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Nutrition and growth of the moderately halophilic bacteria Micrococcus morrhuae K-17 and Micrococcus luteus K-15.

Chemically defined media have been developed for the growth of two moderately halophilic bacteria, Micrococcus morrhuae K-17 and Micrococcus luteus K-15. M. morrhuae K-17 grows well in a synthetic medium (SM-1) which contains a number of salts, 0.21 M KCl, 2 M NaCl, D-mannose, five vitamins and ten amino acids. The synthetic medium (SM-2) for M. luteus K-15 contains a number of salts, 0.21 M KCl, 1 M NaCl, D-fructose, nine vitamins and nine amino acids. Nutritional studies show that M. morrhuae K-17 can utilize a large number of organic compounds as carbon and energy source while the ability of M. luteus K-15 in utilizing the organic compounds is rather limited. The minimum salt requirement is 0.5 M NaCl for both strains when growth at the optimum temperature of 30 degrees C. However, this requirement can be lowered to 0.2 M in M. luteus K-15 when grown at a lower temperature of 25 degrees C. It is concluded that the ability to grow in a wider range of salt concentrations in response to temperature is species specific in moderate halophiles. The salt range for growth to occur can be extended when cells of both strains are grown in complex medium which might provide the amino acids and growth factors that cannot be synthesized by these strains at high salt concentrations.

Amino Acids

The fine structure of Micrococcus radiophilus and Micrococcus radioproteolyticus.

The radiation resistant bacteria Micrococcus radiophilus and M. radioproteolyticus were studied by thin sectioning and freez-etching techniques and the two species were found to be similar in the fine structure. The only significant difference was in the appearance of the surfaces of the cell walls in freeze-etched preparations. Since the two species, together with M. radiodurans, possess a unique cell wall structure and a cell wall peptidoglycan, which is different from that of other micrococci and Gram-positive cocci, it is recommended that they be reclassified into a new genus.

Cell Division

Differentiation of Micrococcus luteus and Micrococcus varians on the basis of catalase isoenzymes.

Crude extracts prepared from four Micrococcus varians strains, 11 M. luteus strains and four laboratory isolates subsequently classified with M. luteus were assayed for catalase activity following electrophoresis on polyacrylamide gels. The enzyme patterns produced from the M. varians strains exhibited three catalase isoenzymes which were distinguished into two types of patterns depending upon the location of the major band. The extracts from all the M. luteus strains produced the same pattern, composed of two catalase isoenzymes of similar electrophoretic mobility. For both species the isoenzyme patterns agreed with the differentiation based on biochemical properties. The catalase activity activity staining method was shown to be a restricted yet reliable assay in the intrageneric but not intraspecies differentiation of yellow-pigmented micrococci.

Catalase

Distribution and persistence of Staphylococcus and Micrococcus species and other aerobic bacteria on human skin.

The districution of Staphylococcus and Micrococcus species and associated coryneform bacteria, Acinetobacter, Klebsiella, Enterobacter, Bacillus, and Streptomyces on skin was determined during October 1971 from samples collected on persons living in North Carolina and New Jersey. Persistence of these organisms on skin was estimated in temporal studies conducted during the period from June 1971 to June 1972 on persons living in North Carolina. Staphylococci and coryneforms were the most predominant and persistent bacteria isolated from the nares and axillae. Staphylococci, coryneforms, micrococci, and Bacillus were the most predominant and persistent bacteria isolated from the head, legs, and arms. Acinetobacters were most frequently isolated during the warmer months of the years. Staphylococcus aureus and S. epidermidis were the most predominant and persistent staphylococci isolated from the nares, whereas S. epidermidis and S. hominis were the most predominant and persistent staphylocicci isolated from the axillae, head, legs, and arms. S. capitis was often isolated from the head and arms and S. haemolyticus was often isolated from the head, legs, and arms. S. simulans, S. xylosus, S. cohnii, S. saprophyticus, S. warneri, and an unclassified coagulase-positive species were only occasionally isolated from skin. Micrococcus luteus was the most predominant and persistent Micrococcus isolated from skin and preferred regions of the head, legs, and arms. M. varians was the second most frequent Micrococcus isolated. M. lylae, M. sedentarius, M. roseus, M. kristinae, and M. nishinomiyaensis were only occasionally isolated from skin. M. lylae was most frequently isolated during the colder months of the years.

Acinetobacter

Comparative immunological study of catalases in the genus Micrococcus.

