Search

Teichuronic acid released from its phosphodiester linkage to peptidoglycan in the cell walls of Micrococcus luteus by mild acid treatment is resolved into a ladderlike series of bands by electrophoresis on polyacrylamide gels in the presence of borate. Each band of the ladder differs from its nearest neighbor by one disaccharide repeat unit, ----4)-2-acetamido-2-deoxy-beta-D-mannopyranuronosyl-(1----6)- alpha-D-glucopyranosyl-(1-. Acid-fragmented teichuronic acid, after conversion to the phenylamine derivative, was fractionated by preparative-scale molecular sieve column chromatography, which produced a series of elution peaks. Fast-atom-bombardment mass spectrometry of the smallest member of the series determined its molecular weight and established its identity as the phenylamine derivative of one disaccharide repeat unit of teichuronic acid. Homologous fractions of the same series were used to index the ladder of bands obtained by polyacrylamide gel electrophoresis from samples containing a more extensive distribution of polymer lengths. Nearly native teichuronic acid consists of polymers with a broad range of molecular sizes ranging from 20 to 55 disaccharide units. The most abundant species are those which have 25 to 40 repeat units. Prolonged treatment of teichuronic acid with the acid conditions used to release it from peptidoglycan causes gradual fragmentation of the teichuronic acid.

Teichuronic acid-peptidoglycan complex isolated from Micrococcus luteus cells by lysozyme digestion in osmotically stabilized medium was treated with mild acid to cleave the linkage joining teichuronic acid to peptidoglycan. This labile linkage was shown to be the phosphodiester which joins N-acetylglucosamine, the residue located at the reducing end of the teichuronic acid, through its anomeric hydroxyl group to a 6-phosphomuramic acid, a residue of the glycan strand of peptidoglycan. 31P nuclear magnetic resonance spectroscopy of the lysozyme digest of cell walls demonstrated the presence of a phosphodiester which was converted to a phosphomonoester by the conditions which released teichuronic acid from cell walls. Reduction of acid-liberated reducing end groups by NaB3H4 followed by complete acid hydrolysis yielded [3H] glucosaminitol from the true reducing end residue of teichuronic acid and [3H]glucitol from the sites of fragmentation of teichuronic acid. The amount of N-acetylglucosamine detected was approximately stoichiometric with the amount of phosphate in the complex. Partial fragmentation of teichuronic acid provides an explanation of the previous erroneous identification of the reducing end residue.

Studies of the lipid composition of the marine bacterium Caulobacter halobacteroides revealed the presence of glycolipid as the predominant lipid constituent. The presence of minor amounts of phospholipid was confirmed with the incorporation of 14C- and 32P-labeled compounds. Other marine caulobacters had similar lipid compositions. Five chromatographically separable glycolipids were detected, two of which were identified as mono- and diglycosyldiglycerides. Glycolipid constituted 90 to 99% of the total extractable lipid based on 14C-acetate incorporation into six marine caulobacter strains. In addition, comparisons were made with the lipid extracts of the nonmarine Caulobacter crescentus and Micrococcus lysodeikticus, which contain substantial amounts of phospholipid. Studies of lipid composition during growth showed the maximum amount of phospholipid during early logarithmic growth (2.9%) with a decrease to 0.3% in the early stationary phase. The finding of a group of organisms in which phospholipid is not a major constituent of the lipid fraction is unique and generates many questions about the lipid requirements for membrane structure and function.

Electron microscopy and microprobe X-ray analysis were used to study metachromatic inclusions of Spirillum itersonii , Corynebacterium diphtheriae, and Micrococcus luteus. In situ metachromatic inclusions were electron dense and contained phosphorus and divalent cations. Metachromatic inclusions isolated by anion-exchange column chromatography and by isoosmolar Metrizamide density gradient centrifugation were similar in composition to in situ inclusions.

The complexity of the genome of Micrococcus radiodurans was determined to be (2.0 +/- 0.3) X 10(9) daltons by DNA renaturation kinetics. The number of genome equivalents of DNA per cell was calculated from the complexity and the content of DNA. A lower limit of four genome equivalents per cell was approached with decreasing growth rate. Thus, no haploid stage appeared to be realized in this organism. The replication time was estimated from the kinetics and amount of residual DNA synthesis after inhibiting initiation of new rounds of replication. From this, the redundancy of terminal genetic markers was calculated to vary with growth rate from four to approximately eight copies per cell. All genetic material, including the least abundant, is thus multiply represented in each cell. The potential significance of the maintenance in each cell of multiple gene copies is discussed in relation to the extreme radiation resistance of M. radiodurans.

