Genome-enabled determination of amino acid biosynthesis in Xanthomonas campestris pv. campestris and identification of biosynthetic pathways for alanine, glycine, and isoleucine by 13C-isotopologue profiling

To elucidate the biosynthetic pathways for all proteinogenic amino acids in Xanthomonas campestris pv. campestris, this study combines results obtained by in silico genome analysis and by (13)C-NMR-based isotopologue profiling to provide a panoramic view on a substantial section of bacterial metabolism. Initially, biosynthesis pathways were reconstructed from an improved annotation of the complete genome of X. campestris pv. campestris B100. This metabolic reconstruction resulted in the unequivocal identification of biosynthesis routes for 17 amino acids in total: arginine, asparagine, aspartate, cysteine, glutamate, glutamine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Ambiguous pathways were reconstructed from the genome data for alanine, glycine, and isoleucine biosynthesis. (13)C-NMR analyses supported the identification of the metabolically active pathways. The biosynthetic routes for these amino acids were derived from the precursor molecules pyruvate, serine, and pyruvate, respectively. By combining genome analysis and isotopologue profiling, a comprehensive set of biosynthetic pathways covering all proteinogenic amino acids was unraveled for this plant pathogenic bacterium, which plays an important role in biotechnology as a producer of the exopolysaccharide xanthan. The data obtained lay ground for subsequent functional analyses in post-genomics and biotechnology, while the innovative combination of in silico and wet lab technology described here is promising as a general approach to elucidate metabolic pathways.     

Selective utilization of exogenous amino acids by Dehalococcoides ethenogenes strain 195 and its effects on growth and dechlorination activity

Bacteria of the genus Dehalococcoides are important members of bioremediation communities because of their ability to detoxify chloroethenes to the benign end product ethene. Genome-enabled studies conducted with Dehalococcoides ethenogenes 195 have revealed that two ATP-binding cassette (ABC)-type amino acid transporters are expressed during its exponential growth stages. In light of previous findings that Casamino Acids enhanced its dechlorination activity, we hypothesized that strain 195 is capable of importing amino acids from its environment to facilitate dechlorination and growth. To test this hypothesis, we applied isotopomer-based dilution analysis with (13)C-labeled acetate to differentiate the amino acids that were taken up by strain 195 from those synthesized de novo and to determine the physiological changes caused by the significantly incorporated amino acids. Our results showed that glutamate/glutamine and aspartate/asparagine were almost exclusively synthesized by strain 195, even when provided in excess in the medium. In contrast, phenylalanine, isoleucine, leucine, and methionine were identified as the four most highly incorporated amino acids, at levels >30% of respective proteinogenic amino acids. When either phenylalanine or all four highly incorporated amino acids were added to the defined mineral medium, the growth rates, dechlorination activities, and yields of strain 195 were enhanced to levels similar to those observed with supplementation with 20 amino acids. However, genes for the putative ABC-type amino acids transporters and phenylalanine biosynthesis exhibited insignificant regulation in response to the imported amino acids. This study also demonstrates that using isotopomer-based metabolite analysis can be an efficient strategy for optimizing nutritional conditions for slow-growing microorganisms.     

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.     

Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and γ-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra × maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and γ-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.     

Identification and characterization of a re-citrate synthase in Dehalococcoides strain CBDB1

The genome annotations of all sequenced Dehalococcoides strains lack a citrate synthase, although physiological experiments have indicated that such an activity should be encoded. We here report that a Re face-specific citrate synthase is synthesized by Dehalococcoides strain CBDB1 and that this function is encoded by the gene cbdbA1708 (NCBI accession number CAI83711), previously annotated as encoding homocitrate synthase. Gene cbdbA1708 was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified. The enzyme catalyzed the condensation of oxaloacetate and acetyl coenzyme A (acetyl-CoA) to citrate. The protein did not have homocitrate synthase activity and was inhibited by citrate, and Mn2+ was needed for full activity. The stereospecificity of the heterologously expressed citrate synthase was determined by electrospray ionization liquid chromatography-mass spectrometry (ESI LC/MS). Citrate was synthesized from [2-(13)C]acetyl-CoA and oxaloacetate by the Dehalococcoides recombinant citrate synthase and then converted to acetate and malate by commercial citrate lyase plus malate dehydrogenase. The formation of unlabeled acetate and 13C-labeled malate proved the Re face-specific activity of the enzyme. Shotgun proteome analyses of cell extracts of strain CBDB1 demonstrated that cbdbA1708 is expressed in strain CBDB1.     

