The uptake of amino acids from a chemically defined medium byFusobacterium species

The uptake of amino acids from a chemically defined medium was determined for various species ofFusobacterium. Reference strains utilized a wider range and higher levels of amino acids than clinical isolates. Among the acidic and basic amino acids, arginine, histidine, and glutamate were used by most species. In general, nonpolar neutral amino acids such as alanine, valine, leucine, isoleucine, proline, and methionine were poorly utilized. Apart from glycine and tyrosine, the neutral but polar amino acids such as serine, cysteine, and asparagine were incorporated at significantly high levels. ThusFusobacterium species, unlike several Gram-negative anaerobic bacteria, have the capacity to utilize both free amino acids and peptides as energy sources and may partly account for the wide distribution of these species in areas of tissue degradation.     

Purification and structure analysis of mycolic acids in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum is widely used for producing amino acids. Mycolic acids, the major components in the cell wall of C. glutamicum might be closely related to the secretion of amino acids. In this study, mycolic acids were extracted from 5 strains of C. glutamicum, including ATCC 13032, ATCC 13869, ATCC 14067, L-isoleucine producing strain IWJ-1, and L-valine producing strain VWJ-1. Structures of these mycolic acids were analyzed using thin layer chromatography and electrospray ionization mass spectrometry. More than twenty molecular species of mycolic acid were observed in all 5 strains. They differ in the length (20–40 carbons) and saturation (0–3 double bonds) of their constituent fatty acids. The dominant species of mycolic acid in every strain was different, but their two hydrocarbon chains were similar in length (14–18 carbons), and the meromycolate chain usually contained double bonds. As the growth temperature of cells increased from 30°C to 34°C, the proportion of mycolic acid species containing unsaturated and shorter hydrocarbon chains increased. These results provide new information on mycolic acids in C. glutamicum, and could be useful for modifying the cell wall to increase the production of amino acids.     

Taxonomic evaluation of amino acid metabolism inLegionella

Legionella utilizes amino acids as the sole source of carbon and energy. We have examined 62 strains of nine species to determine whether their amino acid metabolism correlated with species definition. Neither definitive species differences nor serogroup differences within the speciesLegionella pneumophila were found. Greatest metabolic activity of all strains was observed with glutamate, alanine, aspartate, and proline; anaerobically, the greatest reduction of methylene blue was obtained with glutamate, aspartate, histidine, and lysine. Within the genus, two major groups were observed: those species that showed little endogenous activity and metabolized a select group of amino acids, and those species that showed strong endogenous activity and appeared to actively metabolize virtually all the amino acids.     

The influence of peptides on the uptake of amino acids inFusobacterium; predicted interactions withPorphyromonas gingivalis

A study of the uptake of amino acids and its influence by a peptide source was carried out withFusobacterium varium as a convenient representative of the genus. Reference strains and a clinical isolate had similar amino acid uptake profiles, but most amino acids were incorporated at lower concentrations by the latter. In general, high levels of serine, asparagine, glutamate, cysteine, and arginine were incorporated by all species. Histidine, lysine, threonine, and aspartate were taken up at lower levels, whereas the nonpolar neutral amino acids such as alanine, valine, leucine, isoleucine, glycine, proline, phenylalanine, and methionine were poorly metabolized. Yeast extract, as a source of peptides, stimulated the uptake of several amino acids such as histidine and glutamate, whereas others such as methionine, threonine, and asparagine were repressed. The incorporation of some amino acids such as aspartate, ornithine, lysine, and arginine was unaffected by the presence of peptides. Equimolar nitrogen concentrations of amino acids or ammonia could not replace the peptide requirement, emphasizing the importance of peptides as an energy source. The limited capacity ofFusobacterium spp. to hydrolyze proteins increased approximately 30% in the presence of the proteolytic species,Porphyromonas gingivalis, and may represent one bacterial interaction in which peptides may become available toFusobacterium species in vivo.     

Variations in the content of free and bound amino acids during dormancy and the first cell cycle in Helianthus tuberosus tuber explants

Abstract

Qualitative and quantitative analysis of free and bound amino acids and amides during dormancy and the most important phases of the first cell cycle was carried out in tubers of Helianthus tuberosus.

