Amino acid sequence of the threonine-containing mureins of coryneform bacteria

In a study of the mureins of coryneform bacteria (Arthrobacter, Brevibacterium, Cellulomonas, Corynebacterium, Erysipelothrix), 21 threonine-containing strains were found. In several of the strains the amino acid and amino sugar composition of the murein was muramic acid (Mur), glucosamine (GlcNH(2)), d-Glu, l-Lys, l-Thr, and Ala in a molar ratio of 1:1:1:1:1:4 or 5, and in several other strains it was Mur, GlcNH(2), d-Glu, l-Lys, l-Thr, Ala, and l-Ser in a molar ratio of 1:1:1:1:1:3:1. The amino acid sequence of the mureins was determined by analyzing the oligopeptides derived from partial acid hydrolysates. It was shown that there were five different murein types. The peptide subunits attached to the muramic acid are the same, namely l-Ala-d-GluNH(2)-l-Lys-d-Ala. In one strain, the alpha-carboxyl group of d-Glu is substituted by d-alanine amide. The interpeptide bridges of the different types consist of the peptides l-Ala-l-Thr-l-Ala, l-Ala-l-Thr, l-Ala-l-Ala-l-Thr, l-Ala-l-Ala-l-Ala-l-Thr, or l-Ala-l-Thr-l-Ser which are bound through their C-termini (l-Ala, l-Thr, l-Ser) to the epsilon-amino group of l-Lys of one peptide subunit and by their N-termini (l-Ala) to the C-terminal d-Ala of an adjacent peptide subunit. Determination of the N- and C-terminal groups in the mureins showed that about 15 to 30% of the interpeptide bridges are not cross-linked.     

Murein type and polysaccharide composition of cell walls from Renibacterium salmoninarum

Abstract The primary structure of the murein of Renibacterium salmoninarum ATCC33209 was determined. It contained lysine in the third position of the peptide subunit, a glycyl-alanine interpeptide bridge between lysine and the d -alanine of adjacent peptide subunits and a d -alanine-amide substitution at the α-carboxyl group of d -glutamic acid in position 2 of the peptide side chain.
Cell walls contained a considerable amount of polysaccharide with galactose as major sugar component. In addition N -acetyl-glucosamine, rhamnose and N -acetyl-fucosamine were detected.     

Neutral Lipids in the Study of Relationships of Members of the Family Micrococcaceae

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.     

Endo-N-acetyl-glucosaminidase from Clostridium perfringens, Lytic for Cell Wall Murein of Gram-Negative Bacteria

An endo-N-acetyl-glucosaminidase which degrades the murein (peptidoglycan) sacculi of the cell walls of Escherichia coli and Spirillum serpens, but not those of Micrococcus lysodeikticus and Sarcina lutea, is present as a contaminant in a "phospholipase C" from Clostridium perfringens. The specificity of enzyme action was elucidated by reduction of liberated glycosidic groups with NaBH(4) and identification of glucosaminol as the reduction product. This finding contradicts previous reports associating cell wall breakdown with specific phospholipase action.     

Deoxyribonucleic acid base composition of some micrococci and sarcinae

The strains designated in this paper asMicrococcus lysodeikticus, M. sodonensis, M. flavus, Sarcina flava, S. pelagia, S. variabilis, S. marginata, S. subflava, S. citrea, S. lutea andStaphylococcus afermentans have similar DNA base compositions. The mole % GC (guanine plus cytosine) contents in DNA of these strains ranged from 71.8 to 73.3 as calculated from the denaturation temperature (Tm). They may be, therefore, closely related. However, at variance with Kocur and Martinec (1962) they do not seem to be identical withMicrococcus luteus (Schroeter 1872) Cohn 1872, because the neotype culture of the latter species has a different content of guanine and cytosine in its DNA (GC=66.3%). Sarcina aurantiaca, Micrococcus dentrificans andM. luteus have a similar DNA base composition. However, they are not identical as they differ from each other in several physiological characters. In the strains designated asStaphylococcus roseus andSarcina erythromyxa the content of GC varies within the range 72–72.8%. These species do not differ from each other physiologically. They form a pink pigment, reduce nitrates, do not hydrolyze casein and gelatin, and do not produce urease. They seem, therefore, to be identical, which confirms the conclusion of Kocur and Martinec (1962) who designated them asMicrococcus roseus Flügge 1886. Micrococcus conglomeratus differs significantly in DNA base composition from almost all strains of the groupM. lysodeikticus—Staphylococcus afermentans, also fromMicrococcus luteus, M. roseus andM. denitrificans. It differs fromSarcina aurantiaca only physiologically.     

