Peptidoglycan compositions of a new strain of Arthrobacter crystallopoietes during sphere-rod morphogenesis

Arthrobacter crystallopoieties ATCC 15481 was used to isolate a new strain, designated Arthrobacter crystallopoieties EPSR-16, which had a mass doubling time in brain heart infusion broth and in glucose/salts/yeasts extract medium of 30 min compared to 2.40 h for the parent strain in similar media. The growth rates for the new strain and for the parent were close to 12 h in glucose/salts medium. The new strain formed well-separated cocci and diplococci in glucose/salts medium, and upon nutrient shift-up all the cells in the population gradually changed into well-separated rods of regular shape. In the spherical state the cell wall peptidoglycan of the new strain contained lysine and no diaminopimelic acid. A gradual loss in lysine and a gain in diaminopimelic acid occurred during morphogenesis. Diaminopimelic acid became predominant in the cell wall during balanced growth in the rod state.     

Ratio of Teichoic Acid and Peptidoglycan in Cell Walls of Bacillus subtilis Following Spore Germination and During Vegetative Growth

Cell walls were isolated from cells of Bacillus subtilis strain Marburg during synchronous outgrowth of spores, during the two synchronous cell divisions which followed, and at various times during exponential and early stationary growth. The amounts of teichoic acid and peptidoglycan components were determined in each cell wall preparation. The peptidoglycan is composed of hexosamine, alanine, diaminopimelic acid, and glutamic acid. The ratio of these was relatively constant in the cell walls at each stage of growth. The teichoic acid is composed of glycerol, phosphate, glucose, and ester-linked alanine. With the exception of glucose and ester-linked alanine, the ratios of these components were relatively constant throughout the growth cycle. There was a slight increase in the glucose content of the teichoic acid as the cells aged. There was no correlation between the amount of ester-linked alanine and the stage of growth. The ratio of teichoic acid (based upon phosphate content) to peptidoglycan (based upon diaminopimelic acid content) remained at nearly a constant level throughout the growth cycle. The conclusion is presented that these two cell wall polymers are coordinately synthesized during spore outgrowth and throughout the vegetative growth cycle.     

The effect of substitution of diaminopimelic acid by 4-hydroxy-diaminopimelic acid on the synthesis and degradation of murein inEscherichia coli 173-25

Defects in the formation of the septum and gradually autolysis of cells occur when the dapdependent mutant ofEscherichia coli is grown in a medium with 4-hydroxy-diaminopimelic acid. When the culture grown in the presence of the labelled analogue is supplemented with the non-radioactive diaminopimelic acid a portion of the TCA-soluble radioactivity is released from the cells during 20 min after the addition of diaminopimelic acid. During this time interval the elongated forms formed in the presence of the analogue divide, however, only on the condition that the above forms are not irreversibly damaged. The increased concentration of the analogue in the medium substantially suppresses the irregularities in the development of the septum as well as the degradation of analogue containing cell wall. However, the growth rate in the presence of the analogue is always slightly lower than that in the presence of diaminopimelic acid. The cell wall pulse-labelled with diaminopimelic acid or its analogue for a time interval shorter than 1/4 of the generation time exhibits the same or only slightly higher rate of turnover than the wall labelled with dap during two generations. It can be assumed that 4-hydroxydiaminopimelic acid is probably utilized less effectively for the synthesis of murein than diaminopimelic acid. However, its incorporation into the wall does not result in pronounced damage of the cell.     

Effect of Hydroxylysine on the Biosynthesis of Lysine in Streptococcus faecalis

We were able to show that two lysine-independent mutants of Streptococcus faecalis ATCC 8043 contained the enzymes for the usual bacterial pathway for lysine biosynthesis. Because of this synthetic capacity, one mutant, the Lys(+)OHLys(s) strain, could not grow in the presence of hydroxylysine without a lysine supplement. Both lysine and hydroxylysine inhibited the first enzyme of the pathway, aspartokinase. Unlike the Escherichia coli enzyme, S. faecalis dihydrodipicolinic acid synthetase was not inhibited by either lysine or hydroxylysine. Both amino acids caused the repression of dihydrodipicolinic acid synthetase and diaminopimelic acid decarboxylase. Failure of Lys(+)OHLys(s) strain to grow in hydroxylysine-supplemented medium was caused by the mimicking of lysine control by hydroxylysine. Because hydroxylysine could not completely substitute for lysine and lysine could not be synthesized, the organism did not grow. We tested three lysine analogues and found that they prevented lysine-depletion lysis in the Lsy(-)OHLys(s) strain, as did hydroxylysine. Each analogue seemed to support cell wall mucopeptide synthesis, although ornithine did not. Preliminary data indicated that these analogues like hydroxylysine, have growth-inhibitory action on the Lys(+)OHLys(s) strain, but not the Lys(+)OHLys(r) strain. The nature of the specificity of the lysine-adding enzyme for cell wall mucopeptide synthesis is discussed.     