Double immunodiffusion tests were performed with crude extracts from various Micrococcus species and antisera against catalase of Micrococcus luteus CCM 169. Cell-free extracts of M. lylae ATCC 27566 exhibited good cross-reaction. Cell-free extracts or catalase enriched preparations of M. varians reacted very weakly and no reaction has been found with preparation of M. kristinae, M. nishinomiyaensis, M. roseus and M. sedentarius. The quantitative microcomplement fixation assay also revealed a closer relationship between M. luteus and M. lylae than between M. luteus and M. varians. Strains of other Micrococcus species reacted in the microcomplement assay with M. luteus antiserum just a weakly as non-related strains, e.g. Staphylococcus aureus or Cellulomonas cartalyticum.

Catalase

[Micrococcus luteus: a rare pathogen of valve prosthesis endocarditis].

A rare case of prosthetic valve endocarditis caused by Micrococcus luteus is described and compared with the few cases reported in the literature, as well as the clinical features, microbiological profile, therapy, and prognosis of common prosthetic valve endocarditis. Micrococcus luteus is a constituent of the normal human buccal bacterial flora which forms yellowish colonies and appears as a gram-positive coccus typically arranged in tetrades. Although of low virulence, the germ may become pathogenic in patients with impaired resistance, colonizing the surface of heart valves. In contrast to staphylococci (for which it may easily be mistaken) it is usually penicillin-sensitive. However, the most promising antibiotic regimen proposed for treatment of Micrococcus luteus seems to be a combination of vancomycin, amikacin, and rifampicin. If the infection leads to severe hemodynamic alterations, however, valve replacement may become necessary similar to the situation in prosthetic valve endocarditis caused by more aggressive and highly resistant bacteria.

Aged

Identification of Staphylococcus and Micrococcus species with the STAPHYtest system.

A collection of 216 well-characterized strains of Staphylococcus, Micrococcus and Stomatococcus was examined by a commercially available STAPHYtest system (Lachema, Brno, Czechoslovakia). The results of STAPHYtest agreed with those of conventional tests. The STAPHYtest permitted a clear-cut separation of Staphylococcus from Micrococcus and Stomatococcus strains and correctly identified 104 of 145 (72%) Staphylococcus strains after 24 h of incubation. However, it allowed the identification only of 19 of 29 validly published Staphylococcus species. The STAPHYtest proved to be a simple and rapid system for the separation of staphylococci from micrococci and for the identification of most frequent clinically significant staphylococci.

Bacteriological Techniques

Induction of nutritional mutants of Micrococcus glutamicus and their amino acid accumulation.

Micrococcus glutamicus ATCC 13032, a glutamic acid-producing organism, was treated with 0.2M ethylmethane sulfonate, the auxotrophs isolated showing varied patterns of extracellular amino acids. Eighty auxotrophic strains were obtained, out of which 31 excreted 1.0-4.0 mg threonine per ml and all the auxotrophs required biotin for growth and production of the amino acid. Eleven auxotrophs produced 1.5 to 3.0 mg alanine per ml and these auxotrophs required amino acids for their growth. Other auxotrophs lost their excretion capacity in subsequent fermentation trials. Further mutation of the biotin-requiring auxotroph Micrococcus glutamicus EM with gamma rays resulted in the isolation of 89 auxotrophic strains, out of which 28 excreted threonine (up to 5.0 mg per ml) higher than the parent auxotroph. Exposure to X-rays yielded 97 auxotrophs, out of these 35 producing 1.0-3.0 mg methionine per ml and requiring biotin for growth and production of the amino acid. Other auxotrophs produced alanine (0.5 to 2.0 mg per ml) and threonine (2.0 to 3.3 mg per ml). Irradiation with gamma rays favoured the development of threonine producing auxotrophs while X-rays favoured methionine-producing auxotrophs.

Alanine

Induction of mutation to streptomycin resistance in Micrococcus radiodurans.

No detectable induction of mutation to streptomycin resistance could be used in wild-type Micrococcus radiodurans and its radiation-sensitive and super-resistant mutants by ionizing or UV-radiation. N-methyl-N'-nito-N-nitrosoguanidine (NTG) was mutagenically active. The results suggest that repair of radiation-damaged DNA in Micrococcus radiodurans is mutation-proof.

Cobalt Radioisotopes

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

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

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

Repair of ultraviolet light-induced damage in Micrococcus radiophilus, an extremely resistant microorganism.

