Quantification and efficiency of Lactobacillus sakei strain mixtures used as protective cultures in ground beef.

Lactobacillus sakei is a lactic acid bacterium, naturally associated with long term storage of fresh meat at low temperature. Here we investigated the effect, on the evolution of pathogenic and spoilage microorganisms in ground beef, of L. sakei cocktails used as bioprotective cultures. We selectively developed a real time quantitative PCR method, allowing the quantification of individual L. sakei strains inoculated in ground meat with specific probes. Six cocktails of three strains were tested to evaluate their effect on the growth of Salmonella enterica Typhimurium, Escherichia coli O157:H7 and Brochothrix thermosphacta at 4°C and 8°C, under vacuum or modified atmosphere packaging. Using plating methods to quantify the different bacterial species, one cocktail showed an effect against S. Typhimurium and E. coli under given conditions. Real time quantitative PCR showed that the three inoculated L. sakei strains had a different growth pattern, and that the association of these three strains indeed impaired growth of S. Typhimurium and E. coli.

AGMIAL: implementing an annotation strategy for prokaryote genomes as a distributed system.

We have implemented a genome annotation system for prokaryotes called AGMIAL. Our approach embodies a number of key principles. First, expert manual annotators are seen as a critical component of the overall system; user interfaces were cyclically refined to satisfy their needs. Second, the overall process should be orchestrated in terms of a global annotation strategy; this facilitates coordination between a team of annotators and automatic data analysis. Third, the annotation strategy should allow progressive and incremental annotation from a time when only a few draft contigs are available, to when a final finished assembly is produced. The overall architecture employed is modular and extensible, being based on the W3 standard Web services framework. Specialized modules interact with two independent core modules that are used to annotate, respectively, genomic and protein sequences. AGMIAL is currently being used by several INRA laboratories to analyze genomes of bacteria relevant to the food-processing industry, and is distributed under an open source license.

Intrahepatic CD4 T-Cell apoptosis is related to METAVIR score in patients with chronic hepatitis C virus.

Hepatitis C virus (HCV) infection leads to liver injury, which is thought to be immune-mediated. Apoptosis of hepatic T cells could influence histological damage. We quantified peripheral and intrahepatic T-cell apoptosis in 28 patients with chronic hepatitis C by using cytofluorometric techniques. METAVIR score and HCV plasma viral load were determined. Six liver biopsies, obtained from controls without chronic hepatitis during hepatobiliary surgery, served as controls. In patients, liver T-cell apoptosis was upregulated compared to peripheral T cells: 35 versus 7% for CD4+ and 56 versus 13% for CD8+ T cells (P < 0.001). Liver T-cell apoptosis levels from patients were increased compared to controls for both CD4+ (P = 0.041) and CD8+ T cells (P = 0.007). Nine patients exhibiting METAVIR scores A and F < or = 1 showed higher intrahepatic CD4+ T-cell apoptosis compared to the 19 patients with a higher METAVIR score (P = 0.001) and both histological activity and fibrosis were related to apoptosis level. There was also an inverse relationship between the level of intrahepatic CD8+ T-cell apoptosis and serum transaminase activity (P = 0.023). Our study shows immune compartmentalization, suggesting that the study of peripheral blood lymphocytes may not be fully relevant to the pathophysiology of HCV hepatitis, and that the severity of liver injury is inversely correlated with intrahepatic CD4+ T-cell apoptosis.

Intracellular concentration of protease inhibitors in HIV-1-infected patients: correlation with MDR-1 gene expression and low dose of ritonavir.