一株转化淀粉或麦芽寡糖生成海藻糖的菌株D-97鉴定

由东北大田采集的土样中筛选到菌株D-97,该菌株胞内酶可以利用淀粉或麦芽寡糖合成海藻糖。通过生理、形态、结构特征分析及16SrDNA基因全序列与参比菌株的序列比较,菌株D-97与食尼古丁节杆菌的16SrDNA序列同源性高达97．98％,故将该菌株命名为食尼古丁节杆菌D-97（Arthrobacter nicotinovorus D-97)。我们还将D-97菌株与日本林原公司的海藻糖生产苗——节杆菌Q36的有关生理生化特征进行了比较。     

Chemically defined media for the cultivation of Naegleria: pathogenic and high temperature tolerant species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCC 30100, ATCC 30863, and ATCC 30896) and two strains of N. lovaniensis (ATCC 30467 and ATCC 30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCC 30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysin for ATCC 30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCC 30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCC 30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B12, nicotinamide, and Ca pantothenate) was required.     

Aspartate kinase-independent lysine synthesis in an extremely thermophilic bacterium, Thermus thermophilus: lysine is synthesized via alpha-aminoadipic acid not via diaminopimelic acid

An aspartate kinase-deficient mutant of Thermus thermophilus, AK001, was constructed. The mutant strain did not grow in a minimal medium, suggesting that T. thermophilus contains a single aspartate kinase. Growth of the mutant strain was restored by addition of both threonine and methionine, while addition of lysine had no detectable effect on growth. To further elucidate the lysine biosynthetic pathway in T. thermophilus, lysine auxotrophic mutants of T. thermophilus were obtained by chemical mutagenesis. For all lysine auxotrophic mutants, growth in a minimal medium was not restored by addition of diaminopimelic acid, whereas growth of two mutants was restored by addition of alpha-aminoadipic acid, a precursor of lysine in biosynthetic pathways of yeast and fungi. A BamHI fragment of 4.34 kb which complemented the lysine auxotrophy of a mutant was cloned. Determination of the nucleotide sequence suggested the presence of homoaconitate hydratase genes, termed hacA and hacB, which could encode large and small subunits of homoaconitate hydratase, in the cloned fragment. Disruption of the chromosomal copy of hacA yielded mutants showing lysine auxotrophy which was restored by addition of alpha-aminoadipic acid or alpha-ketoadipic acid. All of these results indicated that in T. thermophilus, lysine was not synthesized via the diaminopimelic acid pathway, believed to be common to all bacteria, but via a pathway using alpha-aminoadipic acid as a biosynthetic intermediate.     

Control of diaminopimelate decarboxylase by L-lysine during growth and sporulation of Bacilluscereus

l-Lysine caused repression of diaminopimelate decarboxylase synthesis in Bacillus cereus when grown in either a minimal defined medium (CDGS medium) or a complex defined medium (a modified lysine assay medium). When cells were grown in either of the two media, variations in the specific activity of the enzyme as a function of time were found to be correlated with the intracellular lysine pool size during growth. From all of the data presented, it seems reasonable to conclude that during growth the synthesis of diaminopimelate decarboxylase is probably regulated by the intracellular lysine pool size. The relationship between lysine pool concentration and the specific activity of the enzyme did not occur in sporulating cells. The specific activity of diaminopimelate decarboxylase started to decrease at the end of exponential growth and continued to decline until it became nondetectable at the time of dipicolinic acid synthesis and development of spore refractility. Throughout this time, the intracellular lysine pool size remained below that which allowed derepression of enzyme synthesis during exponential growth. The mechanism(s) responsible for the observed decrease in the specific activity of the enzyme at the end of exponential growth is unknown. A threefold rise in the intracellular diaminopimelic acid concentration occurred when there was little or no detectable enzyme activity at the time of dipicolinic acid synthesis. This accumulation of diaminopimelic acid may exert positive control on the synthesis of spore peptidoglycan, the major component of the spore cortex.     