Membrane-bound antigens of the respiratory chain of Micrococcus luteus were analyzed by crossed immunoelectrophoresis after growth of the organism in the presence of 59Fe, the flavin adenine dinucleotide-flavin mononucleotide precursor D-[2-14C]riboflavin, or the heme precursor 5-amino-[4-(14)C]levulinic acid. Using zymograms and procedures of selective extraction in conjunction with autoradiography, it was possible to resolve and partially characterize a number of antigens. Succinate dehydrogenase (EC 1.3.99.1) was shown to possess covalently bound flavin and nonheme iron and was possibly present as a complex with cytochrome. Three other dehydrogenases, namely, NADH dehydrogenase, NAD(P)H dehydrogenase (EC 1.6.99.3), and malate dehydrogenase (EC 1.1.1.37), contained flavin in noncovalent linkage, the NAD(P)H dehydrogenase also possessing nonheme iron. Four other discrete antigens (or antigen complexes) containing both iron and heme centers also resolved, as were two minor immunogens possessing iron as the sole detectable prosthetic group.

Walls of the pigmented strain of Micrococcus radiodurans showed several layers in the electron microscope. These layers include an outermost network structure removed by trypsin, a fragile soft layer containing hexagonally packed subunits, and a rigid layer penetrated by numerous holes. The two inner layers were separated by a process of autolysis, trypsin treatment, and gradient centrifugation. The hexagonally packed layer was less dense, pink in color, and it contained carotenoids, lipid, protein, and polysaccharide. The lipid consisted of odd-numbered as well as even-numbered fatty acids, and the polysaccharide contained rhamnose and mannose, but it did not contain heptose. The "holey" layer was white and was composed of a mucopeptide containing glucosamine, muramic acid, and four main amino acids (glutamic acid, alanine, glycine, and l-ornithine, in the ratios of 1:1.7:1.8:1.2, respectively). This layer also contained phosphorus, glucose, and a trace of meso- and ll-diaminopimelic acid. A white mutant, W(1), of M. radiodurans had no pigment or lipid in its walls, but it contained small amounts of the "hexagonal" layer. The holey layer, constituting the bulk of the wall, was similar in morphology and composition to that layer in the pigmented strain. Lysozyme did not remove the lipoprotein-polysaccharide component from the walls of the pigmented strains, and the hexagonally packed structure was not visibly affected, except for change in a minor structure. Most of the mucopeptide layer was solubilized by lysozyme, but a structureless bag-shaped residue was left. This residue contained phosphorus, carbohydrate, and limited amino acids, but it did not contain muramic acid, glucosamine, or ornithine. Aqueous phenol removed a lipoprotein component from strain R(1), which contained limited fatty acids. It also removed meso- and ll-diaminopimelic acid.

An orange carotenoid from Micrococcus roseus was purified by solvent partitioning followed by column and thin-layer chromatography. Absorption spectra, chromatographic mobility, and partition coefficient suggested that the pigment was echinenone (4-keto-beta-carotene). Reduction yielded a pigment with the spectral and polar properties of isocryptoxanthin (4-hydroxy-beta-carotene), the expected product. The orange pigment and its reduction product co-chromatographed with the respective authentic pigments, confirming the original pigment as echinenone. To our knowledge echinenone has not been identified previously as a bacterial pigment.

In addition to canthaxanthin, seven pigment fractions were isolated from Micrococcus roseus. They were purified by solvent partitioning and by column and thin-layer chromatography. Visible absorption spectra, chromatographic behavior, and partition coefficients of the pigments and derivatives prepared from the pigments were used in characterizing them. Both alpha- and beta-carotene derivatives were present. The structure of one pigment was suggested as phoenicoxanthin (3-hydroxy-4,4'-diketo-beta-carotene). Four other pigments were tentatively characterized as a dihydroxy-3,4-dehydro-alpha-carotene, a dihydroxy-alpha-carotene, a diketo-alpha-carotene, and a polyhydroxy-beta-carotene. Two pigments were isolated in trace amounts and could not be characterized. All the pigments studied were isolated as mixtures of cis-trans isomers and all except the diketo-alpha-carotene were isolated as esters from M. roseus. Quantitation of the pigments showed that canthaxanthin (4,4'-diketo-beta-carotene) represented 85% of the pigment recovered from extracts. Three of the other pigments contributed a significant proportion of the remaining pigments, whereas the other four were present in only small amounts. beta-Carotene derivatives comprised 96% and alpha-carotene derivatives 4% of the pigments recovered from extracts.