BACKGROUND: Protease inhibitors (PIs) are substrates for the P-glycoprotein (P-gp/170) encoded by the multi-drug resistance gene (MDR-1). HIV infection is associated with increased expression of P-gp. The role of MDR gene overexpression in clinical pharmacokinetics is not known. METHOD: We determined by HPLC, at trough and peak levels, the current PI concentrations in plasma (P) and in peripheral blood mononuclear cells (PBMCs) (intracellular concentration [IC]) from 49 HIV-infected patients receiving different treatment combinations: nelfinavir ([NFV] n = 12); indinavir ([IDV] n = 10); amprenavir ([APV] n = 5); ritonavir (RTV) 100 bid/IDV 800 mg bid (n = 6); RTV 400 bid/IDV 400 mg bid (n = 3); RTV 100 bid/saquinavir (SQV) 600 mg tid (n = 9); APV 600 bid/RTV 100 mg bid (n = 4). We determined the mean ratio of intracellular/plasma PI concentration for each treatment group. The MDR-1 gene expression was determined by a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). HIV viral load was simultaneously measured. RESULTS: 49 patients (mean age 41 +/- 8.7 years; mean CD4 cell count 418 [57-972]; mean HIV RNA 2.1 +/- 0.8 log(10)) were included in the study. Patients who overexpressed the MDR-1 gene had significantly lower trough intracellular PI levels (p =.02) or lower intracellular accumulation of PI (p =.042). Patients treated with low-dose RTV in combined regimens with detectable RTV intracellular concentration showed lack of MDR-1 gene expression (p =.01). Patients with HIV RNA < 40 copies/mL had significantly higher RTV intracellular accumulation (p =.029). CONCLUSION: In HIV-infected patients, IC of PI is inversely correlated with MDR-1 gene overexpression. Undetectable viral load was associated with the use of low-dose RTV, probably linked to better intracellular accumulation of the drug. Nevertheless, further investigation is needed to confirm these results.

Consensual treatment of plasmodium falciparum malaria does not lead to adequate quinine blood concentrations.

Since 1996, we have a common protocol in the Infectious Diseases Department and the Intensive Care Unit for the administration of quinine in case of Plasmodium falciparum malaria. Patients were classified as uncomplicated form of malaria (UFM) or severe form of malaria (SFM) according to WHO criteria, adding parasitemia >5% as a criteria of SFM. Treatment of SFM should consist of a 4 h infusion of 16 mg/kg quinine-base loading dose, followed by 8 mg/kg every 8 h. Patients with UFM receive quinine-base, 8 mg/kg every 8 h. A therapeutic index of 10-15 mg/l was considered adequate. Hypoglycemia and cardiotoxicity were the two main adverse effects of quinine to be investigated. In order to verify that these modalities for quinine administration are associated with adequate quinine blood concentrations, we have reviewed the pharmacological data and the occurrence of adverse effects. Between April 1996 and December 2000, 95 patients were hospitalised: 25 with SFM and 70 with UFM: 78/95 patients (82%) received adequate treatment and 26/95 (28%) of the patients presented an overdosage of quinine. Six severe adverse effects were observed, even in case of adequate quinine administration. Consensual treatment of malaria does not confer adequate quinine blood concentrations, and toxic effects are still common.

Bay-0 x Shahdara recombinant inbred line population: a powerful tool for the genetic dissection of complex traits in Arabidopsis.

Natural genetic variation in Arabidopsis is considerable, but has not yet been used extensively as a source of variants to identify new genes of interest. From the cross between two genetically distant ecotypes, Bay-0 and Shahdara, we generated a Recombinant Inbred Line (RIL) population dedicated to Quantitative Trait Locus (QTL) mapping. A set of 38 physically anchored microsatellite markers was created to construct a robust genetic map from the 420 F6 lines. These markers, evenly distributed throughout the five chromosomes, revealed a remarkable equilibrium in the segregation of parental alleles in the genome. As a model character, we have analysed the genetic basis of variation in flowering time in two different environments. The simultaneous mapping of both large- and small-effect QTLs responsible for this variation explained 90% of the total genotypic variance. Two of the detected QTLs colocalize very precisely with FRIGIDA and FLOWERING LOCUS C genes; we provide information on the polymorphism of genes confirming this hypothesis. Another QTL maps in a region where no QTL had been found previously for this trait. This confirms the accuracy of QTL detection using the Bay-0 x Shahdara RIL population, which constitutes the largest in size available so far in Arabidopsis. As an alternative to mutant analysis, this population represents a powerful tool which is currently being used to undertake the genetic dissection of complex metabolic pathways.