Changes in plasma amino acid concentrations with increasing age in patients with propionic acidemia

The objective of the study is to analyze plasma amino acid concentrations in propionic acidemia (PA) for the purpose of elucidating possible correlations between propionyl-CoA carboxylase deficiency and distinct amino acid behavior. Plasma concentrations of 19 amino acids were measured in 240 random samples from 11 patients (6 families) with enzymatically and/or genetically proven propionic acidemia (sampling period, January 2001–December 2007). They were compared with reference values from the literature and correlated with age using the Pearson correlation coefficient test. Decreased plasma concentrations were observed for glutamine, histidine, threonine, valine, isoleucine, leucine, phenylalanine and arginine. Levels of glycine, alanine and aspartate were elevated, while values of serine, asparagine, ornithine and glutamate were normal. For lysine, proline and methionine a clear association was not possible. Significant correlations with age were observed for 13 amino acids (positive correlation: asparagine, glutamine, proline, alanine, histidine, threonine, methionine, arginine; negative correlation: leucine, phenylalanine, ornithine, glutamate and aspartate). This study gives new insight over long-term changes in plasma amino acid concentrations and may provide options for future therapies (e.g., substitution of anaplerotic substances) in PA patients.     

Multiple nonidentical reductive-dehalogenase-homologous genes are common in Dehalococcoides

Degenerate primers were used to amplify large fragments of reductive-dehalogenase-homologous (RDH) genes from genomic DNA of two Dehalococcoides populations, the chlorobenzene- and dioxin-dechlorinating strain CBDB1 and the trichloroethene-dechlorinating strain FL2. The amplicons (1,350 to 1,495 bp) corresponded to nearly complete open reading frames of known reductive dehalogenase genes and short fragments (approximately 90 bp) of genes encoding putative membrane-anchoring proteins. Cloning and restriction analysis revealed the presence of at least 14 different RDH genes in each strain. All amplified RDH genes showed sequence similarity with known reductive dehalogenase genes over the whole length of the sequence and shared all characteristics described for reductive dehalogenases. Deduced amino acid sequences of seven RDH genes from strain CBDB1 were 98.5 to 100% identical to seven different RDH genes from strain FL2, suggesting that both strains have an overlapping substrate range. All RDH genes identified in strains CBDB1 and FL2 were related to the RDH genes present in the genomes of Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain BAV1; however, sequence identity did not exceed 94.4 and 93.1%, respectively. The presence of RDH genes in strains CBDB1, FL2, and BAV1 that have no orthologs in strain 195 suggests that these strains possess dechlorination activities not present in strain 195. Comparative sequence analysis identified consensus sequences for cobalamin binding in deduced amino acid sequences of seven RDH genes. In conclusion, this study demonstrates that the presence of multiple nonidentical RDH genes is characteristic of Dehalococcoides strains.     

Changes in protein profile and amino acids in <Emphasis Type="Italic">Cladophora vagabunda</Emphasis> (Chlorophyceae) in response to salinity stress

