Micrococcus Sodonensis Research Articles

L-Arginine Production by Arginine Analog-resistant Mutants of Microorganisms

L-Arginine analog-resistant mutants were derived from Bacillus subtilis, Serratia marcescens, Microbacterium ammoniaphilum, Micrococcus sodonensis, Nocardia corynebacteroides, N. rubra, Saccharomyces cerevisiae and Candida tropicalis.The mutants of all species tested produced an appreciable amount of L-arginine. The arginine productivity of SAH4-7, an L-arginine hydroxamate-resistant L-arginine-producer of B. subtilis,increased stepwisely by successively introducing such characters as pyrimidine analog-, histidine analog-, and tryptophan analog-resistance and then increased resistance to arginine analog. The mutant strain finally selected was KY7690 and it produced ca. 17mg per ml of L-arginine.

L-arginine production by arginine analog-resistant mutants of microorganisms.

L-Arginine analog-resistant mutants were derived from Bacillus subtilis, Serratia marcescens, Microbacterium ammoniaphilum, Micrococcus sodonensis, Nocardia corynebacteroides, N. rubra, Saccharomyces cerevisiae and Candida tropicalis.The mutants of all species tested produced an appreciable amount of L-arginine. The arginine productivity of SAH4-7, an L-arginine hydroxamate-resistant L-arginine-producer of B. subtilis,increased stepwisely by successively introducing such characters as pyrimidine analog-, histidine analog-, and tryptophan analog-resistance and then increased resistance to arginine analog. The mutant strain finally selected was KY7690 and it produced ca. 17mg per ml of L-arginine.

Passive diffusion of nucleosides into Micrococcus sodonensis membrane vesicles.

Nucleoside entry into isolated membrane vesicles of Micrococcus sodonensis (luteus) was studied using 14C-labelled nucleosides: adenosine, inosine, cytidine, uridine, guanosine, and thymidine. All nucleosides were recovered unmetabolized from the vesicles except adenosine and cytidine which were partly deaminated by membrane-bound enzymes. Vesicle preparations actively transported proline but no energy source was found capable of supporting concentrative nucleoside uptake. The entry of nucleosides into M. sodonensis vesicles was not saturable, nor was there competition between the nucleosides studied for entry. It was concluded that nucleoside entry into M. sodonensis vesicles occurs by passive diffusion.

Microbial conversion of DL-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine and L-cystine: screening of microorganisms and identification of products.

Microorganisms able to form L-cysteine from DL-2-amino-delta2-thiazoline-4-carboxylic acid (DL-ATC), a chemical intermediate in the synthesis of DL-cysteine, were isolated from soil samples and classified as Pseudomonas sp., Pseudomonas cohaerens, P. desmolytica, and P. ovalis. Thirteen L-cysteine-producing bacteria were also found in among 463 stock cultures representing 37 genera. These were Achromobacter delmarvae. Alcaligenes denitrificans, Bacillus brevis, Brevibacterium flavum, Enterobacter aerogenes, Erwinia carotovora, Escherichia coli, Micrococcus sodonensis, Myocoplana dimorpha, Sarcina lutea, Serratia marcescens, Flavobacterium acidoficum, and Pseudomonas ovalis. In the presence of intact cells of Pseudomonas sp. AJ 3854, 6.1 mg of L-cysteine and/or L-cystine per ml was produced from 10 mg of DL-ATC-3H2O per ml in a molar yield of 100%. This finding suggests that racemization and asymmetric hydrolysis occurred simultaneously in this incubation mixture. After the complete oxidation of cysteine to cystine by aeration in the presence of ferrous ion, crystalline cystine was isolated; its configuration was the L isomer based on data from X-ray diffraction, microbioassay, and optical rotation studies.

A membrane-associated lipomannan in micrococci.

Membranes of Micrococcus lysodeikticus, Micrococcus flavus and Micrococcus sodonensis contain acidic lipomannans. Lipoteichoic acids could not be detected in these organisms, and the suggestion that they are substituted for by the lipomannans is strengthened by the chemical and physical resemblances between the two polymers. The mannans contain glycerol, ester-linked fatty acids and mono-esterified succinic acid residues, giving them both hydrophobic and charged properties. The M. lysodeikticus mannan has a chain of about 60 hexose units with two branch points, and is joined at its reducing end to the 1-position of a glycerol moiety bearing two fatty acid residues. Succinic acid on the mannan enables it to bind Mg2+ efficiently, and the polymer is firmly associated with the cytoplasmic membrane, probably by intercalation of its fatty acids with those of the membrane lipids.