Cell wall composition in Corynebacterium bovis and some other corynebacteria

The cell wall compositions of two strains of Corynebacterium bovis were found to differ: one contained lysine, rhamnose, mannose, and glucose, the other meso-alpha, epsilon, diaminopimelic acid (DAP), arabinose, galactose, and mannose. The walls of a strain of C. nephridii were characterized by l-DAP and galactose. Those of a strain of C. paurometabolum and of two strains of "lipophilic diphtheroids" contained meso-DAP, arabinose, galactose, and mannose as did walls of a reference strain of C. xerosis. The results are discussed in relation to the taxonomy of the organisms examined.     

Degradation and turnover of bacterial cell wall mucopeptides in growing bacteria

The mucopeptide layer of the cell wall ofBacillus megaterium is broken down into separate components during growth of the cells. The released diaminopimelic acid is partly decarboxylated to lysine, which is incorporated in the proteins and partly used for cell wall resynthesis. The smaller portion of the degraded mucopeptide is released into the medium in the form of non-utilized fragments. The rate of the mucopeptide turnover is a function of the rate of growth of the culture. About 15–20% of the rigid layer of the cell wall is degraded during on cell division. The sensitivity ofBacillus megaterium to lysozyme and the rate of its conversion to protoplasts is also proportionate to the rate of growth of the culture. There is no measurable mucopeptide turnover in non-growing cells, either in the stationary phase of the culture or in starvation in nitrogen-free medium. The resistance of the cell wall to lysozyme also increases during the stationary phase. The rigid component of the cell wall is probably also broken down during growth ofBacillus cereus andEscherichia coli cultures.     

Chemical composition and serological analysis of the cell wall of Peptostreptococcus

Bahn, Arthur N. (Northwestern University, Chicago, Ill.), Patrick C. Y. Kung, and James A. Hayashi. Chemical composition and serological analysis of the cell wall of Peptostreptococcus. J. Bacteriol. 91:1672-1676. 1966.-Chemical and serological analyses were made of the cell wall of Peptostreptococcus to characterize taxonomically this genus of anaerobic streptococci. Cell wall hydrolysates of P. putridus strains 06 and 85, P. intermedius strains 11 and 87, and P. elsdenii strain B-159 were prepared, and the cell wall sugars were measured quantitatively by paper chromatography. Strain 85 contained only glucose, whereas strain 06 contained 93% glucose and 7% mannose. Strain 87 contained only rhamnose, and strain 11 contained approximately equal amounts of glucose and rhamnose. Strain B-159 differed from all the other strains in having a low (3.1%) content of total carbohydrate, consisting of rhamnose, galactose, and glucose. Quantitative amino acid analyses showed that the major amino compounds present in the cell wall were glutamic and aspartic acids, alanine, lysine, muramic acid, glucosamine, and galactosamine. Strains 06 and 85 possessed this complement of amino compounds, but strains 11 and 87 had relatively little aspartic acid. Strain B-159 was markedly different in having a high content of glycine and diaminopimelic acid, with only traces of lysine; it was the only strain in which teichoic acid was found. Serological analyses were made with the use of cell wall extracts as antigenic material and with homologous antisera, as well as streptococcal group antisera for groups A through S. The only strong agglutination was obtained between strain 87 antigen and group C antisera; weak agglutination was obtained with 87 against N, O, and K, and between strain 11 and groups E and F. All other antisera gave negative reactions. It is concluded that strain B-159 does not belong to the genus Peptostreptococcus, that strains 06 and 85 are members of P. putridus, and that strains 11 and 87 may be members of two different genera.     