Structure of peptidoglycan from Thermus thermophilus HB8.

The composition and structure of peptidoglycan (murein) extracted from the extreme thermophilic eubacterium Thermus thermophilus HB8 are presented. The structure of 29 muropeptides, accounting for more than 85% of total murein, is reported. The basic monomeric subunit consists of N-acetylglucosamine-N-acetylmuramic acid-L-Ala-D-Glu-L-Orn-D-Ala-D-Ala, acylated at the delta-NH2 group of Orn by a Gly-Gly dipeptide. In a significant proportion (about 23%) of total muropeptides, the N-terminal Gly is substituted by a residue of phenylacetic acid. This is the first time phenylacetic acid is described as a component of bacterial murein. Possible implications for murein physiology and biosynthesis are discussed. Murein cross-linking is mediated by D-Ala-Gly-Gly peptide cross-bridges. Glycan chains are apparently terminated by (1-->6) anhydro N-acetylmuramic acid residues. Neither reducing sugars nor murein-bound macromolecules were detected. Murein from T. thermophilus presents an intermediate complexity between those of gram-positive and gram-negative organisms. The murein composition and peptide cross-bridges of T. thermophilus are typical for a gram-positive bacterium. However, the murein content, degree of cross-linkage, and glycan chain length for T. thermophilus are closer to those for gram-negative organisms and could explain the gram-negative character of Thermus spp.     

Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600.

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.     

Synergistic antimicrobial effect of nisin whey permeate and lactic acid on microbes isolated from fish

The antimicrobial activity of a combination of lactic acid and whey permeate fermented by a nisin-producing Lactococcus lactis strain was tested by the agar diffusion method using bacteria isolated from fish as test organisms. Lactic acid inhibited all bacterial strains studied, but nisin whey permeate inhibited Gram-positive bacteria only. The combination was more effective than lactic acid alone against Pseudomonas fluorescens and Staphylococcus hominis isolated from fish, and Pseudomonas aeruginosa ATCC9721 and Micrococcus luteus ATCC9341.     

Molecular characterization of a novel hepcidin (HepcD) from Camelus dromedarius. Synthetic peptide forms exhibit antibacterial activity

Hepcidin is a cysteine‐rich peptide widely characterized in immunological processes and antimicrobial activity in several vertebrate species. Obviously, this hormone plays a central role in the regulation of systemic iron homeostasis. However, its role in camelids' immune response and whether it is involved in antibacterial immunity have not yet been proven. In this study, we characterized the Arabian camel hepcidin nucleotide sequence with an open reading frame of 252 bp encoding an 83‐amino acid preprohepcidin peptide. Eight cysteine key residues conserved in all mammalian hepcidin sequences were identified. The model structure analysis of hepcidin‐25 peptide showed a high homology structure and sequence identity to the human hepcidin. Two different hepcidin‐25 analogs manually synthesized by SPPS shared significant cytotoxic capacity toward the Gram‐negative bacterium Escherichia coli American Type Culture Collection (ATCC) 8739 as well as the Gram‐positive bacteria Bacillus subtilis ATCC 11779 and Staphylococcus aureus ATCC 6538 in vitro. The three disulfide bridges hepcidin analog demonstrated bactericidal activity, against B. subtilis ATCC 11779 and S. aureus ATCC 6538 strains, at the concentration of 15 μM (50 µg/ml) or above at pH 6.2. This result correlates with the revealed structural features suggesting that camel hepcidin is proposed to be involved in antibacterial process of innate immune response. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Murein structure of Acetobacterium woodii

The isolated cell walls of Acetobacterium woodii contain a murein of the crosslinkage type B. d-Orinithinyl residues function as interpeptide bridges between the -carboxyl group of d-glutamic acid and the carboxyl group of the terminal d-analyl residue of an adjacent peptide subunit. The usual l-alanyl residue in position 1 of the peptide subunit is replaced by a l-seryl residue. As yet this murein type was only found in Eubacterium limosum, an organism which was supposed to be related to Acetobacterium because of some metabolic similarities.     