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.

Studies of the mechanisms of radiosensitization of bacterial and mammalian cells by Diamide.

Diamide sensitizes bacterial and mammalian cells to radiation by at least two mechanisms. Sensitization of V79-GL1 Chinese Hamster cells is due mainly to a reduction of a reduction of the survival-curve shoulder, is observed both in oxygen and in hypoxia,and is additive to the sensitization of hypoxic cells by some nitroimidazoles. In contrast, sensitization of the radioresistant organism, Micrococcus sodonensis, which has apronounced shoulder, is entirely dose-modifying. In a rapid-mix study using Serratia marcescens, two mechanisms of sensitization have been identified. After 4 to 40 msec preirradiation contact with Daimide, the enhancement ratio is constant at 1.6; after 10min contact, it is 2.5. With V79-GL1 cells, no sensitization occurs in the short-time-range; likewise non is seen when Diamide is added to bacteria or mammalian cells 4 msec after irradiation. It is concluded that Diamide sensitizes by at least two mechanisms, one of which resembles that of the electron-affinic type.

An extracellular 5′-nucleotidase with both monoesterase and diesterase activity from Micrococcus sodonensis

A 5′-nucleotidase which has both monoesterase and diesterase activity has been isolated from the growth medium of Micrococcus sodonensis . The enzyme can be separated electrophoretically into three isoenzymes. The enzyme exhibited a pH optimum of 8.7, a molecular weight of approximately 115,000, and requires Mn 2+ for stability. It catalyzes the hydrolysis of the 5′-phosphate bond in both mononucleotides and dinucleotides, but has negligible activity with nucleic acids. The enzyme also catalyzes the hydrolysis of the synthetic substrate, p -nitrophenyl thymidine-5′-phosphate, to p -nitrophenyl phosphate and thymidine, the reaction used in the spectrophotometric assay of the enzyme. Its substrate specificity differentiates it from other nucleotidases which have been described, and indicates that it may be of use in the sequencing of oligonucleotides.

The Role of Polysaccharide in the Secretion of Protein by Micrococcus sodonensis

Abstract Cell suspensions of Micrococcus sodonensis (ATCC 11880) secrete 7 to 10 individual proteins including an alkaline phosphatase, a nuclease, and a protease. The appearance of enzyme activities in the extracellular medium is dependent on the co-secretion of at least one of several polysaccharides also elaborated by these cells. This conclusion is based on the following observations: (a) bacitracin, an inhibitor of polysaccharide biosynthesis, proportionately inhibits polysaccharide and protein secretion at concentrations which have no effect on intracellular protein synthesis; (b) a mutant, impaired in its ability to secrete proteins, is also impaired in its ability to secrete a glucosamine-rich polysaccharide; (c) alkaline phosphatase secretion by mutant protoplasts is enhanced in the presence of exogenously supplied polysaccharide; (d) inclusion of 10 mm glucosamine or 2-deoxyglucose (a variety of other monosaccharides were ineffective) in the medium suppresses the secretion of all three enzymes as well as the glucosamine-containing polysaccharide; and (e) restoration of alkaline phosphatase secretion in glucosamine-inhibited protoplasts is achieved by the addition of purified preparations of the glucosamine-containing polysaccharide. The inhibition of enzyme secretion by either glucosamine or 2-deoxyglucose can be completely prevented or reversed by addition of equimolar concentrations of glucose. The addition of albumin or casein to glucosamine-inhibited cells markedly enhances the rate of alkaline phosphatase secretion. These results suggest that during secretion polypeptide chains are vulnerable to proteolytic degradation and that co-secretion of polysaccharide protects the protein from proteolysis.

Metabolism and transport of purine nucleosides by membrane preparations of Micrococcus sodonensis.