Regulation of lysine and dipicolinic acid biosynthesis in Bacillus brevis ATCC 10068: significance of derepression of the enzymes during the change from vegetative growth to sporulation

Lysine biosynthetic pathway enzymes of Bacillus brevis ATCC 1068 were studied as a function of stage of development (growth and sporulation). The synthesis of aspartic-2-eemialdehyde dehydrogenase (ASA-dehydrogenase), dihydrodipicolinate synthase (DHDPA-synthase), DHPA-reductase and diaminopimelate decarboxylase (DAP-decarboxylase) was found not to be co-regulated, since lysine was not a co-repressor for these enzymes. Unlike the aspartokinase isoenzymes, the other enzymes of the lysine pathway were not derepressed in thiosine-resistant, lysine-excreting mutants. Thus, the aspartokinase isoenzymes were the key enzymes during growth and regulation of lysine biosynthesis through restriction of l-ASA synthesis via feedback control by lysine on the aspartokinases was therefore suggested.In contrast to other Bacillus species, the levels of the lysine biosynthetic pathway enzymes of strain ATCC 10068 were not derepressed during the change from vegetative growth to sporulation. Two control mechanisms, enabling the observed preferential channelling of carbon for the synthesis of spore-specific diaminopimelic acid (DAP) and dipicolinic acid (DPA) were a) loss of DAP-decarboxylase, b) inhibition of DHDPA-reductase by DPA. Increase in the level of the DAP pool during sporulation, as a consequence of the loss of DAP-decarboxylase, and its relevance to the non-enzymatic formation of DPA has been discussed.Abbreviations l-ASA l-aspartic-2-semialdehyde - DAP diaminopimelic acid - DPA dipicolinic acid - DHDPA dihydrodipicolinate - AGM aspargine-glycerol medium - PY peptone-yeast extract - NB+NSM nutrient broth plus nutrient sporulation medium     

Biochemical and phylogenetic analyses of psychrophilic isolates belonging to the Arthrobacter subgroup and description of Arthrobacter psychrolactophilus, sp. nov.

During our work on psychrophilic microorganisms we obtained a large collection of new isolates. In order to identify six of these, we examined their growth properties, cell wall compositions, and their 16S rRNA gene sequences. The results showed that all of the isolates are gram-positive, aerobic, contain lysine in their cell walls, and belong to the high mol% G+C Arthrobacter subgroup. Phylogenetic analysis of the 16S rRNA genes grouped five isolates obtained from a small geographical region into a monophyletic clade. Isolate B7 had a 16S rRNA sequence that was 94.3% similar to that of Arthrobacter polychromogenes and 94.4% similar to that of Arthrobacter oxydans. Primary characteristics that distinguish isolate B7 from the Arthrobacter type strain (Arthrobacter globiformis) and A. polychromogenes include lack of growth at 37 degrees C, growth at 0-5 degrees C, the ability to use lactose as a sole carbon source, and the absence of blue pigments. Because of these differences, isolate B7 was chosen as a type strain representing a new Arthrobacter species, Arthrobacter psychrolactophilus. The sixth isolate, LV7, differed from the other five because it did not have the rod/ coccus morphological cycle and was most closely related to Arthrobacter agilis.     

Chemically Defined Media for the Cultivation of Naegleria: Pathogenic and High Temperature Tolerant Species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCCr?30100, ATCCr?30863, and ATCCr?30896) and two strains of N. lovaniensis (ATCCr?30467 and ATCCr?30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCCr?30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysine for ATCCr?30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCCr?30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCCr?30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B₁₂, nicotinamide, and Ca pantothenate) was required.     

Effects of lemongrass oil on the morphological characteristics and peptidoglycan synthesis of Escherichia coli cells

The antibacterial effect of lemongrass oil, obtained from the aerial part of Cymbopogon citratus, on cells of Escherichia coli was investigated by electron microscopy and by measuring cell wall formation. Two strains of E. coli K-12 were used, one of which required diaminopimelic acid in the growth medium for its murein formation. Lemongrass oil was found to elicit morphological changes like filamentation, inhibition of septum formation, spheroplast formation, production of 'blisters', 'bulges' or mesosomes, as well as lysis and development of abnormally shaped cells. The incorporation of radioactively labelled diaminopimelic acid into the cell wall murein of strain W7, was inhibited by lemongrass oil in a dose dependent way. The sequence of changes induced by lemongrass oil on bacterial cell morphology and also its interference with murein synthesis in E. coli cells were interpreted to involve the penicillin binding proteins PBP 2 and PBP 3.     