Murein hydrolase (N-acetyl-muramyl-l-alanine amidase) in human serum

An enzyme was identified in human serum which unlike lysozyme cleaved the amide bond between N-acetyl-muramic acid and l-alanine of the peptide side chain of the rigid layer (murein) of Escherichia coli. The N-acetylmuramyl-l-alanine amidase released all of the peptide side chains including those to which the lipoprotein is bound. A portion of the peptide side chains of the Micrococcus lysodeikticus murein was also hydrolysed from the polysaccharide chains. E. coli, M. lysodeikticus, Bacillus subtilis and Staphylococcus aureus were not killed by the amidase. Treatment of E. coli with EDTA or osmotic shock rendered the cells sensitive to the amidase and they were killed. Possible biological functions of the amidase are discussed.The enzyme was separated from lysozyme in human serum. Gel permeation chromatography indicated a molecular weight of the active enzyme of 82,000 while gel electrophoresis in the presence of sodium dodecyl sulfate revealed a molecular weight of 75,000. Thus, the enzyme probably consists of a single polypeptide chain. Incubation with neuraminidase rendered the amidase more basic suggesting the release of sialic acid residues. The modified glycoprotein disclosed an increased activity to murein. Enzyme activity was inhibited by p-chloromercuribenzene sulfonate and ethyleneglycol-bis(2-aminomethyl) tetraacetate (EGTA) at 1 and 0.2 mM concentration, respectively, whereas EDTA up to 5 mM was without effect. The amidase was also inactivated by agents that reduce disulfide bridges.     

A New Broad-Spectrum Peptide Antibiotic Produced by <Emphasis Type="Italic">Bacillus brevis</Emphasis> Strain MH9 Isolated from Margalla Hills of Islamabad,Pakistan

The antimicrobial peptide from a bacterial strain is isolated from soil sample of Margalla Hills of Islamabad, Pakistan. The peptide is found to significantly inhibit the growth of both Gram-positive (Staphylococcus aureus ATCC 6538 and Micrococcus luteus ATCC 10240) and Gram-negative (Escherichia coli ATCC 25922 and Salmonella typhi ATCC 14028) bacteria as compared to gramicidin as standard. The bacterium is identified as Bacillus brevis strain MH9 based on phenotype and phylogenetic analysis. The antibacterial polypeptide was produced optimally at 35 °C after 48 h of growth, precipitated by 50 % ammonium sulphate, and further purified using HPLC. The sequential steps of purification decrease the peptide contents with prominent antibacterial activity. The peptide composed of 11 amino acid was further characterized by FT-IR and NMR. Results suggested that the peptide molecule is a novel antibacterial agent that is effective against both Gram-positive and Gram-negative bacteria. This study may have important implications for new peptide antibiotic that could be a new addition to treat infections.     

Inhibition of Swarming in Proteus spp. by Tannic Acid

Swarming in all 27 strains of Proteus spp. tested was inhibited by the presence of 0.02% (w/v) tannic acid in the nutrient medium. Cells from colonies on this medium were nearly all short forms but were motile and piliated. The swarm-inhibition effect was not reversed by the addition of calcium chloride. The growth of other bacterial species was inhibited to varying extents by tannic acid: Gram positive cocci ( Micrococcus, Sarcina , and Staphylococcus spp.) were particularly sensitive. The relative resistance of Gram negative bacteria and the swarm-inhibition of Proteus spp. could be due to binding of tannic acid to proteins in the outer membrane of the cell wall.     

Secondary structure and phylogeny of Staphylococcus and Micrococcus 5S rRNAs

Nucleotide sequences of 5S rRNAs from four bacteria, Staphylococcus aureus Smith (diffuse), Staphylococcus epidermidis ATCC 14990, Micrococcus luteus ATCC 9341 and Micrococcus luteus ATCC 4698, were determined. The secondary structural models of S. aureus and S. epidermidis sequences showed characteristics of the gram-positive bacterial 5S rRNA (116-N type [H. Hori and S. Osawa, Proc. Natl. Acad. Sci. U.S.A. 76:381-385, 1979]). Those of M. luteus ATCC 9341 and M. luteus ATCC 4698 together with that of Streptomyces griseus (A. Simoncsits, Nucleic Acids Res. 8:4111-4124, 1980) showed intermediary characteristics between the gram-positive and gram-negative (120-N type [H. Hori and S. Osawa, 1979]) 5S rRNAs. This and previous studies revealed that there exist at least three major groups of eubacteria having distinct 5S rRNA and belonging to different stems in the 5S rRNA phylogenic tree.     