The organisms studied were those of the family Micrococcaceae which cannot participate in genetic exchange with Micrococcus luteus and those whose biochemical and physiological characteristics appear to bridge the genera Staphylococcus and Micrococcus. The hydrocarbon compositions of M. luteus ATCC 4698 and Micrococcus sp. ATCC 398 were shown to be similar to those previously reported for many M. luteus strains, consisting of isomers of branched monoolefins in the range C25 to C31. However, Micrococcus sp. ATCC 398 differed somewhat by having almost all C29 isomers (approximately 88% of the hydrocarbon composition). Micrococcus spp. ATCC 401 and ATCC 146 and M. roseus strains ATCC 412, ATCC 416, and ATCC 516 contained the same type of hydrocarbon patterns, but the predominant hydrocarbons were within a lower distribution range (C23 to C27), similar to Micrococcus sp. ATCC 533 previously reported. The chromatographic profile and carbon range of the hydrocarbons of an atypical strain designated M. candicans ATCC 8456 differed significantly from the hydrocarbon pattern presented above. The hydrocarbons were identified as branched and normal olefins in the range C16 to C22. Studies of several different strains of staphylococci revealed that these organisms do not contain readily detectable amounts of aliphatic hydrocarbons. The members of the family Micrococcaceae have been divided into two major groups based on the presence or absence of hydrocarbons. With the exception of M. candicans ATCC 8456, this division corresponded to the separation of these organisms according to their deoxyribonucleic acid compositions.

The carotenoid 4'-hydroxyechinenone (4'-hydroxy-beta, beta-carotene-4-one) was isolated from Micrococcus roseus. It is proposed as an intermediate between echinenone and canthaxanthin.

The occurrence of succinic dehydrogenase [succinic:(acceptor) oxidoreductase, EC 1.3.99.1] in membrane fractions of Micrococcus lysodeikticus was investigated. The enzyme could be purified 10-fold, by deoxycholate treatment. Butanol extraction of membranes yielded an active fraction, nonsedimentable at 130,000 x g for 2 hr and altered in its phospholipid content relative to membranes. The activity of the enzyme in particulate preparations was decreased in the presence of competitive inhibitors and by compounds known to react with iron, sulfhydryl groups, and flavine. In this respect, the bacterial succinic dehydrogenase is similar to the enzyme derived from yeast and mammalian sources. In certain membrane fractions, Ca(2+) and Mg(2+) exhibited inhibitory effects whereas Triton X-100 caused activation. The enzyme could also be activated by substrate. In the phenazine reductase assay, incomplete reduction of electron acceptor was observed upon addition of divalent cations and iron binding agents.

Cessation of exponential growth of Micrococcus radiodurans heightens X-radioresistance while either increasing or decreasing the deoxyribonucleic acid-protein ratio.

The phospholipid composition of Micrococcus denitrificans was unusual in that phosphatidyl choline (PC) was a major phospholipid (30.9%). Other phospholipids were phosphatidyl glycerol (PG, 52.4%), phosphatidyl ethanolamine (PE, 5.8%), an unknown phospholipid (5.3%), cardiolipin (CL, 3.2%), phosphatidyl dimethylethanolamine (PDME, 0.9%), phosphatidyl monomethylethanolamine (PMME, 0.6%), phosphatidyl serine (PS, 0.5%), and phosphatidic acid (0.4%). Kinetics of (32)P incorporation suggested that PC was formed by the successive methylations of PE. Pulse-chase experiments with pulses of (32)P or acetate-1-(14)C to exponentially growing cells showed loss of isotopes from PMME, PDME, PS, and CL with biphasic kinetics suggesting the same type of multiple pools of these lipids as proposed in other bacteria. The major phospholipids, PC, PG, and PE, were metabolically stable under these conditions. The fatty acids isolated from the complex lipids were also unusual in being a simple mixture of seven fatty acids with oleic acid representing 86% of the total. Few free fatty acids and no non-extractable fatty acids associated with the cell wall or membrane were found.

