Concentrations Of L-alanine Research Articles

L-Serine Decreases Taurine Concentration in the Extracellular Fluid of Brain Slices

L-Serine is considered a functional amino acid in the central nervous system, and induces sedation and hypnotic effects in some animal models of acute and chronic stress. Accordingly, while L-serine is a candidate anti-stress factor, the central mechanism of L-serine is not clear. The present study clarifies the action of L-serine using acute chick brain slices. We investigated the changes in some extracellular fluid amino acid concentrations in response to L-serine perfusion. Taurine concentration decreased while L-alanine concentration increased following L-serine perfusion. To examine the involvement of the taurine transporter, the effect of L-serine on the taurine concentration in the presence and absence of Na+ was also investigated. Na+ had no effect on taurine concentration induced by L-serine perfusion. These results suggest that L-serine has an ability to promote L-alanine synthesis facilitating the catabolism of taurine. In conclusion, L-serine modifies the metabolism of taurine and L-alanine in the extracellular space in chick brain.

Deficiency inl-Serine Deaminase Interferes with One-Carbon Metabolism and Cell Wall Synthesis inEscherichia coliK-12

Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes L-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S L-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of L-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C(1) units and interferes with cell wall synthesis. We suggest that at high concentrations, L-serine decreases synthesis of UDP-N-acetylmuramate-L-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high L-serine is overcome in several ways: by a large concentration of L-alanine, by overproducing MurC together with a low concentration of L-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide.

Characterization of Bacterial Spore Germination Using Integrated Phase Contrast Microscopy, Raman Spectroscopy, and Optical Tweezers

We present a methodology that combines external phase contrast microscopy, Raman spectroscopy, and optical tweezers to monitor a variety of changes during the germination of single Bacillus cereus spores in both nutrient (l-alanine) and non-nutrient (Ca-dipicolinic acid (DPA)) germinants with a temporal resolution of approximately 2 s. Phase contrast microscopy assesses changes in refractility of individual spores during germination, while Raman spectroscopy gives information on changes in spore-specific molecules. The results obtained include (1) the brightness of the phase contrast image of an individual dormant spore is proportional to the level of CaDPA in that spore; (2) the end of the first Stage of germination, revealed as the end of the rapid drop in spore refractility by phase contrast microscopy, precisely corresponds to the completion of the release of CaDPA as revealed by Raman spectroscopy; and (3) the correspondence between the rapid drop in spore refractility and complete CaDPA release was observed not only for spores germinating in the well-controlled environment of an optical trap but also for spores germinating when adhered on a microscope coverslip. Using this latter method, we also simultaneously characterized the distribution of the time-to-complete-CaDPA release (T(release)) of hundreds of individual B. cereus spores germinating with both saturating and subsaturating concentrations of l-alanine and with CaDPA.

Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

BackgroundThe first step of the bacterial lifecycle is the germination of bacterial spores into their vegetative form, which requires the presence of specific nutrients. In contrast to closely related Bacillus anthracis spores, Bacillus cereus spores germinate in the presence of a single germinant, inosine, yet with a significant lag period.Methods and FindingsWe found that the initial lag period of inosine-treated germination of B. cereus spores disappeared in the presence of supernatants derived from already germinated spores. The lag period also dissipated when inosine was supplemented with the co-germinator alanine. In fact, HPLC-based analysis revealed the presence of amino acids in the supernatant of germinated B. cereus spores. The released amino acids included alanine in concentrations sufficient to promote rapid germination of inosine-treated spores. The alanine racemase inhibitor D-cycloserine enhanced germination of B. cereus spores, presumably by increasing the L-alanine concentration in the supernatant. Moreover, we found that B. cereus spores lacking the germination receptors gerI and gerQ did not germinate and release amino acids in the presence of inosine. These mutant spores, however, germinated efficiently when inosine was supplemented with alanine. Finally, removal of released amino acids in a washout experiment abrogated inosine-mediated germination of B. cereus spores.ConclusionsWe found that the single germinant inosine is able to trigger a two-tier mechanism for inosine-mediated germination of B. cereus spores: Inosine mediates the release of alanine, an essential step to complete the germination process. Therefore, B. cereus spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination.

