A novel mode of sensory transduction in archaea: binding protein-mediated chemotaxis towards osmoprotectants and amino acids

Directly upstream of the Halobacterium salinarum transducer genes basT and htpIV we identified two open reading frames (orfs) with significant homologies to genes encoding binding proteins for amino acids and compatible solutes, respectively. Behavioral testing of deletion mutants indicates that halobacterial chemotaxis towards branched-chain amino acids as well as compatible osmolytes of the betaine family requires both a binding and a transducer protein. We therefore named the binding/transducer proteins BasB/BasT for branched-chain and sulfur-containing amino acids and CosB/CosT for compatible solutes. Our data support a signaling mechanism with the binding proteins functioning as lipid-anchored receptors interacting with the extracellular domain of their cognate transducers. Inspection of the halobacterial genome suggests that BasB and CosB exclusively mediate chemotaxis responses without any additional role in transport, which is in contrast to bacterial binding proteins, which are always part of ABC transport systems. The CosB/CosT system is the first instance of a chemotaxis signaling pathway for organic osmolytes in the living world and natural abundance 13C-NMR analysis of cytoplasmic extracts suggests that H.salinarum utilizes these solutes for osmotic adaptation.     

Receptors for chemotaxis in Bacillus subtilis.

At least three receptors for chemotaxis toward L-amino acids in Bacillus subtilis could be found with the aid of taxis competition experiments. They are called the asparagine receptor, which detects asparagine and glutamine, the isoleucine receptor, which detects isoleucine, leucine, valine, phenylalanine, serine, threonine, cysteine, and methionine, and the alanine receptor, which detects alanine and proline. Histidine and glycine could not be assigned to one of these receptors. Cysteine and methionine were found to be general inhibitors of chemotaxis and serine was found to be a general stimulator of chemotaxis. Some structural analogues of amino acids were tested for chemotactic activity. The chemotactic activity of B. subtilis is compared with that of Escherichia coli.     

Protein content and amino acids composition of bee-pollens from major floral sources in Al-Ahsa,eastern Saudi Arabia

Protein content and amino acids composition of bee-pollens from major pollen floral sources in Al-Ahsa, Saudi Arabia were determined to investigate the nutritive value of pollen protein relative to requirements of honeybees and adult humans. The major pollen sources were alfalfa, date palm, rape, summer squash, and sunflower. Bee-pollens from alfalfa and date palm showed high content of crude protein and amino acid concentrations. Bee-pollen from sunflower had low content of those components. Eighteen amino acids were found in bee-pollens from the five major floral sources. The highest concentrations of individual amino acids valine, leucine, isoleucine, phenylalanine and proline were obtained from alfalfa bee-pollen; lysine, arginine, cysteine, tryptophan and tyrosine from date palm; methionine, histidine, glycine and alanine from summer squash; threonine, serine and glutamic acid from sunflower; and aspartic acid from rape bee-pollen. The amino acid composition obtained from sunflower bee-pollen showed the lowest concentrations of the essential amino acids: isoleucine, leucine, methionine, phenylalanine and valine. Apart from methionine, arginine and isoleucine, the essential amino acids of bee-pollen from alfalfa, date palm, summer squash and rape exceeded the honeybees’ requirements. Methionine was the limiting amino acid in bee-pollens from the five selected sources. Concentrations of essential amino acids in the tested bee-pollens were variable and significantly correlated to their botanical origin of pollen. Bee-pollens from alfalfa, date palm and summer squash was found to be rich source of protein and amino acids for bees and for humans.     

Novel methyl transfer during chemotaxis in Bacillus subtilis

If Bacillus subtilis is incubated in radioactive methionine in the absence of protein synthesis, the methyl-accepting chemotaxis proteins (MCPs) become radioactively methylated. If the bacteria are further incubated in excess nonradioactive methionine ("cold-chased") and then given the attractant aspartate, the MCPs lose about half of their radioactivity due to turnover, in which lower specific activity methyl groups from S-adenosylmethionine (AdoMet) replace higher specific activity ones. Due to the cold-chase, the specific activity of the AdoMet pool is reduced at least 2-fold. If, later, the attractant is removed, higher specific activity methyl groups return to the MCPs. Thus, there must exist an unidentified methyl carrier that can "reversibly" receive methyl groups from the MCPs. In a similar experiment, labeled cells were transferred to a flow cell and exposed to addition and removal of attractant and of repellent. All four kinds of stimuli were found to cause methanol production. Bacteria with maximally labeled MCPs were exposed to many cycles of addition and removal of attractant; the maximum amount of radioactive methanol was evolved on the third, not the first, cycle. This result suggests that there is a precursor-product relationship between methyl groups on the MCPs and on the unidentified carrier, which might be the direct source of methanol. However, since no methanol was produced when a methyltransferase mutant, whose MCPs were unmethylated, was exposed to addition and removal of attractant or repellent, the methanol must ultimately derive from methylated MCPs.     