In the dormant tuber arginine was confirmed to be the most abundant amino acid. A high amount of asparagine was also present; on the contrary glutamine was found in very low concentrations. During the progression of dormancy, all the free amino acids and amides declined while aspartic and glutamic acid increased.

During the G₁ phase of the first cell cycle induced by 2,4-D, all the free amino acids and amides decreased with the exception of glutamic acid.

At 18, 20, 24 h of activation with 2,4-D, corresponding to the S phase and the beginning of mitosis, bound amino acids were also determined. In these phases of the cell cycle they increased reaching a maximum at 20 h; on the other hand the free amino acid and amide content, especially aspartic acid, asparagine and arginine, decreased with the exception of glutamic acid, alanine and phenylalanine.     

Studies on utilization of 2-ketoglutarate,glutamate and other amino acids by the unicellular alga Cyanidium caldarium

Two strains of Cyanidium caldarium which possess different biochemical and nutritional characteristics were examined with respect to their ability to utilize amino acids or 2-ketoglutarate as substrates.One strain utilizes alanine, glutamate or aspartate as nitrogen sources, and glutamate, alanine or 2-ketoglutarate as carbon and energy sources for growth in the dark. The growth rate in the dark on 2-ketoglutarate is almost twice as high or higher than that on glutamate or alanine. During growth or incubation of this alga on amino acids, large amounts of ammonia are formed; however, ammonia formation is strongly inhibited by 2-ketoglutarate. The capacity of the alga to form ammonia from amino acids is inducible and develops fully only when the cells are grown or incubated in the presence of glutamate.By contrast, the other strain of Cyanidium caldarium cannot utilize alanine or aspartate as nitrogen sources. It utilizes glutamate only very poorly and does not excrete ammonia into the external medium. This strain is unable to utilize amino acids or 2-ketoglutarate as carbon and energy sources for heterotrophic growth.Cell-free extracts were tested for the occurrence of enzymes which could account for amino acid metabolism and ammonia formation.     

Genetic Changes of Regulatory Mechanisms Occurred in Leucine and Valine Producing Mutants Derived from Brevibacterium lactofermentum 2256

The regulatory mechanisms in branched-chain amino acid synthesis were compared between 2-thiazolealanine (2-TA) resistant l-leucine and l-valine producing mutants and the 2-TA sensitive original strains of Brevibacterium lactofermentum 2256.

In the original strains, sensitive to 2-TA, α-isopropylmalate (IPM) synthetase, the initial enzyme specific for l-leucine synthesis, is sensitive to feedback inhibition and to repression by l-leucine, and α-acetohydroxy acid (AHA) synthetase, the common initial enzyme for synthesis of l-isoleucine, l-valine as well as l-leucine, is sensitive to feedback inhibition by each one of these amino acids, and to repression by them all. In strain No. 218, a typical l-leucine producer resistant to 2-TA, IPM synthetase was found to be markedly desensitized and derepressed, and AHA synthetase remained unaltered. On the contrary, in strain No. 333, l-valine producer resistant to 2-TA, AHA synthetase was found to be desensitized and partially derepressed, and IPM synthetase remained unaltered.

The genetic alteration of these regulatory mechanisms was discussed in connection with the accumulation pattern of amino acids.     

Optimized syntheses of Fmoc azido amino acids for the preparation of azidopeptides