X-ray Diffraction Studies of Cell Walls and Peptidoglycans from Gram-positive Bacteria

THE cell walls of Gram-positive bacteria consist principally of a water-insoluble polymer and peptidoglycan (synonyms, murein, mucopeptide, glycosaminopeptide), which in some cases accounts for as much as 90% of the cell wall. After other components (teichoic acid, teichuronic acid, polysaccharide or protein) have been gently removed from the cell walls, peptidoglycan remains as a cell-shaped structure at least 100 Å thick. We report here results of X-ray diffraction observations on whole cell walls and peptidoglycans of Staphylococcus aureus, Bacillus licheniformis and Micrococcus lysodeikticus. Chemical data shows that all the muramic acid residues in the glycan chains of the peptidoglycan of S. aureus are substituted with the peptide L Ala-D GluNH₂-L Lys-D Ala and that there is extensive cross linking by pentaglycine bridges between peptides on adjacent glycan chains^1,3. Such a peptidoglycan might be expected to have an ordered crystalline structure. On the contrary, peptidoglycans of the bacilli, in which the cross linking between peptides is direct and considerably less^4,5 might be expected to have a less ordered structure. The mode of packing of the glycan and peptide moieties has been considered by Kelemen and Rogers⁶. When the glycan chains are stacked in pairs, as in the analogous polysaccharide chitin⁷, the muramic acid residues are orientated in such a way as to allow a three-dimensional structure to be built. If the bulk of the peptides are then arranged in a pseudo β configuration, calculations show that the expected dimensions of the cell wall calculated from the model are of the right order and also such a model allows for the existence of extensive stabilizing hydrogen bonds between adjacent peptide chains.     

Identification of Micrococcaceae in Clinical Bacteriology

The cellular morphology, identifying physiological characteristics, and a key to the human genera of Micrococcaceae are presented with flow charts for identification of aerobic and anaerobic isolates. These flow charts can be amended as desired, depending upon the degree of accuracy desired. Micrococcaceae isolates in a 350-bed private general hospital during a 15-week period are tabulated to show relative numbers of the different genera and species, with their probable relationship to infection or contamination. Only 11 of the 220 Micrococcaceae isolates were not Staphylococcus; no Sarcina or Peptococcus were isolated. Of the Staphylococcus isolates, 61% were S. epidermidis. Almost 18% of the S. aureus isolates were coagulase-negative. Of the S. aureus isolates, 80% of the coagulase-positive isolates were infecting agents, as were 67% of the coagulase-negative S. aureus isolates, compared to only 48% of S. epidermidis isolates. Two of four Gaffkya isolates but only one of seven Micrococcus isolates were infecting agents. If coagulase production is used as the sole criterion for speciation of staphylococci, and Micrococcus is not differentiated from Staphylococcus, the term "coagulase-negative staphylococci" does not differentiate three distinct levels of pathogenicity. Coagulase-negative S. aureus is more virulent than S. epidermidis or Gaffkya, which are more virulent than Micrococcus or Sarcina.     

Die Aminosäuresequenz von 2,4-Diaminobuttersäure enthaltenden Mureinen bei verschiedenen coryneformen Bakterien und Agromyces ramosus

Zusammenfassung Bie 17 Stämmen von coryneformen Organismen wurde 2,4-Diaminobuttersäure als Bestandteil des Mureins gefunden. In 15 Fällen ergab die genauere Analyse die gleiche Aminosäuresequenz, wie sie schon früher von Perkins (1968) bei Corynebacterium insidiosum beschrieben wurde. In diesem Falle ist die L-2,4-Diaminobuttersäure ein Bestandteil der Peptiduntereinheit, während die D-2,4-Diaminobuttersäure die Quervernetzung zwischen dem Glutaminsäurerest und dem C-terminalen Alanin zweier benachbarter Peptiduntereinheiten herstellt. Das Murein gehört demnach zur Gruppe B nach Schleifer u. Kandler (1972). Die -Aminogruppe der L-2,4-Diaminobuttersäure ist in einigen Fällen acetyliert, in anderen Fällen ist sie frei.Das Murein der beiden anderen Stämme unterscheidet sich in seiner Primärstruktur dadurch, daß nur L-2,4-Diaminobuttersäure vorkommt. Im Falle von C. bovis ist wie bei einigen coryneformen pflanzenpathogenen Stämmen die Diaminosäure der Peptiduntereinheit durch Homoserin ersetzt und die Quervernetzung erfolgt durch das Dipeptid -Gly-L-Dab zwischen Glutaminsäure und D-Alanin. Dieses Murein gehört demnach ebenfalls zur Gruppe B. Dagegen ist das Murein von Arthrobacter sp. Ar 22 eine neue Variante der Gruppe A. Die L-2,4-Diaminobuttersäure ist hier ein Glied der Peptiduntereinheit und die Quervernetzung zwischen der -Aminogruppe der 2,4-Diaminobuttersäure und dem D-Alaninrest einer benachbarten Peptiduntereinheit wird durch das Pentapeptid -L-Asp-L-Ala-Gly-L-Ala-L-Ala gebildet. Außerdem ist die Position 1 der Peptiduntereinheit nicht mit L-Alanin, sondern mit Glycin besetzt. Letzteres ist bisher nur bei Mureinen der Gruppe B, aber nicht bei denen der Gruppe A gefunden worden. Ebenfalls neu ist das Vorkommen von L-Asparaginsäure anstelle der bisher gefundenen D-Form.