The aim of the present study was to detect organic substances functioning as osmoticants that are used by the intertidal alga, Cladophora vagabunda (L.) Hoek (Chlorophyceae), to adapt to a wide range of salinity. The major constituents of the amino acid pool were aspartate, glutamate, glycine, valine, lysine, histidine, arginine, and proline. There were concomitant increases in the acidic amino acids: aspartate and the glutamate and the basic amino acids: lysine, histidine and arginine in response to salinity stress. The appearance of proline at hypersalinity alone showed that it acts as an osmoticant. As salinity increased, there was a progressive shift in the electrophoretic pattern of protein bands. New peptide bands appeared under hyposalinity (10‰) and hypersalinity (65‰) stress conditions in addition to the usual bands which appeared in the control (35‰). Glycine betaine, which has been considered a novel organic osmolyte in a number of organisms, has also been observed in C. vagabunda in response to salinity stress. The synthesis of the compatible solute glycine betaine and the amino acid proline with increasing salinity illustrates the contention that marine algae establish an osmotic equilibrium primarily by the synthesis of organic compounds. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Formation of the intermediate products of the tricarboxylic acid cycle and ammonia from free amino acids in anoxic heart muscle

Glutamate and aspartate showed the highest rate of catabolism in oxygenated isolated rat heart with the formation of glutamine, asparagine and alanine. Under anoxia, the catabolism of branch chained amino acids and that of lysine, proline, arginine and methionine was inhibited. However, glutamate and aspartate catabolized at a higher rate as compared with oxygenation. Alanine was the product of their excessive degradation. During oxygenation, 70% of ammonia were produced via deamination of amino acids. Under anaerobic conditions the participation of amino acids in ammoniagenesis decreased to 4%; the principal source of ammonia was the adenine nucleotide pool. The total pool of the tricarboxylic acid cycle intermediates increased 2.5-fold due to accumulation of succinate. The data obtained suggest that the constant influx of intermediates into the cycle from amino acids is supported by coupled transamination of glutamate and aspartate. This leads to the formation of ATP and GTP in the tricarboxylic acid cycle during blocking of aerobic energy production.     

Glycerol affects the quantification of aspartate and glutamate in acid-hydrolyzed proteins

Summary Glycerol is widely used in protein isolation pathways to improve folding and solubility of the proteins of interest. Amino acid composition analysis of protein samples hydrolyzed in the presence of glycerol resulted in underestimation of aspartate and glutamate, when compared to hydrolysis in the absence of glycerol. Quantification of free asparagine, aspartic acid, glutamine and glutamic acid hydrolyzed with hydrochloric acid or methanesulfonic acid in the presence of glycerol resulted in poor recoveries of aspartate and glutamate (between 6 and 66%). Gas chromatography-mass spectrometry analysis of the hydrolyzates revealed, as expected, the presence of esterification products. The esters were formed between the primary and secondary hydroxyl groups of the glycerol and both carboxyl groups of the amino acids. Protein samples intended for compositional analysis should be free of glycerol.Abbreviations EI electron impact - GC-MS gas chromatography-mass spectrometry - MS mass spectrum - MSA methanesulfonic acid - BSTFA bis(trimethylsilyl)trifluoroacetamide - TMS trimethylsilyl     

Chemotaxis toward amino acids in Escherichia coli

Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis.     

Biochemical mutants of Actinomyces griseus, a producer of the antibiotic grisin. The isolation of the mutants and a study of the level of grisin antibiotic synthesis]

Biochemical mutants of Actinomyces griseus producing grisin were obtained under the action of chemical mutagens. The mutants were divided into 2 groups. The mutants with impaired synthesis of amino acids of the aspartic acid family, i.e. lysine, homoserine and methionine were included into the 1st group. The 2nd group included the mutants with impaired synthesis of the other amino acids, i.e. histidine, arginine, tyrosine, phenylalanine and valine. The antibiotic production level in the biochemical mutants was studied. It was found that the level of the antibiotic production was decreased in most of the biochemical mutants. A necessity for increased fonds of lysine and arginine for biosynthesis of grisin was shown.     