Lactobacillus sakei: recent developments and future prospects.

Lactobacillus sakei is one of the most important bacterial species involved in meat preservation and meat fermentation. In the last fifteen years, numerous studies have focused on this species due to its important role in food microbiology. The present paper reviews current knowledge of this emerging species in the fields of taxonomy, phylogeny and physiology, and metabolism. Recent developments in genetic tools and molecular genetics will also be emphasized to evaluate future prospects.

Transport of D-xylose in Lactobacillus pentosus, Lactobacillus casei, and Lactobacillus plantarum: evidence for a mechanism of facilitated diffusion via the phosphoenolpyruvate:mannose phosphotransferase system.

We have identified and characterized the D-xylose transport system of Lactobacillus pentosus. Uptake of D-xylose was not driven by the proton motive force generated by malolactic fermentation and required D-xylose metabolism. The kinetics of D-xylose transport were indicative of a low-affinity facilitated-diffusion system with an apparent K(m) of 8.5 mM and a V(max) of 23 nmol min(-1) mg of dry weight(-1). In two mutants of L. pentosus defective in the phosphoenolpyruvate:mannose phosphotransferase system, growth on D-xylose was absent due to the lack of D-xylose transport. However, transport of the pentose was not totally abolished in a third mutant, which could be complemented after expression of the L. curvatus manB gene encoding the cytoplasmic EIIB(Man) component of the EII(Man) complex. The EII(Man) complex is also involved in D-xylose transport in L. casei ATCC 393 and L. plantarum 80. These two species could transport and metabolize D-xylose after transformation with plasmids which expressed the D-xylose-catabolizing genes of L. pentosus, xylAB. L. casei and L. plantarum mutants resistant to 2-deoxy-D-glucose were defective in EII(Man) activity and were unable to transport D-xylose when transformed with plasmids containing the xylAB genes. Finally, transport of D-xylose was found to be the rate-limiting step in the growth of L. pentosus and of L. plantarum and L. casei ATCC 393 containing plasmids coding for the D-xylose-catabolic enzymes, since the doubling time of these bacteria on D-xylose was proportional to the level of EII(Man) activity.

[Prospective comparative study of HCV serologic status in and AIDS population]. / Etude pronostique comparative d'une population SIDA en fonction du statut sérologique VHC.

OBJECTIVE: Study the influence of hepatitis C virus (HCV) serology on the course of HIV disease in AIDS patients. PATIENTS AND METHODS: A prospective study of survival prognosis in HIV infected patients who had reached the AIDS stage was conducted in the Saint-Etienne, Clermond-Ferrand and Lyons infectious disease centers to compare patients with positive and negative HCV serology. Data were collected using the clinico-epidemiological software DMI II. The effect of HCV ìco-infectionî defined by RIBA II or III confirmed seropositivity, was studied using Kaplan-Meier survival plots. RESULTS: Among the 1,005 HIV-infected subjects included in the study, 219 had AIDS and 43 of them (19.6%) were HCV positive. Survival curves in HIV/HCV positive patients with AIDS were not significantly different from those of HCV-negative AIDS patients (median 17.8 versus 18.6 months respectively, p = 0.93). This result was confirmed by univariate Kaplan-Meier analysis. Only 2 patients were treated with interferon and no deaths were attributed to liver disease. CONCLUSION: HCV positivity in AIDS patients does not appear to influence survival. The longer survival obtained with the new anti-retroviral treatments may have an effect on the HIV-HCV interaction.

Molecular cloning and functional expression in lactobacillus plantarum 80 of xylT, encoding the D-xylose-H+ symporter of Lactobacillus brevis.