The rise of CuI‐catalyzed click chemistry has initiated an increased demand for azido and alkyne derivatives of amino acid as precursors for the synthesis of clicked peptides. However, the use of azido and alkyne amino acids in peptide chemistry is complicated by their high cost. For this reason, we investigated the possibility of the in‐house preparation of a set of five Fmoc azido amino acids: β‐azido l ‐alanine and d ‐alanine, γ‐azido l ‐homoalanine, δ‐azido l ‐ornithine and ω‐azido l ‐lysine. We investigated several reaction pathways described in the literature, suggested several improvements and proposed several alternative routes for the synthesis of these compounds in high purity. Here, we demonstrate that multigram quantities of these Fmoc azido amino acids can be prepared within a week or two and at user‐friendly costs. We also incorporated these azido amino acids into several model tripeptides, and we observed the formation of a new elimination product of the azido moiety upon conditions of prolonged couplings with 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate/DIPEA. We hope that our detailed synthetic protocols will inspire some peptide chemists to prepare these Fmoc azido acids in their laboratories and will assist them in avoiding the too extensive costs of azidopeptide syntheses. Experimental procedures and/or analytical data for compounds 3 – 5 , 20 , 25 , 26 , 30 and 43 – 47 are provided in the supporting information. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.     

Screening and Identification of the Phospholipase D-producing Streptomyces

About 2,000 strains of streptomycetes and 200 strains of molds were tested for egg yolk-degrading activity. A strain isolated from soil was found to produce the strong activity in the culture medium.

The egg yolk-degrading substance was found to be phospholipase D and the producing strain was identified as Streptomyces hachijoensis. It was found out that the strains producing egg yolk-degrading substance appeared with high frequency in whirl-forming species in genus Streptomyces.     

Studies on the Amino Acids Fermentation of Pentose Materials

Fundamental studies on the cultural conditions for the amino acids production from pentoses and hexoses employing a strain of our new isolates named Brevibacterium pentoso-aminoacidicum nov. sp. were carried out.

As a result of these experiments, it became possible to obtain about 30% of alanine and 10% of L-glutamic acid based on xylose, and alanine from glucose with a yield of about 40% in the proper conditions.     

Species distinctions among closely related strains of Eustigmatophyceae (Stramenopiles) emphasizing ITS2 sequence-structure data: Eustigmatos and Vischeria

ABSTRACT

There is an increasing interest in the Eustigmatophyceae, a class of stramenopile microalgae, because they offer a variety of high-value health-beneficial compounds, e.g. polyunsaturated fatty acids (PUFAs), while concomitantly producing high biomass. Clarification of the taxonomy of these organisms at the species level is important in order to achieve reproducible results and constant yields of valuable compounds in their exploitation. Here the distinction of the, so far exclusively, morphologically defined species of the genera Eustigmatos and Vischeria was tested. Distinctions inferred from almost full 18S and ITS2 rRNA as well as plastid-encoded rbcL gene sequences were evaluated following a morphological investigation. The ITS2 secondary-structure-based phylogenies separated independent lineages (species) with long internal branches. This recommends ITS2 as a promising marker for a DNA metabarcoding approach (culture-independent biodiversity assessment). In contrast, the 18S V4 region which is commonly used in metabarcoding was almost invariant, whereas the almost full length sequences distinguished eight groups/types of strains. Monophyly of the species was supported by shared ITS2 secondary structure features, making them distinct from other eustigmatophyte lineages in concordance with phylogenetic analyses. No groups of strains were congruently supported by all three markers. Consequently, the previous distinction of two genera on the basis of morphology cannot be retained and the species should be accommodated in a single genus, Vischeria. Taxonomic changes among the species with the definition of epitypes, on the basis of cryopreserved strains, are recommended. Two findings point to a more complex evolutionary history of the species. The rbcL and nuclear markers resulted in disparate groupings of strains. In three species divergent intragenomic ITS2 paralogues were revealed. Therefore, a still broader taxon sampling, in conjunction with a deep sequencing approach, is needed for a more comprehensive understanding of the complex evolution of eustigmatophyte species.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

We have found that some straight-chained α-amino acids are converted by yeast to the alcohols with correspondingly longer carbon chains in the alcoholic fermentation contrary to F. Ehrlich’s scheme, i.e., isobutyl alcohol from alanine and active amyl alcohol from α-amino-n-butyric acid or threonine.

In this report, we confirmed this fact in the alcoholic fermentation of many aliphatic amino acids by 2 yeast strains using gas chromatography. Moreover, n-propyl alcohol was proved to come from α-amino-n-butyric acid or threonine. Small quantities of n-propyl, isobutyl, active amyl and isoamyl alcohols were found in all the fermented solutions. There was some difference in the composition of higher alcohols of the alcoholic solutions fermented by different yeasts.     