The preparation, germination properties and stability of superdormant spores ofBacillus cereus

To determine yields, germination and stability of superdormant Bacillus cereus spores. Superdormant B. cereus spores were isolated by germination with high concentrations of inosine or L-alanine in 2-5% yield and did not germinate with high concentrations of either of these germinants, but germinated like starting spores with Ca-DPA, dodecylamine, L-alanine plus inosine or concentrated complete medium. Yields of superdormant spores from germinations with low inosine concentrations were higher, and these spores germinated poorly with low inosine, but relatively normally with high inosine. Yields of superdormant spores were also higher when nonheat-activated spores were germinated. Superdormant spores stored at 4 degrees C slowly recovered some germination capacity, but recovery was slowed significantly at -20 degrees C and -80 degrees C. Factors that influence levels of superdormant B. cereus spores and the properties of such spores are similar to those in B. megaterium and B. subtilis, suggesting there are common mechanisms involved in superdormancy of Bacillus spores. Superdormant spores are a major concern in the food industry, because the presence of such spores precludes decontamination strategies based on triggering spore germination followed by mild killing treatments. Studies of the properties of superdormant spores may suggest ways to eliminate them.

Molecular Insight into the Synergism between the Minor Allele of Human Liver Peroxisomal Alanine:Glyoxylate Aminotransferase and the F152I Mutation

Human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that converts glyoxylate into glycine. AGT deficiency causes primary hyperoxaluria type 1 (PH1), a rare autosomal recessive disorder, due to a marked increase in hepatic oxalate production. Normal human AGT exists as two polymorphic variants: the major (AGT-Ma) and the minor (AGT-Mi) allele. AGT-Mi causes the PH1 disease only when combined with some mutations. In this study, the molecular basis of the synergism between AGT-Mi and F152I mutation has been investigated through a detailed biochemical characterization of AGT-Mi and the Phe(152) variants combined either with the major (F152I-Ma, F152A-Ma) or the minor allele (F152I-Mi). Although these species show spectral features, kinetic parameters, and PLP binding affinity similar to those of AGT-Ma, the Phe(152) variants exhibit the following differences with respect to AGT-Ma and AGT-Mi: (i) pyridoxamine 5'-phosphate (PMP) is released during the overall transamination leading to the conversion into apoenzymes, and (ii) the PMP binding affinity is at least 200-1400-fold lower. Thus, Phe(152) is not an essential residue for transaminase activity, but plays a role in selectively stabilizing the AGT-PMP complex, by a proper orientation of Trp(108), as suggested by bioinformatic analysis. These data, together with the finding that apoF152I-Mi is the only species that at physiological temperature undergoes a time-dependent inactivation and concomitant aggregation, shed light on the molecular defects resulting from the association of the F152I mutation with AGT-Mi, and allow to speculate on the responsiveness to pyridoxine therapy of PH1 patients carrying this mutation.

Oral Glutamine Dipeptide Prevents against Prolonged Hypoglycemia Induced by Detemir Insulin in Rats

The role of glutamine dipeptide (GDP) to prevent against prolonged insulin induced hypoglycemia (IIH) in overnight fasted rats was investigated. The glycemia was measured 0, 2, 4, 8, and 10 h after an intraperitoneal (i.p.) injection (1 U/kg) of Detemir insulin. Because the lowest glycemia was obtained 4 h after insulin administration, the experiments were done at this time. The hypoglycemia obtained 4 h after insulin injection was partially prevented with increasing oral doses of GDP (1.56, 3.12, 6.25, 12.5 mg/kg). The best result was obtained with 12.5 mg/kg. However, from this dose (25.0, 50.0, 100.0 mg/kg), the values of glycemia progressively decreased (p<0.05). The effect of GDP to prevent prolonged IIH was mediated, partly at least, by an intensification of liver gluconeogenesis. Moreover, the increased portal availability of GDP, L-alanine and L-glutamine after GDP administration also contributed to the IIH prevention, since the rate of gluconeogenesis progressively augmented with the infusion of increasing concentrations of these substances. However, after getting the maximal value, the rate of liver gluconeogenesis decreased (p<0.05) if a more elevated concentration of L-alanine or L-glutamine was infused. On the other hand, the liver gluconeogenesis during the infusion of increasing concentrations of GDP was unchanged. Because, GDP did not directly inhibit liver gluconeogenesis, but an inhibition of this metabolic pathway was observed with low ammonia concentrations (from 0.062 mM) it is possible that the ammonia from the catabolism of GDP by extra hepatic tissues could inhibit liver gluconeogenesis. This mechanism could help to explain the lower glycemia obtained with more elevated doses of oral GDP.