Molecular cloning and characterization of a chemotactic transducer gene in Pseudomonas aeruginosa.

A Pseudomonas aeruginosa mutant, defective in taxis toward L-serine but responsive to peptone, was selected by the swarm plate method after N-methyl-N'-nitrosoguanidine mutagenesis. The mutant, designated PCT1, was fully motile but failed to show chemotactic responses to glycine, L-serine, L-threonine, and L-valine. PCT1 also showed weaker responses to some other commonly occurring L-amino acids than did the wild-type strain PAO1. A chemotactic transducer gene, denoted pctA (Pseudomonas chemotactic transducer A), was cloned by phenotypic complementation of PCT1. Nucleotide sequence analysis showed that the pctA gene encodes a putative polypeptide of 629 amino acids with a calculated mass of 68,042. A hydropathy plot of the predicted polypeptide suggested that PctA may be an integral membrane protein with two potential membrane-spanning regions. The C-terminal domain of PctA showed high homology with the enteric methyl-accepting chemotaxis proteins (MCPs). The most significant amino acid sequence similarity was found in the region of MCPs referred to as the highly conserved domain. The pctA gene was inactivated by insertion of a kanamycin resistance gene cassette into the wild-type gene, resulting in the same observed deficiency in taxis toward L-amino acids as PCT1. In vivo methyl labeling experiments with L-[methyl-3H]methionine showed that this knockout mutant lacked an MCP with a molecular weight of approximately 68,000.     

凤眼莲根分泌物氨基酸组分对根际肠杆菌属F2细菌的趋化作用

凤眼莲(Eichhornia crassipes)的根分泌物中含有Met等多种氨基酸,其中Met、GABA、Gly、Ala、Asp、Ser、Val和Leu(10^-7～10^-2mol·L^-1)均对凤眼莲的根际肠杆菌属F₂(Enterobacter sp.F₂)细菌有强烈的正趋化作用;Glu、Thr和His(10^-7～10^-3mol·L^-1)也对该菌有一定的正趋化作用;而Lys、Cys、Arg、Tyr、Pro、Asn、Gln、Ile、Phe和Typ则对该菌表现出一定的负趋化作用.对细菌的正趋化作用存在一个趋化物的最适浓度范围.具有正趋化作用的氨基酸在凤眼莲根际的浓度都较高,而具有负趋化作用的浓度则较低,这正是凤眼莲与该根际细菌结合为根际微生态系统的原因之一.     

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.     

Rapid attractant-induced changes in methylation of methyl-accepting chemotaxis proteins in Bacillus subtilis

In Bacillus subtilis, addition of chemotactic attractant causes an immediate change in distribution of methyl groups on methyl-accepting chemotaxis proteins (MCPs), whereas in Escherichia coli, it causes changes that occur throughout the adaptation period. Thus, methylation changes in B. subtilis are probably related to excitation, not adaptation. If labeled cells are exposed to excess nonradioactive methionine, then attractant causes immediate 50% delabeling of the MCPs, suggesting that a flux of methyl groups through the MCPs occurs. Methanol is given off at a high rate during the adaptation period and probably reflects demethylation of some substance to bring about adaptation. The fact that many radioactive methyl groups are lost immediately from the MCPs but only slowly arise as methanol is consistent with the hypothesis that they are transferred from the MCPs to a carrier from which methanol arises. Demethylation of this carrier may cause adaptation.     

Transport of Biosynthetic Intermediates: Regulation of Homoserine and Threonine Uptake in Escherichia coli

Homoserine is transported by a single system that it shares with alanine, isoleucine, leucine, phenylalanine, threonine, valine and perhaps cysteine, methionine, serine, and tyrosine. We investigated the regulation of this transport system and found that alanine, isoleucine, leucine, methionine, and valine each repress the homoserine-transporting system. From the concentration resulting in 50% repression of this transport system and the maximal amount of repression, we ranked the amino acids according to their effectiveness in repressing homoserine transport (in decreasing order): leucine>methionine>alanine>valine>isoleucine. The exponential rate of decrease in transport capacity after leucine addition equals the exponential growth rate of the culture, and protein synthesis is necessary for the derepression seen when leucine is removed. Threonine, in addition to using the above system, is transported by a second system shared with serine. We present further evidence for this serine-threonine transport system and show that it is not regulated like the homoserine-transporting system.     