Analysis of peptidoglycan as a means in diagnostics of the genusActinomyces

Analysis of the peptidoglycan component of the cell wall was used as a means for identification of the genusActinomyces. The peptidoglycan ofActinomyces meyeri was of the lysine type, whereas that ofCorynebacterium granulosum isolated from the same infectious material contained diaminopimelic acid. Analysis according to Park and Hancock was found to be sufficient for taxonomic purposes. In samples containing both diaminopimelic acid and lysine the presence of lysine indicated contamination of the wall with residues of proteins. Dedicated to the 60th birthday of Dr. L. Bíla, Director of the Thomayer Hospital     

l-threonine production by polyauxotrophic mutants ofArthrobacter globiformis

A number of methionine, methionine+lysine-, and (methionine+lysine+isoleucine)-auxotrophic mutants producing threonine have been isolated from a glutamate-producing strain ofArthrobacter globiformis by a three-step mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine. The best double mutant ML24 requiring methionine and lysine for growth produced 3.2 gl-threonine per L in the synthetic Alföldi medium (200 mmol/L) glucose, 80 mmol/L ammonium nitrate) supplemented with 5 μg biotin per L and optimum (0.5 mmol/L) methionine and lysine concentrations.     

耐铵型产琥珀酸放线杆菌的选育及铵离子对其生长代谢的影响

经硫酸二乙酯(DES)诱变,在含61～242mmol/LNH4+梯度平板中,筛选到一株耐铵型突变株YZ25,该菌株在含121mmol/LNH4+发酵培养基中,琥珀酸产量达32.68g/L,转化率为65.4%,比出发菌提高了180.5%。进一步考察了不同形态铵盐对YZ25生长的影响,结果表明添加少量铵盐能够提高突变菌的生长速率,但当超过一定量后菌株生长受到抑制,不同铵盐对菌株的抑制程度不同,硫酸铵、碳酸氢铵、氯化铵和硝酸铵对突变株YZ25的半抑制浓度分别为:215mmol/L、265mmol/L、235mmol/L、210mmol/L。为了考察铵离子对YZ25发酵产琥珀酸的影响过程,在3.0L发酵罐以氨水作为pH的调控剂发酵,结果表明在稳定期前菌株生长基本不受铵离子抑制,生物量能够达到正常水平,但是进入稳定期后铵离子抑制作用越来越明显,导致菌株生长提前结束,耗糖不完全,产酸受阻。最后结合产琥珀酸放线杆菌Actinobacillus succinogenes代谢途径分析了铵离子对菌株抑制作用的机理。     

Influence of antibiotics and trace salts on lysine production by arthrobacter globiformis

A hydrocarbon utilizing strain of Arthrobacter globiformis Lb isolated from local soil has been found to yield lysine 3.4 g l^?1, keeping the medium optimal for pH, C- and N-sources. Addition of antibiotics and micronutrients to that optimal media stimulated cell growth and enhanced lysine yield.     

Fermentation products, amino acid utilization, maintenance energies and growth yields for the fibrillar Streptococcus salivarius HB and a non-fibrillar mutant HB-B grown in continuous culture under glucose limitation

The fibrillar strain Streptococcus salivarius HB and a non-fibrillar mutant, strain HB-B, were grown in a defined medium under glucose limitation in a chemostat. Fermentation balances were produced for both strains in batch culture and at growth rates between 0.1/h and 1.1/h. In batch culture both strains fermented glucose to lactate, but in continuous culture glucose was fermented to formate, acetate and ethanol with increasing amounts of lactate as the growth rate was increased. Lactate never became the major fermentation product even at the highest growth rate. Amino acid analysis showed that only lysine was more than 50% utilized, while proline and tyrosine showed net production. The non-fibrillar strain HB-B showed, in general, a reduced utilization of amino acids compared with the fibrillar strain HB. Calculated growth yields and maintenance energies for the two strains showed that there was a reduction in the true growth yield and the maintenance energy coefficient of the non-fibrillar strain HB-B when compared with the fibrillar strain HB. The increase in the maintenance energy of the fibrillar strain HB (1.382 mmol/g/h) when compared with the non-fibrillar strain HB-B (0.546 mmol/g/h) of 153% is proposed to be the energy required for the maintenance of the fibrillar surface of the cell.