Forced swimming and imipramine modify plasma and brain amino acid concentrations in mice

The relationships between monoamine metabolism and forced swimming or antidepressants have been well studied, however information is lacking regarding amino acid metabolism under these conditions. Therefore, the aim of the present study was to investigate the effects of forced swimming and imipramine on amino acid concentrations in plasma, the cerebral cortex and the hypothalamus in mice. Forced swimming caused cerebral cortex concentrations of l-glutamine, l-alanine, and taurine to be increased, while imipramine treatment caused decreased concentrations of l-glutamate, l-alanine, l-tyrosine, l-methionine, and l-ornithine. In the hypothalamus, forced swimming decreased the concentration of l-serine while imipramine treatment caused increased concentration of β-alanine. Forced swimming caused increased plasma concentration of taurine, while concentrations of l-serine, l-asparagine, l-glutamine and β-alanine were decreased. Imipramine treatment caused increased plasma concentration of all amino acid, except for l-aspartate and taurine. In conclusion, forced swimming and imipramine treatment modify central and peripheral amino acid metabolism. These results may aid in the identification of amino acids that have antidepressant-like effects, or may help to refine the dosages of antidepressant drugs.

Viscosity studies of ( l-alanine-, l-proline, l-valine, l-leucine + aqueous KCl/KNO 3) solutions at different temperatures

Viscosity coefficients of ( l-alanine-, l-proline, l-valine, l-leucine + 2.0 M aqueous KCl/KNO 3) solutions have been determined as a function of amino acid concentration at different temperatures: (298.15, 303.15, 308.15, 313.15, 318.15, and 323.15) K. The trends of variation of viscosity values with increase in the concentration of l-alanine, l-proline, l-valine, and l-leucine in 2.0 M aqueous KCl and 2.0 M aqueous KNO 3 solutions, and temperature have been ascribed to the solute–solvent interactions operative in the solutions.

Contribution of hepatic glycogenolysis and gluconeogenesis in the defense against short-term insulin induced hypoglycemia in rats

In this study, the contribution of liver glycogenolysis and gluconeogenesis in the defense against short-term insulin induced hypoglycemia (IIH) was investigated. For this purpose, we used an experimental model in which IIH was obtained by administering an IP injection of a pharmacological dose (1 U/kg) of regular insulin to rats that had been deprived of food for a period of six hours. This experimental model is suitable to study the simultaneous participation of glycogen breakdown and gluconeogenesis in the defense against IIH. The livers of IIH rats showed insignificant changes in the glycogen concentration, total phosphorylase, active phosphorylase, and percent of active phosphorylase. Our results also indicated that the livers of IIH rats that received the concentration of l-alanine, l-glutamine, l-lactate, or glycerol found in the blood during IIH (basal values) showed negligible glucose production. Nonetheless, glucose, urea, and pyruvate production increased ( P < 0.05) if the livers were perfused with a saturating concentration of gluconeogenic precursors. In agreement with these results, IIH rats that received intragastric l-alanine, l-glutamine, or l-lactate showed increased ( P < 0.05) glycemia 30 min after the administration of these substances. However, when using glycerol, higher glycemia ( P < 0.05) was observed at 2 and 5 min, but not 30 min after the administration of this hepatic gluconeogenic precursor. Thus, we can conclude that the oral availability of gluconeogenic precursors could allow for their use as important antidote in the defense against IIH.

Monitoring the Kinetics of Bacillus subtilis Endospore Germination via Surface-Enhanced Raman Scattering Spectroscopy

Mirror sandwich SERS substrates (M3S) were used to monitor the kinetics of Bacillus subtilis endospore germination. The sandwich configuration of the substrates allows real-time observation of germination in samples that contained only several hundred endospores. The enhancement provided by the substrates is attributed to the enhanced local electromagnetic field that originates from coupling between the Ag nanoparticles and the underlying metal film as well as from coupling between the Ag nanoparticles themselves. The germination kinetics at varying concentrations of l-alanine and different temperatures were studied by monitoring the intensity and growth of the Raman peak at 1010 cm(-1), which is characteristic of dipicolinic acid. A total of four concentrations (50, 75, 100, and 150 mM) of l-alanine and three different temperatures (30, 37, and 55 degrees C) were investigated.