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The amino acid sequence of 2,4-diaminobutyric acid containing mureins of various coryneform bacteria and Agromyces ramosus

Summary In 17 strains of coryneform bacteria, 2,4-diaminobutyric acid was found to be a component of the murein (peptidoglycan). A detailed analysis showed that 15 strains contain a murein with the same amino acid sequence as that found in Corynebacterium insidiosum by Perkins (1968). In this case the L-2,4-diaminobutyric acid is a component of the peptite subunit while the D-2,4-diaminobutyric acid serves as interpetide bridge between D-glutamatic and the C-terminal D-alanine residue. Therefore this murein belongs to group B according to Schleifer and Kandler (1972). The -amino group of L-2,4-diaminobutyric acid is in some species acetylated, in others free.The murein of the remaining two strains differs by the lack of D-2,4-diaminobutyric acid. Only L-2,4-diaminobutyric acid is found. In the case of C. bovis, the diamino acid of the peptide subunit is replaced by L-homoserine as found in various plant pathogenic coryneform bacteria. The interpeptide bridge consists of the dipeptide -Gly-2,4-Dab. It connects the D-glutamic acid of one peptide subunit with the C-terminal D-alanine residue of an adjacent peptide subunit. Therefore this murein belongs also to group B.The murein of Arthrobacter sp. Ar 22 is a new varition of group A, however. Here the L-2,4-diaminobutyric acid is a component of the peptide subunit. The interpeptide bridge consists of the pentapeptide -L-Asp-L-Ala-Gly-L-Ala-L-Ala. It connects the -amino group of L-2,4-diaminobutyric acid and the C-terminal D-alanine residue of two peptide subunits. Position 1 of the peptide subunit is occupied by glycine instead of L-alanine as found in all the other mureins of group A so far. Another new feature of this murein is the occurrence of the L-form instead of the D-form of aspartic acid.

     

Amino acid sequence of the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis

The complete nucleotide sequence of the gene encoding the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis, and the derived amino acid sequence are presented. The nucleotide sequence of the gene reveals the existence of a typical bacterial signal peptide of 20 amino acid residues which is not found in the mature cytochrome subunit. The gene encoding the cytochrome subunit is preceded by the gene encoding the M subunit. Both genes overlap by 1 bp. The mature cytochrome subunit consists of 336 amino acid residues; 73% of its amino acid sequence was confirmed by protein sequencing work. The mol. wt of the cytochrome subunit including the covalently bound fatty acids and the bound heme groups is 40 500. The internal sequence homology is low, despite the symmetric structure of the cytochrome subunit previously shown by X-ray crystallographic analysis of the intact photosynthetic reaction centre. Sequence homologies to other cytochromes were not found.     

Deoxyribonucleic acid base composition of some members of the Micrococcaceae

Auletta, Angela E. (Catholic University, Washington, D.C.), and E. R. Kennedy. Deoxyribonucleic acid base composition of some members of the Micrococcaceae. J. Bacteriol. 92:28-34. 1966.-Thirty-seven strains from the genera Micrococcus, Staphylococcus, Gaffkya, and Sarcina were examined for deoxyribonucleic acid base composition and biochemical activity. Organisms were tested for production of catalase, coagulase, deoxyribonuclease, oxidase, phosphatase, hydrogen sulfide, indole, and acetoin; nitrate reduction; gelatin, starch, and urea hydrolysis; citrate and ammonium phosphate utilization; NaCl tolerance; growth at 10 and 45 C, and growth in litmus milk. They were tested for production of acid from dextrose and mannitol under anaerobic conditions, and for aerobic production of acid from dextrose, mannitol, lactose, sucrose, raffinose, maltose, xylose, and glycerol. Organisms could be divided into two groups on the basis of guanine-cytosine (GC) content. Group I had an average GC content of 32%, and included all organisms which produced acid from dextrose. Group II had an average GC content of 62%, and included those organisms incapable of producing acid from dextrose under anaerobic conditions. Sarcina ureae had a GC content of 43%.     

Induction of nutritional mutants ofMicrococcus 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.