Car: a cytoplasmic sensor responsible for arginine chemotaxis in the archaeon Halobacterium salinarum

A new metabolic signaling pathway for arginine, both a chemoeffector and a fermentative energy source, is described for Halobacterium salinarum. Systematic screening of 80+ potentially chemotactic compounds with two behavioral assays identified leucine, isoleucine, valine, methionine, cysteine, arginine and several peptides as strong chemoattractants. Deletion analysis of a number of potential halobacterial transducer genes led to the identification of Car, a specific cytoplasmic arginine transducer which lacks transmembrane helices and was biochemically shown to be localized in the cytoplasm. Flow assays were used to show specific adaptive responses to arginine and ornithine in wild-type but not Deltacar cells, demonstrating the role of Car in sensing arginine. The signaling pathway from external arginine to the flagellar motor of the cell involves an arginine:ornithine antiporter which was quantitatively characterized for its transport kinetics and inhibitors. By compiling the chemotactic behavior, the adaptive responses and the characteristics of the arginine:ornithine antiporter to arginine and its analogs, we now understand how the combination of arginine uptake and its metabolic conversion is required to build an effective sensing system. In both bacteria and the archaea this is the first chemoeffector molecule of a soluble methylatable transducer to be identified.     

Attenuation of the editing activity of the Escherichia coli leucyl-tRNA synthetase allows incorporation of novel amino acids into proteins in vivo

The fidelity of translation is dependent on the specificity of the aminoacyl-tRNA synthetases (aaRSs). The aaRSs that activate the hydrophobic amino acids leucine, isoleucine, and valine employ a proofreading mechanism that hydrolyzes noncognate aminoacyl adenylates and misaminoacylated tRNAs. Discrimination between structurally similar amino acids by these AARSs is believed to operate by a double-sieve principle, wherein a separate editing domain governs hydrolysis on the basis of the size and hydrophilicity of the amino acid side chain. Leucyl-tRNA synthetase (LeuRS) relies on its editing function to correct misaminoacylation of tRNA(Leu) by isoleucine and methionine. Thr252 of Escherichia coli LeuRS has been shown previously to be important in defining the size of the editing cavity. Here we report the isolation and characterization of three LeuRS mutants with point mutations at this position (T252Y, T252L, and T252F). The proofreading activity of the synthetase is significantly impaired when an amino acid bulkier than threonine is introduced. The rate of misaminoacylation of tRNA(Leu) by isoleucine and valine increases with the increasing size of the amino acid substituent at position 252, and the noncognate amino acids norvaline and norleucine are inserted efficiently at the leucine sites of recombinant proteins under conditions of constitutive overexpression of the T252Y mutant in E. coli. In addition, the unsaturated amino acids allylglycine, homoallylglycine, homopropargylglycine, and 2-butynylalanine all support protein synthesis in E. coli hosts harboring the mutant synthetase. These results demonstrate that programmed manipulation of the editing cavity can allow in vivo incorporation of novel protein building blocks.     

The role of free amino acids in semen of rainbow trout Oncorhynchus mykiss and carp Cyprinus carpio

The present study investigated (1) the free amino acid (FAA) composition in semen of rainbow trout Oncorhynchus mykiss and carp Cyprinus carpio, (2) enzyme systems involved in amino acid metabolism and (3) the effect of amino acids on sperm viability under in vitro storage conditions. In the seminal plasma of O. mykiss, the main FAAs were arginine, glutamic acid, isoleucine, leucine, methionine and proline, in spermatozoa cysteine, arginine and methionine. In the seminal plasma of C. carpio, the main FAAs were alanine, arginine, cysteine, glutamic acid, histidine, leucine, lysine, methionine and proline, in spermatozoa arginine, glutamic acid, histidine, leucine and lysine. When spermatozoa were incubated for 48 h together with the seminal plasma, the quantitative amino acid pattern changed in both species indicating their metabolism. In spermatozoa and seminal plasma of O. mykiss and C. carpio, the following enzymes were found to be related to amino acid metabolism: transaminases (specific for alanine, aspartate, isoleucine and leucine), decarboxylases (specific for valine and lysine), glutamate dehydrogenase and α‐keto acid dehydrogenases (substrates: 3‐methyl‐2‐oxovaleric acid and 4‐methyl‐2‐oxovalerate). These data demonstrate that amino acid catabolism by transamination, decarboxylation and oxidative deamination can occur in semen of the two species. Also activity of methionine sulphoxide reductase was detected, an enzyme which reduces methionine sulphoxide to methionine. This reaction plays an important role in antioxidant defence. To determine the effect of FAAs on the sperm viability, C. carpio and O. mykiss spermatozoa were incubated in sperm motility inhibiting saline solution containing different amino acids. Methionine had a positive effect on the sperm viability in both species. Taken together this result with the in vivo occurrence of methionine and of methionine reductase in semen, it can be assumed that this amino acid plays an important role in antioxidant defence. Also isoleucine in O. mykiss and leucine in C. carpio had a positive effect on sperm viability. As seminal plasma and spermatozoa of the two species exhibit enzyme activities to catabolize leucine and isoleucine, they might serve as additional energy resources especially during prolonged incubation and storage periods.     