Modelling the effect of a heat shock and germinant concentration on spore germination of a wild strain of Bacillus cereus

The effect of different concentrations of l-alanine on the germination kinetics of a strain of Bacillus cereus isolated from liquid egg after heat shock during sporulation was studied. Germination at 30 °C and was followed by spectrophotometry. The higher the concentration of l-alanine within the range 100–1 mM the faster was the germination obtained. On the other hand, the application of a heat shock had an effect on the germination producing a diminution on the germination kinetic rates. The Weibull distribution function, a model that has been used for describing inactivation kinetics [Collado, J., Fernández, A., Cunha, L., Ocio, M.J., Martínez, 2003. Improved model based on the Weibull distribution to describe the combined effect of pH and temperature on the heat resistance of three strains of Bacillus cereus in carrot juice. Journal of Food Protection 66 (6), 978–984; Loss, C.R., Hotchkiss, S.H., 2004. Determination of thermal inactivation kinetics of microorganisms with continuous micro flow apparatus. Journal of Food Protection 67 (11) 2560–2564; Chen, H., Hoover, D.G., 2004. Use of Weibull model to describe and predict pressure inactivation of Lysteria monocytogenes Scott A in whole milk. Innovative Food Science and Emerging Technologies 5 (3) 269–276; Guerrero, S., Tognom, M., Alzamora, S.M., 2005. Response of Saccharomyces cerevisiae to the combined action of ultrasound and low weight chitosan. Food Control 16 (2) 131–139] , was used for modelling germination experimental data. Results indicated that the Weibull distribution function model produced a good description of experimental data.

A microtiter fluorometric assay to detect the germination of Bacillus anthracis spores and the germination inhibitory effects of antibodies

Bacillus anthracis spore germination is usually detected in vitro by alterations in spore refractility, heat resistance, and stainability. We developed a more quantitative, sensitive, and semi-automated procedure for detecting germination by using a microtiter kinetic reader for fluorescence spectrophotometry. The procedure was based on the increase in fluorescence of spores with time during their incubation in germination medium containing a fluorescent nucleic acid-binding dye which stained germinated B. anthracis but not ungerminated (UG) spores. Spore germination in the presence of several germinants was characterized. Although l-alanine and inosine alone stimulated rapid germination in this assay, a medium containing optimal concentrations of l-alanine, adenosine, and casamino acids gave low background fluorescence, stimulated germination completely, and at a reasonable rate. Suspensions of heat-activated, UG spores of B. anthracis strain Ames were preincubated with antibodies (Abs) against whole spores to assess their effect on germination. Analyses of the germination data obtained revealed significant differences between spores pretreated with these Abs and those treated with non-immune sera or IgG. Germination inhibitory activity (GIA) was detected for several polyclonal rabbit anti-spore Ab preparations. These included anti-Ames strain spore antisera, IgG purified from the latter, and spore affinity-purified Abs from antisera elicited against four strains of B. anthracis. Abs elicited against UG as well as completely germinated Ames spores inhibited germination. Abs were ranked according to their GIA, and those specific for UG spores usually exhibited greater GIA. Direct binding to spores of these Abs was detected by an ELISA with whole un-germinated Ames spores. Although specific binding to spores by the anti-spore Abs was shown, their titers did not correlate with their GIA levels. Current efforts are focused on identifying the spore antigens recognized by the anti-spore Abs, characterizing the role of these targeted antigens in disease pathogenesis, and evaluating the ability of specific anti-spore Abs to protect against infection with B. anthracis.

Exercise and recovery metabolism in the Pacific spiny dogfish (Squalus acanthias).

