect in the Toll-like receptor 4 (TLR4) gene, both in vivo and in vitro, similarly to LPS (T. Monodane, Y. Kawabata, S. Yang, S. Hase, and H. Takada, J. Med. Microbiol. 50:4-12, 2001). In this study, we found that purified TUA (p-TUA) induced tumor necrosis factor alpha (TNF-alpha) in murine monocytic J774.1 cells but not in mutant LR-9 cells expressing membrane CD14 at a lower level than the parent J774.1 cells. The TNF-alpha-inducing activity of p-TUA in J774.1 cells was completely inhibited by anti-mouse CD14 monoclonal antibody (MAb). p-TUA also induced interleukin-8 (IL-8) in human monocytic THP-1 cells differentiated to macrophage-like cells expressing CD14. Anti-human CD14 MAb, anti-human TLR4 MAb, and synthetic lipid A precursor IV(A), an LPS antagonist, almost completely inhibited the IL-8-inducing ability of p-TUA, as well as LPS, in the differentiated THP-1 cells. Reduced p-TUA did not exhibit any activities in J774.1 or THP-1 cells. These findings strongly suggested that M. luteus TUA activates murine and human monocytic cells in a CD14- and TLR4-dependent manner, similar to LPS.

Animals↗

Halophilic amylase from a moderately halophilic Micrococcus.

A moderately halophilic Micrococcus sp., isolated from unrefined solar salt, produced a considerable amount of extracellular dextrinogenic amylase when cultivated aerobically in media containing 1 to 3 m NaCl. The Micrococcus amylase had maximal activity at pH 6 to 7 in 1.4 to 2 m NaCl or KCl at 50 C. Calcium ion and a high concentration of NaCl or KCl were essential for activity and stability of the amylase. The salt response of the amylase depended greatly on the pH and temperature of the enzyme assay.

Aerobiosis↗

Isolation and properties of a recombination-deficient mutant of Micrococcus radiodurans.

A mutant of micrococcus radiodurans which is deficient in recombination has been isolated after treatment of the wild type with N-methyl-N'-nitro-N-nitrosoguanidine. We have called this mutant Micrococcus radiodurans rec30. The efficiency of recombination in this mutant, as measured by transformation, is less than 0.01% that of the wild type. It is 15 times more sensitive to the lethal action of ultraviolet radiation, 120 times more sensitive to ionizing radiation, and 300 times more sensitive to mitomycin C (MMC) than the wild type. It is probably inactivated by a single MMC-induced deoxyribonucleic acid cross-link per genome. The excision of ultraviolet-induced pyrimidine dimers is normal. There is no radiation-induced degradation of deoxyribonucleic acid. All spontaneous revertants selected for resistance to low levels of MMC had wild-type resistance to radiation and MMC, and the same efficiency of recombination as the wild type, suggesting that the recombination deficiency of the strain is due to a single mutation. Deoxyribonucleic acid from this mutant can transform M. radiodurans UV17 presumed deficient in an exr type gene to wild type.

Acriflavine↗