Alteration in the Amino Acid Content of Yeast During Growth Under Various Nutritional Conditions

Yeast cells grown under optimal and suboptimal concentrations of biotin were analyzed for the amino acid content of their soluble pool and cellular protein. Optimally grown yeast cells exhibited a maximum amino acid content after 18 hr of growth. Biotin-deficient cells were depleted of all amino acids at 26 and 43 hr, with alanine, arginine, aspartate, cysteine, glutamate, isoleucine, leucine, lysine, methionine, serine, threonine, and valine being present in less than half the concentration observed in biotin-optimal cells. At early time intervals, the amino acid pool of biotin-deficient yeast contained lower concentrations of all amino acids except alanine. After more prolonged incubation, several amino acids accumulated in the pool of biotin-deficient yeast, but citrulline and ornithine accumulated to appreciable levels. The addition of aspartate to the growth medium resulted in a decrease in the amino acid content of biotin-optimal cells but caused a marked increase in the concentration of amino acids in biotin-deficient cells. The pools of biotin-deficient yeast grown in the presence of aspartate displayed a marked reduction in every amino acid with the exception of aspartate itself. These data provide evidence that the amino acid content of yeast cells and their free amino acid pools are markedly affected by biotin deficiency as well as by supplementation with aspartate, indicating that aspartate plays a major role in the nitrogen economy of yeast under both normal as well as abnormal nutritional conditions.     

Identification and validation of eukaryotic aspartate and glutamate methylation in proteins

Methylation of lysine and arginine is known to be critical in cellular processes. However, methylation of other amino acidic residues has been largely overlooked. Here, we report a systematic screening for methylation of side chains of aspartate and glutamate (D/E-methylation), involving exhaustive nano-HPLC/MS/MS, a protein sequence database search, and manual verification. The putative D/E-methylated peptides were confirmed by MS/MS of synthetic peptides. Our analysis identified several D/E-methylation substrate proteins and their modification sites in human and yeast cells. To our knowledge, this is the first report conclusively identifying in vivo D/E-methylation substrates and their modification sites in eukaryotic cells, demonstrating that D/E-methylations are abundant protein modifications. The substrate proteins identified here provide a stepping stone for future biochemical characterization of protein methylation pathways.     

Guanidination of Soluble Lysine-Rich Cyanophycin Yields a Homoarginine-Containing Polyamide

Soluble cyanobacterial granule polypeptide (CGP), especially that isolated from recombinant Escherichia coli strains, consists of aspartic acid, arginine, and a greater amount of lysine than that in insoluble CGP isolated from cyanobacteria or various other recombinant bacteria. In vitro guanidination of lysine side chains of soluble CGP with o-methylisourea (OMIU) yielded the nonproteinogenic amino acid homoarginine. The modified soluble CGP consisted of 51 mol% aspartate, 14 mol% arginine, and 35 mol% homoarginine. The complete conversion of lysine residues to homoarginine was confirmed by (i) nuclear magnetic resonance spectrometry, (ii) coupled liquid chromatography-mass spectrometry, and (iii) high-performance liquid chromatography. Unlike soluble CGP, this new homoarginine-containing polyamide was soluble only under acidic or alkaline conditions and was insoluble in water or at a neutral pH. Thus, it showed solubility behavior similar to that of the natural insoluble polymer isolated from cyanobacteria, consisting of aspartic acid and arginine only. Polyacrylamide gel electrophoresis revealed similar degrees of polymerization of the native (12- to 40-kDa) and modified (10- to 35-kDa) polymers. This study showed that the chemical structure and properties of a biopolymer could be changed by in vitro introduction of a new functional group after biosynthesis of the native polymer. In addition, the modified CGP could be digested in vitro using the cyanophycinase from Pseudomonas alcaligenes strain DIP1, yielding a new dipeptide consisting of aspartate and homoarginine.     

Salmonella typhimurium LT-2 mutants with altered glutamine synthetase levels and amino acid uptake activities.