The uptake and metabolism of adenosine-8-14C was studied using isolated membrane vesicles prepared from Micrococcus sodonensis. When the adenosine concentration was 1.0 mM, a concentration that caused prolonged bacteriostasis in growing cells, the membranes vesicles initially accumulated adenosine and inosine to similar levels but rapidly became enriched with inosine. However, when the external adenosine concentration was lowered to 0.1 mM, the vesicle contents were almost entirely inosine. No compounds other than adenosine and inosine were detected. These effects were shown to be the result of a membrane-bound adenosine deaminase and are in marked contrast to the phosphoribosyltransferase-linked mechanisms of purine nucleoside transport in other bacterial systems.

Production and control of extracellular enzymes in Micrococcus sodonensis.

Micrococcus sodonensis has been shown to produce several extracellular enzymes: an alkaline phosphatase, at least two forms of phosphodiesterase, a 5′-nucleotidase, and an alkaline proteinase. The quantitative release of these enzymes into the culture medium during logarithmic growth under all the various culture conditions tested indicates that these enzymes are truly extracellular in nature. Inorganic phosphate repressed the production of the alkaline phosphatase in synthetic as well as in complex media, whereas, the repression of the production of active diesterase and 5′-nucleotidase by inorganic phosphate was partly reversed by the addition of supplemental organic nutrients to the culture medium. Proteinase production was independent of the culture conditions used. A mutant strain of M. sodonensis with an altered production of diesterase was obtained; the other extracellular enzymes were unaffected. These results suggest that the extracellular enzymes of M. sodonensis are not produced in a pleiotropic fashion since the level of one of the enzymes can be changed without affecting a corresponding change in the levels of the other enzymes. An extracellular high molecular weight carbohydrate fraction was shown to be produced by M. sodonensis in synthetic medium. The fraction was also shown to contain glycoprotein.

Adenosine metabolism in Micrococcus sodonensis.

Enzymes required for the synthesis of purine ribonucleotides from exogenous adenosine, inosine, and guanosine were shown to be present constitutively in Micrococcus sodonensis. An indirect pathway for the synthesis of adenine nucleotides from adenosine was suggested by the high intracellular adenosine deaminase activity. Concentrations of intracellular adenine nucleotides were elevated in the presence of exogenous adenosine and under those conditions direct measurements of the activity of the pathway for de novo purine ribonucleotide biosynthesis indicated that the activity of that pathway was almost completely inhibited.

Adenosine-induced bacteriostasis in Micrococcus sodonensis.

In a synthetic growth medium at a concentration of 1.0 mM, adenosine had a marked bacteriostatic effect on the growth of Micrococcus sodonensis. With the exceptions of hypoxanthine and inosine, which were slightly inhibitory, other purine bases and ribonucleosides did not inhibit the growth of the organism. When incubation of adenosine-supplemented cultures was prolonged, the organism was able to recover from the growth inhibition after a period of time which varied directly with the initial adenosine concentrations. Chromatographic analysis of the supernatants of those cultures revealed that exogenous adenosine concentrations declined steadily and the termination of the inhibited phase of growth correlated with the exhaustion of exogenous adenosine.It was observed that de novo biosynthesis of thiamine was inhibited in adenosine-supplemented cultures and that this inhibition was also relieved upon the exhaustion of exogenous adenosine. Bacteriostasis could be prevented by including either thiamine or its pyrimidine precursor (B1-pyrimidine) in adenosine-supplemented cultures and it was concluded that the observed growth inhibition reflected a depletion of the intracellular thiamine pool resulting from an adenosine-associated inhibition of B1-pyrimidine biosynthesis.

Effect of growth conditions on peptidoglycan structure and susceptibility to lytic enzymes in cell walls of Micrococcus sodonensis.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEffect of growth conditions on peptidoglycan structure and susceptibility to lytic enzymes in cell walls of Micrococcus sodensisKenneth G. Johnson and J. N. CampbellCite this: Biochemistry 1972, 11, 2, 277–286Publication Date (Print):January 18, 1972Publication History Published online1 May 2002Published inissue 18 January 1972https://doi.org/10.1021/bi00752a020RIGHTS & PERMISSIONSArticle Views35Altmetric-Citations15LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