Intact cells of Streptococcus faecalis and Micrococcus lysodeikticus were found to have high-frequency electric conductivities of 0.90 and 0.68 mho/m, respectively. These measured values, which reflect movements of ions both within the cytoplasm and within the cell wall space, were only about one-third of those calculated on the basis of determinations of the amounts and types of small ions within the cells. Concentrated suspensions of bacteria with damaged membranes showed similarly large disparities between measured and predicted conductivities, whereas the conductivities of diluted suspensions were about equal to predicted values. Thus, the low mobilities of intracellular ions appeared to be interpretable in terms of the physicochemical behavior of electrolytes in concentrated mixtures of small ions and cell polymers. In contrast to the low measured values for conductivity of intact bacteria, values for intracellular osmolality measured by means of a quantitative plasmolysis technique were higher than expected. For example, the plasmolysis threshold for S. faecalis cells indicated an internal osmolality of about 1.0 osmol/kg, compared with a value of only 0.81 osmol/liter of cell water calculated from a knowledge of the cell content and the distribution of small solutes. In all, our results indicate that most of the small ions within vegetative bacterial cells are free to move in an electric field and that they contribute to cytoplasmic osmolality.

Fatty acids derived from Micrococcus cerificans growing at the expense of odd- and even-carbon normal alkanes were studied. Results demonstrated that cultures grown with a variety of nonhydrocarbon substrates serving as sole carbon and energy source yielded only even-carbon fatty acids. Even-chain alkanes, dodecane through octadecane serving as sole carbon source, resulted in even-carbon fatty acids with direct correlation between carbon number of the major fatty acid species and carbon number of the alkane substrate. Odd-carbon alkanes, undecane through heptadecane serving as sole carbon source, yielded both odd- and even-carbon fatty acids. A transitional shift from even-carbon fatty acids to odd-carbon fatty acids was observed as the carbon number of the alkane substrate increased. Unsaturated fatty acids were found to comprise a significant percentage of all profiles. Analysis of unsaturated fatty acids showed all odd- and even-carbon acids analyzed were Delta(9) monounsaturated fatty acids.

The ribosomal proteins from nine species of prokaryotes have been compared by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The stained gels were scanned spectrophotometrically, the weight and number average molecular weights were calculated, and the detailed distribution of the proteins as a function of molecular weight was determined. By all of these criteria, the ribosomal proteins from all the species closely resembled each other, despite differences in the pattern of protein bands by conventional disc-gel electrophoresis. Therefore, it is suggested that the structural requirements for the assembly of ribosomal subunits have imposed limitations on the evolution of the ribosomal proteins and that their size has been highly conserved.

By use of the staphylolytic enzyme lysostaphin, a method was devised for isolating and purifying highly polymerized deoxyribonucleic acid (DNA) from lysostaphinsusceptible Micrococcaceae. Staphylococcus aureus DNA isolated by this procedure gave an estimated molecular weight of ca. 2 x 10(8) and a residual protein content of 2.3%. The mole percentage of guanine + cytosine (GC) present in the DNA from 21 strains of aerobic Micrococceae was determined by buoyant density in cesium chloride. DNA from 12 biochemically typical members of the genus Staphylococcus gave a mean GC composition of 35.2 +/- 0.5 mole per cent. Four biochemically atypical Staphylococcus strains and one biochemically typical strain of the genus Micrococcus (M. candicans) were found to be susceptible to lysostaphin and gave typical Staphylococcus spp. GC base ratios. One biochemically atypical member of the genus Micrococcus (M. varians) was not susceptible to lysostaphin and gave a typical Micrococcus spp. GC base ratio. Lysostaphin susceptibility is an easy test to perform, and the results of this test appear to correlate with GC base ratio studies of the genera of Micrococcaceae.

The distribution of cellular fatty acids in defined lipid classes was analyzed in Micrococcus cerificans after growth on specified hydrocarbons. Neutral lipid, phospholipid, and cell residue fatty acids were qualitatively and quantitatively determined for M. cerificans grown on nutrient broth, tetradecane (C(14)), pentadecane (C(15)), hexadecane (C(16)), and heptadecane (C(17)), respectively. Percentage of total cellular fatty acid localized in defined lipid classes from cells grown on the above growth substrates was (i) neutral lipid-11.8, 1.81, 7.74, 23.1, and 2%; (ii) phospholipid-74.5, 65, 66.43, 62.1, and 86%; (iii) cell residue lipid-13.5, 33.29, 25.82, 14.78, and 11.9%. Phospholipid fatty acid chain length directly reflected the carbon number of the alkane substrate, with 40, 84, 98, and 77% of the fatty acids being 14, 15, 16, and 17 carbons when cells were grown on C(14), C(15), C(16), and C(17)n-alkanes, respectively. The bound lipids of the cell residue after chloroform-methanol extraction were characterized by 2-hydroxydodecanoic and 2-hydroxytetradecanoic acids plus a broad spectrum of fatty acids ranging from C(10) to C(17) chain length. An increase in total unsaturated fatty acid localized in the phospholipids was noted from cells grown on alkanes greater than 15 carbons long. An extracellular accumulation of free fatty acid (FFA) was demonstrated in hexadecane-grown cultures that was not apparent in non-hydrocarbon-grown cultures. Identification of extracellular FFA demonstrated direct derivation from hexadecane oxidation. Studies supporting inhibition of de novo fatty acid biosynthesis in relationship to extracellular FFA and hexadecane oxidation are described. The ability to alter the fatty acid composition of membrane polar lipids in a predictable manner by the alkane carbon source provides an excellent model system for the investigation of membrane structure-function relationships in M. cerificans.