We examined the effects of exhaustive exercise and post-exercise recovery on white muscle substrate depletion and metabolite distribution between white muscle and blood plasma in the Pacific spiny dogfish, both in vivo and in an electrically stimulated perfused tail-trunk preparation. Measurements of arterial-venous lactate, total ammonia, beta-hydroxybutyrate, glucose, and L-alanine concentrations in the perfused tail-trunk assessed white muscle metabolite fluxes. Exhaustive exercise was fuelled primarily by creatine phosphate hydrolysis and glycolysis as indicated by 62, 71, and 85% decreases in ATP, creatine phosphate, and glycogen, respectively. White muscle lactate production during exercise caused a sustained increase (approximately 12 h post-exercise) in plasma lactate load and a short-lived increase (approximately 4 h post-exercise) in plasma metabolic acid load during recovery. Exhaustive exercise and recovery did not affect arterial PO2, PCO2, or PNH3 but the metabolic acidosis caused a decrease in arterial HCO3- immediately after exercise and during the first 8 h recovery. During recovery, lactate was retained in the white muscle at higher concentrations than in the plasma despite increased lactate efflux from the muscle. Pyruvate dehydrogenase activity was very low in dogfish white muscle at rest and during recovery (0.53 +/- 0.15 nmol g wet tissue(-1) min(-1); n=40) indicating that lactate oxidation is not the major fate of lactate during post-exercise recovery. The lack of change in white muscle free-carnitine and variable changes in short-chain fatty acyl-carnitine suggest that dogfish white muscle does not rely on lipid oxidation to fuel exhaustive exercise or recovery. These findings support the notion that extrahepatic tissues cannot utilize fatty acids as an oxidative fuel. Furthermore, our data strongly suggest that ketone body oxidation is important in fuelling recovery metabolism in dogfish white muscle and at least 20% of the ATP required for recovery could be supplied by uptake and oxidation of beta-hydroxybutyrate from the plasma.

Isozymic nature of spore coat-associated alanine racemase of Bacillus subtilis.

Spore coat-associated alanine racemase of Bacillus subtilis, which converts L-alanine to D-alanine, that is, the germinant to the competitive inhibitor, to regulate spore germination for survival of the organism under unfavorable growth conditions, was examined. The dormant spores, L-alanine-initiated germination of which is inhibited by diphenylamine, were used to characterize the enzyme in the native form because of its unextractablility from dormant spores. The presence of isozymes, Enz-I and Enz-II with Km for L-alanine of about 20mM and 50mM and optimum activity at around 40 degrees C and 65 degrees C, respectively, was proposed. The enzymes were selectively used depending on the L-alanine concentration and the temperature. The pH profiles of the activity (optimun at pH 9.0) and the stability (stable between pH 6-11 at 60 degrees C) were similar, but Enz-II was more heat-stable than Enz-I and the denaturation curve demonstrated a two-domain structure for Enz-II. Sensitivity to D-penicillamine, hydroxylamine and HgCl2 was similar between Enz-I and Enz-II, while that to D-cycloserine, L- and D-aminoethylphosphonic acid, monoiodoacetate and N-ethylmaleimide was different; HgCl2 was the most effective inhibitor among these compounds.

Seasonal change and prolonged anoxia affect the kinetic properties of phosphofructokinase and pyruvate kinase in oysters

The effects of seasonal change, November versus July, and prolonged anoxia (96 h under N2 gas) on the properties of phosphofructokinase and pyruvate kinase from five tissues (gill, mantle, hepatopancreas, phasic adductor, catch adductor) of the oyster, Crassostrea virginica were investigated. Both enzymes showed tissue-specific and season-specific changes in kinetic properties; for pyruvate kinase this correlated with seasonal differences in enzyme elution patterns on hydroxylapatite chromatography. Kinetic properties of both enzymes in winter were consistent with primarily catabolic roles in glycolysis with responsiveness to cellular energy demands, whereas in summer these enzymes may be more closely regulated with respect to the biosynthetic and gluconeogenic functions of the tissues. Anoxia-induced changes in phosphofructokinase properties were relatively minor but anoxia stimulated changes in pyruvate kinase properties and elution profiles on hydroxylapatite in all tissues except mantle, with much greater effects seen for the enzyme from winter versus summer animals. For example, anoxia-induced changes in pyruvate kinase from winter gill included a fourfold rise in the substrate affinity constant for phosphoenolpyruvate, a sevenfold increase in the concentration of fructose-1,6-bisphosphate needed to activate the enzyme by 50%, and a 50% decrease in the concentration of L-alanine that inhibits activity by 50%. Changes in pyruvate kinase kinetics and hydroxylapatite elution patterns during prolonged anoxia are consistent with covalent modification of pyruvate kinase but contrary to results for many other mollusc species, anoxia exposure appears to induce a dephosphorylation of the enzyme.

Cellular uptake of valine by lactating porcine mammary tissue.