Application of the profiles of amino acid utilization as the sole carbon and nitrogen sources for pseudomonad taxonomy

Profiles of utilization of 20 amino acids were determined for 218 strains of 10 Pseudomonas species (P. aeruginosa, P. putida, P. fluorescens, P. stutzeri, P. alcaligenes, P. pseudoalcaligenes, P. luteola, P. oryzihabitans, P. mendocina, and P. chlororaphis), including the type strains of these species. Amino acid utilization was determined on minimal salt agar with an amino acid as the sole source of nitrogen and carbon. All the investigated pseudomonad species had species-specific profiles of amino acid utilization. For the type strains of all species, Jaccard’s coefficients of community were different (Sj = 0.31–0.82), in accordance with the interspecies differences. The similarity between the intraspecies variants of the profiles and that of the type strain was high; for 98% of P. aeruginosa strains, Sj = 0.85−1.0; for 100% of P. putida, P. stutzeri, and P. alcaligenes strains, Sj was 0.87–1.0, 0.90, and 0.86–1.0, respectively. Only for P. fluorescens and P. pseudoalcaligenes were low Sj of the intraspecies profiles revealed, in accordance with the known phenotypic heterogeneity of these species. These results agree with the known pseudomonad classification, and the method is therefore valid for identification of known species and for determination of the new members of the genus Pseudomonas.     

Studies on Amino Acid Sequence Determination from N-Terminal of Peptide by Methylthiohydantoin

Methylisothiocyanate reacts with the amino group of amino acid in alkaline solution to give methylthiocarbamyl amino acid. This is converted to the methylthiohydantoin derivative (MTH-amino acid) by subsequent acidification.

Gas chromatographical identification of 20 amino acids were examined by making MTH-amino acids. Among them, ten amino acids (glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, serine and threonine) were successfully identified. Aspartic acid and glutamic acid could be identified as the methyl esters of the MTH-amino acids.     

CHEMOSENSORY CAPABILITIES IN THE PHAGOTROPHIC DINOFLAGELLATE GYMNODINIUM FUNGIFORME11

The non-photosynthetic, phagotrophic dinoflagellate, Gymnodinium fungiforme Anissimova is attracted to a variety of amino acids and other organic compounds. Glycine, taurine and serine attracted the dinoflagellates at a threshold detection level of 10^?8 M fallowed by dextrose (10^?7 M) and alanine, proline and threonine (10^?6 M). Glycine, taurine and alanine are three of the most abundant free amino acids found in invertebrates and protozoa which are major food sources of this dinoflagellate. Three additional species of cultured heterotrophic dinoflagellates were exposed to the water soluble fraction of a shrimp extract known to attract G. fungiforme. All three species responded to the extract, but one species, Oxyrrhis marina, did so only after changing its food source. It is suggested that chemosensory behavior may be suppressed or expressed depending on culture conditions.     

Studies on Biosynthesis of Biotin by Microorganisms

During the course of the study on the production of biotin from desthiobiotin by microorganisms, the present authors have found that some strains of molds produced an unknown biotin-vitamer (BS-factor) from desthiobiotin. The present investigation was undertaken to clarify the characteristics of the unknown vitamer. The unknown vitamer produced from desthiobiotin was isolated in crystalline form from culture filtrate of Aspergillus oryzae. The compound isolated was identified as 4-methyl-5-(ω-carboxybutyl)-imidazolidone-2 by the physico-chemical procedures.

The biosynthesis of biotin-vitamers by resting cell system of Bacillus sphaericus was studied.

It was found that pimelic acid was essential substrate in biosynthesis of biotin-vitamers and that some amino acids and organic acids stimulated the biosynthesis of biotin-vitamers from pimelic acid. Alanine was found to be most effective. It was assumed that, in the presence of pimelic acid, some amino acids, especially alanine, and some organic acids play an important role in the biosynthesis of biotin-vitamers.