Repair of ultraviolet radiation damage was examined in an extremely radioresistant organism, Micrococcus radiophilus. Measurement of the number of thymine-containing dimers formed as a function of ultraviolet dose suggests that the ability of this organism to withstand high doses of ultraviolet radiation (20,000 ergs/mm2) is not related to protective screening by pigments. M. radiophilus carries out a rapid excision of thymine dimers at doses of ultraviolet light up to 10,000 ergs/mm2. Synthesis of deoxyribonucleic acid is reduced after irradiation, but after removal of photodamage the rate approaches that in unirradiated cells. A comparison is drawn with Micrococcus luteus and M. radiodurans. We conclude that the extremely high resistance to ultraviolet irradiation in M. radiophilus is at least partly due to the presence of an efficient excision repair system.

Cell-Free System

Comparison of the cell envelope proteins of Micrococcus cryophilus with those of Neisseria dnd Branhamella species.

In an attempt to elucidate the relation between Micrococcus cryophilus, Neisseria caviae, Neisseria ovis, and Branhamella catarrhalis, fractions derived from outer membranes of a strain of each organism were examined for protein composition by SDS - polyacrylamide gel electrophoresis. Micrococcus cryophilus outer membrane protein showed extensive similarities to that of N. ovis and contained a heat-modifiable protein which behaved almost identically with the corresponding bands previously shown to exist in N. caviae and N. ovis. Branhamella catarrhalis protein was distinctly different from those of M. cryophilus and the two 'false neisserias' N. caviae and N. ovis.

Bacterial Proteins

Inhibition of diptheroid esterase by Micrococcus luteus.

Micrococcus luteus produced a diffusible, esterase inhibitory factor (EIF) which inhibited the activity of cutaneous diphtheroid esterases on Tween 80-CaCl2 agar media. Esterases of Staphylococcus, Micrococcus, Bacillus, and Serratia were not susceptible. EIF did not appear to combine with the substrate or to prevent enzyme synthesis; it was unable to reverse the precipitation of calcium oleate. The composition of the medium, especially peptones, influenced the production of EIF. EIF was synthesized in the absence of diphtheroids, but production required the presence of Tween. The interaction was observed on agar medium of pH 5.5-8.5, at 25-43 degrees C, under an atmosphere of 10-20% CO2, in the presence of urea, but not after the addition of NaCl or dextrose. Supernatants of broth cultures had to be concentrated to detect EIF. Crude dialyzed and concentrated preparations of EIF withstood 60 degrees C for 60 min but were inactivated after 100 degrees C for 10 min. EIF may possibly be associated with a lipoid substance, since it did not precipitate in ethanol.

Bacillus

Flocculation and adsorption of enzymes during growth of a moderate halophile, Micrococcus varians var. Halophilus.

Flocculation of a moderate halophile, Micrococcus varians ATCC 2197, occurred during growth in complex medium containing 3 M NaCl and a concentration of MgSO4 and KH2PO4 greater than 40 and 14 mM, respectively. Extracellular nuclease activity was absent in the flocculated cultures. Repeated washing of flocs by Mg2+-free Tris buffer containing 3 M NaCl, lowering of pH value of floc suspension below 6.3, or addition of ethylenediaminetetraacetic acid resulted in complete dissociation of the flocs and release of Mg2+ ions as well as nuclease and amylase. Inhibition of extracellular enzyme production accompanied by flocculation appeared to be the result of adsorption of enzyme proteins to surfaces of the flocs, but not of inhibition of biosynthesis. Floc formation could also occur in media containing 18 mM CaCl2 and 3.0 mM KH2PO4, but the Ca flocs were not deflocculated by washing with Ca2+-free buffer, suggesting that the affinity of Ca2+ for cell envelopes was stronger than that of Mg2+. It was also observed that most halophilic Planococcus and Micrococcus flocculated in the presence of MgSO4 and phosphate but halophilic Pseudomonas, Acinetobacter, and Bacillus did not.

Adsorption

Purification and some properties of the soluble and membrane-bound adenosine deaminases of Micrococcus sodonensis ATCC 11880 and their distribution within the family Micrococcacea.

In Micrococcus sodonensis and some other Micrococcus species, adenosien deaminase is present both as a membran-bound and a soluble enzyme; The membran-bound adenosine deaminase can be extracted with n-butanol, and may account for up to 5% of the total cellular adenosine deaminase activity. In a number oc comparative tests, no differences between the two enzyme forms could be found, thus they are believed to be similar molecular species; The purified membran-bound or soluble enzyme had a molecular weight, obtained by gel-filtration, of 130 000 and was inactive toward adenine and adenine mononucleotides. It appears, therefore, to be more closely related to the calf-intestine enzyme than the Aspergillus oryzae form in respect to size and substrate specificity; Attempts to correlate membrane-bound adenosine deaminase activity with adenosine transport in isolated membrane vesicles of M. sodonensis indicated no obvious relationship between the two activities.

Adenosine