The cellular uptake of branched-chain amino acids in mammary tissue is important for understanding their role in milk synthesis in the sow. This study characterized the kinetic properties and substrate specificity of the valine uptake system in the porcine mammary gland. Mammary tissue was collected from lactating sows at slaughter and tissue explants were incubated in media containing isosmotic salt and amino acids of interest, plus [3H]valine tracer. Valine uptake was time-dependent and was dependent on the presence of sodium, as indicated by a reduction in uptake when sodium in the medium was replaced by choline. The valine transport system in porcine mammary tissue had a Km of 0.64 mM, a Vmax of 1.84 mmol-kg cell water(-1) 30 min(-l), and a Kd (diffusion constant) of 1.16 L x kg cell water(-1) x 30 min(-1). Valine uptake was inhibited by leucine and alpha-aminoisobutyric acid and by high concentrations of L-alanine, L-lysine, cycloleucine, L-glutamine, and L-methionine, but not by 2-(methyl-amino)-isobutyric acid. This transport system is the primary system responsible for uptake of valine, and probably other branched-chain amino acids, in lactating sow mammary tissue. Physiological concentrations of valine in the blood are below the Km of the specific valine transport system and well below the diffusion uptake capabilities. The kinetic parameters of this valine transport system should not be limiting to valine uptake for milk protein synthesis. However, competition of valine uptake with branched-chain amino acids, as well as with other amino acids, may affect valine uptake in lactating tissue.

Lysine uptake by mammary gland tissue from lactating sows.

Kinetic properties and substrate specificity of the lysine transport system in porcine mammary gland were studied using mammary tissue explants from nine lactating sows. Sodium dependence of lysine uptake was determined by replacing sodium in the medium with choline. Kinetic parameters of lysine uptake were determined using lysine concentrations from 5 microM to 5.12 mM. Competition of lysine uptake by other amino acids was determined using the cationic amino acids, arginine and ornithine, and using other essential amino acids. Transport of lysine was time-dependent and was unaffected by replacing sodium with choline. Lysine uptake occurred by a transport mechanism with a Km of approximately 1.4 mM and a Vmax of 7.9 mmol x kg cell water(-1) x 30 min(-1). Lysine uptake was inhibited by arginine and ornithine and by high concentrations of L-alanine, L-methionine, L-leucine, cycloleucine, and D-lysine, but not by 2-(methylamino)-isobutyric acid. This transport mechanism is the primary system responsible for uptake of cationic amino acids in lactating sow mammary tissue. The relatively high Km, compared with physiological blood concentrations of lysine, indicates that the kinetic properties of the lysine transport system should not be limiting to milk protein synthesis. Transmembrane transport of lysine by lactating sow mammary tissue should be a direct function of plasma concentrations. However, interactions of other amino acids with the uptake system may affect lysine uptake.

Role of the gerI operon of Bacillus cereus 569 in the response of spores to germinants.

Bacillus cereus 569 (ATCC 10876) germinates in response to inosine or to L-alanine, but the most rapid germination response is elicited by a combination of these germinants. Mutants defective in their germination response to either inosine or to L-alanine were isolated after Tn917-LTV1 mutagenesis and enrichment procedures; one class of mutant could not germinate in response to inosine as a sole germinant but still germinated in response to L-alanine, although at a reduced rate; another mutant germinated normally in response to inosine but was slowed in its germination response to L-alanine. These mutants demonstrated that at least two signal response pathways are involved in the triggering of germination. Stimulation of germination in L-alanine by limiting concentrations of inosine and stimulation of germination in inosine by low concentrations of L-alanine were still detectable in these mutants, suggesting that such stimulation is not dependent on complete functionality of both these germination loci. Two transposon insertions that affected inosine germination were found to be located 2.2 kb apart on the chromosome. This region was cloned and sequenced, revealing an operon of three open reading frames homologous to those in the gerA and related operons of Bacillus subtilis. The individual genes of this gerI operon have been named gerIA, gerIB, and gerIC. The GerIA protein is predicted to possess an unusually long, charged, N-terminal domain containing nine tandem copies of a 13-amino-acid glutamine- and serine-rich sequence.

Amperometric biosensors for L-alanine and pyruvate assays in biological fluids

Two amperometric electrochemical biosensors have been developed and used to determine the concentrations of L-alanine and pyruvate in biological fluids (blood, fermentation media) without any prior treatment of the sample. The proposed biosensors have response times lower than 5 min, a precision of 2% and the measured current is proportional to the substrate concentration in the range from 1×10 −6 to 2×10 −3 mol l −1. They have an operating life of one month.