Regulation of branched-chain amino acid transport in Escherichia coli.

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyl-transfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.     

Nutritional factors affecting nisin production by Lactococcus lactis subsp. actis NIZO 22186 in a synthetic medium

L. DE VUYST. 1995 A minimal synthetic medium (SM8) for nisin-producing Lactococcus lactis subsp. lactis strains has been designed; it consists of eight growth-stimulating amino acids (glutamic acid, methionine, valine, leucine, threonine, arginine, isoleucine and histidine), five vitamins (biotin, calcium pantothenate, nicotinic acid, pyridoxine and riboflavin) and the mineral salts dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, magnesium sulphate and trisodium citrate. Nisin biosynthesis is strongly dependent on the presence of a sulphur source, either an inorganic salt (magnesium sulphate or sodium thiosulphate) or the amino acids methionine, cysteine or cystathionine. The amino acids serine, threonine and cysteine highly stimulate nisin production without affecting the final cell yield, indicating their precursor role during nisin biosynthesis.     

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.     

Effects of amino acids on Thiobacillus acidophilus. I. Growth studies with special reference to valine.

The heterotrophic growth of Thiobacillus acidophilus was inhibited by branched-chain amino acids; valine, isoleucine, and leucine. The inhibition by valine and leucine were partially reversed by isoleucine, and the inhibition by isoleucine was partially reversed by valine. Inhibitions by methionine or threonine were partially reversed when both amino acids were present in the growth medium. Inhibition by tyrosine was increased by phenylalanine or tryptophan. Cystine completely inhibited growth. Other amino acids tested produced little or no inhibition. Acetohydroxy acid synthetase (AHAS) activity was demonstrated in crude extracts of T. acidophilus. In crude extracts the optimum pH was 8.5 with a shift to 9.0 in the presence of valine. Valine was the only branched-chain amino acid which inhibited the AHAS activity. The presence of only one peak of AHAS activity upon centrifugation in linear glycerol density gradients demonstrated that the AHAS activity sediments as one component.     

Relationships between amino acid catabolism and protein anabolism in the ruminant

The observed relation found in sheep between the flux rate of an amino acid and the proportion found in whole-body protein suggests that the major immediate fate of an amino acid is its incorporation into tissue protein. This may be true even for dispensable amino acids. In ruminants, there is substantial utilization of several amino acids (serine, glycine, threonine, histidine, and methionine) for the synthesis of methyl groups; the use of these amino acids for gluconeogenesis is limited. There is little evidence that demands of gluconeogenesis limit the availability of amino acids for protein synthesis. Most amino acids are catabolized in the liver but there may be significant catabolism of alanine, aspartate, and glutamate in peripheral tissues, especially muscle. Normally, peripheral catabolism of branched-chain amino acids is significantly less in ruminants than other species. Nevertheless, there is some oxidation of leucine by muscle and this may be substantially increased in the diabetic state. Catabolism of leucine (and perhaps isoleucine and valine) might be inversely related to use for protein synthesis, but there is no evidence of such a relation for other amino acids.     

Effects of branched-chain amino acids on plasma amino acids in amyotrophic lateral sclerosis

Summary Although the cause of amyotrophic lateral sclerosis (ALS) remains unknown, biological findings suggest that the excitatory amino acid glutamate contributes to the pathogenesis of ALS. In previous studies of ALS, the therapeutic effect of the branched-chain amino acids (BCAAs) leucine, valine and isoleucine has been evaluated. The present study aimed at investigating the acute effect of BCAAs on plasma glutamate levels in ALS patients. Following two oral doses of BCAAs, significantly increased plasma levels were seen for valine (500%), isoleucine (1,377%) and leucine (927%), however the plasma level of glutamate was not affected. The plasma level of several other amino acids (tryptophan, tyrosine, phenylalanine and methionine) were found decreased after oral BCAAs, which may indicate a diminution in the rate of degradation of muscle protein and/or an increase in tissue disposal of amino acids.     

Metabolism of amino acids in Ascaridia galli: transamination

Ascaridia galli, using 2-oxoglutarate as an acceptor, transaminates alanine and aspartate at significantly high rates. Among other amino acids valine, phenylalanine, leucine, isoleucine, arginine, tyrosine and methionine are metabolised at moderate rates while lysine, serine, threonine, cysteine, glycine, histidine, tryptophan, DOPA and GABA appear to be inert in this respect. Body parts mimic the whole worm in the pattern of transamination of various amino acids with the exception of methionine. Alanine, aspartate and glutamate may transfer their amino group also to pyruvate and oxaloacetate. Alanine and aspartate: 2-oxoglutarate transaminases are located mainly in the cytosol and mitochondrial fractions.