Studies on the Extracellular Alkaline Phosphatase of Micrococcus sodonensis: II. FACTORS AFFECTING SECRETION

The secretion of alkaline phosphatase by Micrococcus sodonensis has been studied by observing the production and accumulation of extracellular enzyme by log phase cells which have been resuspended in fresh growth medium. The accumulation of extracellular alkaline phosphatase activity is the result of a selective permeation process and is totally dependent upon the presence of divalent cation. Although calcium is an actual component of the enzyme and is required for both catalytic activity and enzyme stability, magnesium appears to be the divalent cation required for the actual synthesis or release (or both) of alkaline phosphatase from the cell. Studies with various inhibitors of protein synthesis suggest that the bacterial cell may possess at least two distinct sites of protein synthesis, one site functioning to provide intracellular and cell-associated proteins and another for the synthesis of extracellular proteins. The over-all process of enzyme secretion is markedly temperature-dependent and is characterized by an activation energy of 55 kcal per mole. This observation suggests that the rate-limiting step in alkaline phosphatase secretion is probably not an enzyme-catalyzed event. A relatively small but significant amount of alkaline phosphatase activity exists in a cell-bound form and can be quantitatively released from cells by treatment with lysozyme. Although lysozyme-sensitive cell wall components are involved in the binding of alkaline phosphatase to the cell, the cell wall is not an obligatory component of the secretion process since lysozyme-prepared, sucrose-stabilized protoplasts are capable of extracellular enzyme production.

Studies on the Extracellular Alkaline Phosphatase of Micrococcus sodonensis: I. ISOLATION AND CHARACTERIZATION

Abstract An orthophosphate-repressible extracellular alkaline phosphatase has been isolated from cultures of Micrococcus sodonensis. The enzyme is pure as judged by molecular sieve chromatography, disc gel electrophoresis, and ultracentrifugation, and possesses the following properties: (a) molecular weight, 80,000; (b) pH optimum, pH 9 to 9.5; (c) competitive inhibitors, orthophosphate, arsenite, and arsenate; (d) substrate specificity, hydrolyzes a wide variety of phosphorylated compounds including phosphomonoesters, nucleotide mono-, di-, and triphosphates, and inorganic pyrophosphate. Enzymatic activity is dependent upon the presence of free sulfhydryl group(s); derivatization of the enzyme with 1 eq of p-chloromercuribenzoate results in the loss of approximately 75% of its catalytic activity. Activity of the enzyme exhibits an absolute requirement for divalent cation with the following relative reactivations of ethylenediaminetetraacetate-inactivated enzyme: Ca++, 90; Mn++, 22; Co++, 15; Sr++, 13; and Cu++, Mg++, Zn++, l1. The Km for Ca++ was estimated to be 1.5 x 10-5 m and the Kdissociation = 1.5 x 10-5 m as determined by equilibrium dialysis. Assuming a molecular weight of 80,000, the Ca++ content of the enzyme was estimated to be 8 g atoms per mole. Removal of Ca++ from the enzyme resulted in a marked increase in susceptibility of the enzyme to inactivation by heat and proteolytic digestion.

The extracellular nuclease activity of Micrococcus sodonensis: IV. Physical studies, characterization as a glycoprotein and involvement with the cell wall