The amino acid composition and amino acid sequence of the murein (peptidoglycan) of 10 strains of planococci were studied. It is shown that the peptide subunit consists of muramyl-l-alanyl-gamma-d-glutamyl-l-lysyl-d -alanine. The cross-linking of two adjacent peptide subunits is mediated by d-glutamic acid which is bound to the epsilon-amino group of lysine by its gamma-carboxyl group and to the carboxyl group of d-alanine of an adjacent peptide subunit by its amino group. About 20 to 25% of the peptide subunits are not cross-linked. The murein structure of the different species and strains of Micrococcus, Staphylococcus, and Sarcina are compared. It is evident that the murein structure is a very good criterion for grouping the micrococci. In addition, some of these groups are fairly well defined by physiological properties as well as by their guanine + cytosine content of the deoxyribonucleic acid e.g., Micrococcus, Staphylococcus, Planococcus, Sarcina ureae. Other groups, represented by a single or a few strains only, such as M. varians NTCC 7281, M. radiodurans, M. freudenreichii ATCC 407, and M. luteus ATCC 398, need further investigation.

The amino acid composition of ferredoxins from Chlorobium thiosulfatophilum 8327 and Chloropseudomonas ethylicum, like C. thiosulfatophilum Tassajara, resembled ferredoxins from nonphotosynthetic anaerobes rather than Chromatium; the terminal sequences, however, more closely resembled Chromatium ferredoxin.

Analysis by dinitrophenylation techniques revealed the occurrence of significant amounts of glucosamine residues with free amino groups in the peptidoglycan component of cell walls isolated from Bacillus cereus, Bacillus subtilis, and Bacillus megaterium. A close correlation was demonstrated between the content of N-unacetylated glucosamine residues in the peptidoglycan component and the resistance of the cell walls to lysozyme. These lysozyme-resistant cell walls and peptidoglycan were converted into a lysozyme-sensitive form by means of N-acetylation with acetic anhydride. Thus, the occurrence of the N-unacetylated glucosamine residues in the peptidoglycan component accounts for the resistance of these cell walls to lysozyme. The N-unacetylated glucosamine residues were not found in a significant amount in the cell walls of Micrococcus lysodeikticus, Staphylococcus aureus, Streptococcus faecalis, Lactobacillus casei, or Lactobacillus arabinosus.

The utilization of 1-alkenes by Micrococcus cerificans was investigated with respect to characteristic fatty acid profiles resulting from growth at the expense of these substrates. Saturated fatty acids containing even numbers of carbon atoms were produced from 1-dodecene and 1-tetradecene. Unsaturated fatty acids related to the parent alkene were not detected. The fatty acid profile from 1-pentadecene utilization resulted in the identification of 14-pentadecenoic acid, indicating preferential methyl-group attack. Studies with 1-hexadecene and 1-octadecene indicated simultaneous methyl-group and double-bond attack. Omega-Unsaturated fatty acids related to carbon number of parent alkene and odd-carbon fatty acids one carbon less than the substrate molecule were identified. A mechanism involving double bond epoxidation and oxidative cleavage was supported by measuring the release of formaldehyde. It appears that a dichotomous mechanism is functional in the assimilation of higher carbon number alkenes.

Granular structures containing inorganic polyphosphate were found in Micrococcus lysodeikticus. These structures were isolated by fractionation of the bacterial extract obtained by lysing the organisms with lysozyme. The composition of the fraction which was enriched with these structures was found to be: protein, 24%; lipids, 30%; and polyphosphate, 27%. This fraction also contained small amounts of ribonucleic acids, carbohydrate, and polyvalent cations. The effect of different reagents and enzymes on the integrity of the granules was examined. It was noticed that they accumulate in the bacteria during the logarithmic phase of growth but disappear gradually during the stationary phase.