To determine whether Salmonella typhimurium has a nitrogen control response, we have examined the regulation of nitrogen utilization in two mutants with fivefold and threefold elevations in their glutamine synthetase activities. The mutants do not require glutamine for growth on glucose--ammonia medium but do have altered growth on other nitrogen sources. They grow better than an isogenic control on media containing arginine or asparate, but more slowly with proline or alanine as nitrogen sources. This unusual growth pattern is not due to altered regulation of the ammonia assimilatory enzymes, glutamate dehydrogenase and glutamate synthase, or to changes in the enzymes for aspartate degradation. However, transport for several amino acids may be affected. Measurement of amino acid uptake show that the mutants with high glutamine synthetase levels have increased rates for glutamine, arginine, aspartate, and lysine, but a decreased rate for proline. The relationship between glutamine synthetase levels and uptake was examined in two mutants with reduced, rather than increased, glutamine synthetase production. The uptake rates for glutamine and lysine were lower in these two glutamine auxotrophs than in the Gln+ controls. These results show a correlation between the glutamine synthetase levels and the uptake rates for several amino acids. In addition, the pleiotropic growth of the mutants with elevated glutamine synthetase activities suggests that a nitrogen control response exists for S. typhimurium and that it can be altered by mutations affecting glutamine synthetase regulation.     

Analysis of energetic amino acid metabolism in Acyrthosiphon pisum: A multidimensional approach to amino acid metabolism in aphids

Aphids are highly specialized insects that feed on the phloem-sap of plants, the amino acid composition of which is very unbalanced. Amino acid metabolism is thus crucial in aphids, and we describe a novel investigation method based on the use of ¹⁴C-labeled amino acids added in an artificial diet. A metabolism cage for aphids was constructed, allowing for the collection and analysis of the radioactivity incorporated into the aphid body, expired as CO₂, and rejected in the honeydew and exuviae. This method was applied to the study of the metabolism of eight energetic amino acids (aspartate, glutamate, glutamine, glycine, serine, alanine, proline, and threonine) in the pea aphid, Acyrthosiphon pisum. All these amino acids except threonine were subject to substantial catabolism as measured by high ¹⁴CO₂ production. The highest turnover was displayed by aspartate, with 60% of its carbons expired as CO₂. For the first time in an aphid, we directly demonstrated the synthesis of three essential amino acids (threonine, isoleucine, and lysine) from carbons of common amino acids. The synthesis of these three compounds was only observed from amino acids that were previously converted into glutamate. This conversion was important for aspartate, and lower for alanine and proline. To explain the quantitative results of interconversion between amino acids, we propose a compartmentation model with the intervention of bacterial endosymbiotes for the synthesis of essential amino acids and with glutamate as the only amino acid supplied by the insect to the symbiotes. Moreover, proline exhibited partial conversion into arginine, and it is suggested that proline is probably indirectly involved in excretory nitrogen metabolism. © 1995 Wiley-Liss, Inc.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.     

Utilization of milk amino acids for body gain in suckling mink (Mustela vison) kits

The efficiency of utilization of milk amino acids for body gain in suckling mink kits from small (n = 3), medium (n = 6) and large litters (n = 9) was investigated by using 36 mink dams and their litters for measurements during lactation weeks 1 through 4. Measurements on each dam and litter were performed once, hence three dams per litter size each week (n = 9). Individual milk intake of kits was determined, milk samples were collected and kits were killed for determination of amino acid composition. The most abundant amino acids in milk were glutamate, leucine and aspartate making up about 40% of total amino acids. Branched chained amino acids made up slightly more than 20% and sulphur containing amino acids less than 5% of total milk amino acids. In kit bodies the sum of glutamate, aspartate and leucine made up about 32% of amino acids, branched chain amino acids about 16% and sulphur containing amino acids about 4%. The amino acid composition of both milk and bodies changed as lactation progressed with decreasing proportions of essential amino acids. The ratio between body and milk amino acids was constantly over 1 only for lysine, suggesting that it was the most limiting amino acid in mink milk. Milk amino acids were efficiently utilized during week 1, ranging from 74.7% (lysine) to 42.1% (leucine), with an average for essential amino acids of 58.4%. Tendencies for improved utilization of lysine (74.7-78.2%), phenylalanine (61.0-70.0%), histidine (62.4-68.8%), arginine (61.3-70.4%) and all essential amino acids (58.4-60.2%) from week 1 to week 2 were recorded. During weeks 3 and 4, the efficiency declined, and for all essential amino acids the average utilization was 38.1% during week 4.     

Comparison of hup trait and intrinsic antibiotic resistance for assessing rhizobial competitiveness axenically and in soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.