The main component of the biotin-vitamers synthesized by the resting cell system was identified as desthiobiotin. The existence of a small amount of unknown biotin-vitamer, an avidin-uncombinable substance, which was assumed to be 7-keto-8-amino-pelargonic acid, was also observed. True biotin was hardly observed in any conditions tested.     

The Effect of Several Protein Amino Acids and Some Inorganic Nitrogen Sources on the Growth of Sphagnum nemoreum

The nitrogen metabolism of a bog moss, Sphagnum nemoreum Scop., has been studied in aseptic cultures. The effect of several protein amino acids, especially those found in peat, has been investigated. NH₄NO₃ (1.25 mM) was the best nitrogen source but NH₄⁺ ions were more effectively utilized than NO₃⁻ ions when given as the only nitrogen source. Some of the amino acids (2.5 mM) allowed fairly satisfactory growth (arginine and alanine) when given as the only nitrogen source, but some of them were not utilized at all (leucine, lysine, isoleucine and methionine). Given at low concentrations (0.001 and 0.25 mM) together with NH₄NO₃ (2.5 mM), most of the protein amino acids failed to reveal any growth-promoting or -inhibiting effect. Only lysine (0.25 mM) clearly inhibited growth under these conditions. The nitrogen metabolism of Sphagnum nemoreum seems to be rather flexible and this species is more tolerant of organic nitrogen, especially hydroxyproline, than the higher plants.     

Purification of the fengycin synthetase multienzyme system from Bacillus subtilis b213

The purification of the multienzyme system producing the lipodecapeptide fengycin in Bacillus subtilis b213 was investigated. By gel filtration of a cell free extract of this organism three enzyme fractions were obtained from which five multifunctional components of fengycin synthetase were separated by high resolution anion-exchange FPLC procedures. These proteins were characterized by their thioester formation activities with ¹⁴C-labeled substrate amino acids and by N-terminal sequencing. Correlation of these data with the DNA sequences of the pps (fen) operons in three B. subtilis strains provided detailed knowledge on the structural and functional organization of fengycin synthetase.     

Endophytic and rhizospheric enterobacteria isolated from sugar cane have different potentials for producing plant growth-promoting substances

Background and aims

Endophytic and rhizospheric environments differ in many respects, leading to the presence of different bacterial communities at each site. However, microorganisms such as enterobacteria can be found both within plants and in the surrounding soil. Bacteria must present differences in the traits that affect such environments in order to successfully colonise them. The present study compared the plant growth-promoting potential of diazotrophic enterobacteria isolated from the rhizosphere and from within surface-disinfected plants.

Methods

A total of 46 diazotrophic enterobacterial strains (21 rhizospheric and 25 putatively endophytic) belonging to the Klebsiella and Enterobacter genera, which are prevalent in sugar cane plantations, were isolated from the rhizosphere and from surface-disinfected plants. Their ability to synthesise amino acids using combined nitrogen obtained from nitrogen fixation, and their ability to synthesise indole-3-acetic acid (IAA) were determined by high performance liquid chromatography. Endogenous ethylene production by the bacteria was measured using gas chromatography, and biocontrol of phytopathogenic fungi was determined qualitatively using a dual culture technique.

Results

The putative endophytes released significantly higher amounts of amino acids than the rhizospheric bacteria, whilst the latter produced higher quantities of ethylene and were more actively antagonistic to fungi. Both types of bacteria released similar amounts of IAA.

Conclusion

Endophytic and rhizospheric bacteria differ in their capacity to release plant growth-promoting substances, which may be a reflection of their adaptations and an indication of their potential impact on their natural environment.

     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

We have found that in the alcoholic fermentation of amino acids by yeast isobutyl alcohol is produced from alanine and n-propyl and active amyl alcohols are formed from α-amino-n-butyric acid or threonine contrary to the F. Ehrlich’s scheme. These results suggest the close relationship among the formation of these higher alcohols and biosynthesis of valine from alanine and biosynthesis of isoleucine from α-amino-n-butyric acid or threonine.

In this report, we studied the formation of n-propyl alcohol and active amyl alcohol from α-amino-n-butyric acid using washed yeast cells.