1.1. The molecular weight and s20,w of Micrococcus sodonensis nuclease were found to be concentration dependent and extrapolation to zero concentration yielded values of 500 000 and 2.2 S, respectively. The concentration dependence and behaviour of the peak in sedimentation velocity experiments are indicative of an asymmetric molecule.2.2. The enzyme was shown to be a glycoprotein consisting of 21% carbohydrate covalently linked to the protein moiety. Glucosamine, glucose, galactose and rhamnose were identified as the component sugars and were present in a ratio of 4:2:1:1. A serine-glucosamine linkage involving at least 80% of the serine residues was demonstrated.3.3. Amino acid analyses showed the enzyme to be low in cysteic acid residue and to contain relatively high amounts of serine, threonine, glycine and proline. Unexpected high values for tryptophan were shown to be a result of the presence of amino sugar.4.4. The carbohydrate moiety of purified enzyme was shown to be related both chemically and immunologically to the cell wall carbohydrate and active enzyme was found bound in a stable configuration to the cell wall.5.5. The possible role of the wall in enzyme formation is discussed. 1. The molecular weight and s20,w of Micrococcus sodonensis nuclease were found to be concentration dependent and extrapolation to zero concentration yielded values of 500 000 and 2.2 S, respectively. The concentration dependence and behaviour of the peak in sedimentation velocity experiments are indicative of an asymmetric molecule. 2. The enzyme was shown to be a glycoprotein consisting of 21% carbohydrate covalently linked to the protein moiety. Glucosamine, glucose, galactose and rhamnose were identified as the component sugars and were present in a ratio of 4:2:1:1. A serine-glucosamine linkage involving at least 80% of the serine residues was demonstrated. 3. Amino acid analyses showed the enzyme to be low in cysteic acid residue and to contain relatively high amounts of serine, threonine, glycine and proline. Unexpected high values for tryptophan were shown to be a result of the presence of amino sugar. 4. The carbohydrate moiety of purified enzyme was shown to be related both chemically and immunologically to the cell wall carbohydrate and active enzyme was found bound in a stable configuration to the cell wall. 5. The possible role of the wall in enzyme formation is discussed.

The extracellular nuclease activity of Micrococcus sodonensis: III. Kinetic studies and control of production

Abstract 1. 1. Kinetic analyses of the purified nuclease of Micrococcus sodonensis yielded K m values of 2.9 · 10 −6 and 2.83 · 10 −5 M for RNA and AMP, respectively. 2. 2. AMP was inhibitory to ribonuclease activity and apparent non-competitive kinetics were obtained with a K i of 2.9 · 10 −3 M . 3. 3. Both diesterase and monoesterase activities were stimulated by chelating agents such as glycine, histidine and EDTA, and by sulfhydryl compounds such as mercaptoethanol, cysteine and GSH. p- Chloromercuribenzoate was inhibitory to both activities as were certain of the purine nucleotide analogues such as inosine 5′-chloromethylphosphonate. 4. 4. Studies with metabolite indicated that enzyme synthesis was controlled by the intracellular pyrimidine nucleotide concentration. A cytidine nucleotide is implicated. 5. 5. The data presented in the paper add additional support to the “one protein-two activities” concept for this enzyme.

Sensitization of Bacteria to X-Ray by Neoarsphenamine

Neoarsphenamine (NARS), an organic trivalent arsenical compound, is a sulfhydryl-binding agent whose pharmalogical action in humans is well documented. Because it is a sulfhydryl-binding agent, the possibility that it would sensitize anoxic cells to ionizing radiation was investigated with the idea that such a sensitization could overcome the relative protection that severely hypoxic cells enjoy in certain parts of most human cancers. Micrococcus sodonensis and Escherichia coli K-12 AB1157 were both tested. Though both bacteria were sensitized, the latter is especially interesting because no sensitization occurred when NARS was used in an oxygenated medium. A sensitization factor [defined as the ratio of the slopes, where the slope of the curve refers to the relationship of the surviving fraction (ordinate) to the dose in kilorads (abscessa)] of approximately 2.2 was obtained with AB1157. The effect was reversed by an excess of cysteine added immediately before x-irradiation but was not lost by pre-x-ray ...

Growth and macromolecular biosynthesis by Micrococcus sodonensis during the utilization of glucose and lactate.

Micrococcus sodonensis can metabolize glucose to carbon dioxide and water via both the hexose monophosphate shunt and glycolysis but cannot use it as a sole source of carbon and energy. When lactate-grown cells of M. sodonensis are replaced on glucose there is a 50% increase in cell population. The synthesis of deoxyribonucleic acid, ribonucleic acid, and protein continues for about 1.5 to 2.0 h before it ceases. Cells replaced in lactate medium under the same conditions double in number in approximately 4 h. Cells replaced in glucose do not synthesize phosphorus-containing compounds as ascertained by inorganic phosphate uptake while lactate-utilizing cells take up phosphate at a significant and continuous rate. The results suggest that the inability to utilize glucose as a sole source of carbon and energy is related to this incapability of M. sodonensis to gain sufficient energy from glucose oxidation. Results with related strains of micrococci suggest this phenomenon may be widespread in this group of microorganisms.