A 3-kb region, located downstream of the Lactobacillus brevis xylA gene (encoding D-xylose isomerase), was cloned in Escherichia coli TG1. The sequence revealed two open reading frames which could code for the D-xylulose kinase gene (xylB) and another gene (xylT) encoding a protein of 457 amino acids with significant similarity to the D-xylose-H+ symporters of E. coli, XylE (57%), and Bacillus megaterium, XylT (58%), to the D-xylose-Na+ symporter of Tetragenococcus halophila, XylE (57%), and to the L-arabinose-H+ symporter of E. coli, AraE (60%). The L. brevis xylABT genes showed an arrangement similar to that of the B. megaterium xylABT operon and the T. halophila xylABE operon. Southern hybridization performed with the Lactobacillus pentosus xylR gene (encoding the D-xylose repressor protein) as a probe revealed the existence of a xylR homologue in L. brevis which is not located with the xyABT locus. The existence of a functional XylR was further suggested by the presence of xylO sequences upstream of xylA and xylT and by the requirement of D-xylose for the induction of D-xylose isomerase, D-xylulose kinase, and D-xylose transport activities in L. brevis. When L. brevis was cultivated in a mixture of D-glucose and D-xylose, the D-xylose isomerase and D-xylulose kinase activities were reduced fourfold and the D-xylose transport activity was reduced by sixfold, suggesting catabolite repression by D-glucose of D-xylose assimilation. The xylT gene was functionally expressed in Lactobacillus plantarum 80, a strain which lacks proton motive force-linked D-xylose transport activity. The role of the XylT protein was confirmed by the accumulation of D-xylose in L. plantarum 80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of D-glucose. The apparent affinity constant of XylT for D-xylose was approximately 215 microM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight). Furthermore, of a number of sugars tested, only 6-deoxy-D-glucose inhibited the transport of D-xylose by XylT competitively, with a Ki of 220 microM.

Functional expression in Lactobacillus plantarum of xylP encoding the isoprimeverose transporter of Lactobacillus pentosus.

The xylP gene of Lactobacillus pentosus, the first gene of the xylPQR operon, was recently found to be involved in isoprimeverose metabolism. By expression of xylP on a multicopy plasmid in Lactobacillus plantarum 80, a strain which lacks active isoprimeverose and D-xylose transport activities, it was shown that xylP encodes a transporter. Functional expression of the XylP transporter was shown by uptake of isoprimeverose in L. plantarum 80 cells, and this transport was driven by the proton motive force generated by malolactic fermentation. XylP was unable to catalyze transport of D-xylose.

Cloning, sequence analysis, and characterization of the genes involved in isoprimeverose metabolism in Lactobacillus pentosus.

Two genes, xylP and xylQ, from the xylose regulon of Lactobacillus pentosus were cloned and sequenced. Together with the repressor gene of the regulon, xylR, the xylPQ genes form an operon which is inducible by xylose and which is transcribed from a promoter located 145 bp upstream of xylP. A putative xylR binding site (xylO) and a cre-like element, mediating CcpA-dependent catabolite repression, were found in the promoter region. L. pentosus mutants in which both xylP and xylQ (LPE1) or only xylQ (LPE2) was inactivated retained the ability to ferment xylose but were impaired in their ability to ferment isoprimeverose (alpha-D-xylopyranosyl-(1,6)-D-glucopyranose). Disruption of xylQ resulted specifically in the loss of a membrane-associated alpha-xylosidase activity when LPE1 or LPE2 cells were grown on xylose. In the membrane fraction of wild-type bacteria, alpha-xylosidase could catalyze the hydrolysis of isoprimeverose and p-nitrophenyl-alpha-D-xylopyranoside with apparent Km and Vmax values of 0.2 mM and 446 nmol/min/mg of protein, and 1.3 mM and 54 nmol/min/mg of protein, respectively. The enzyme could also hydrolyze the alpha-xylosidic linkage in xyloglucan oligosaccharides, but neither methyl-alpha-D-xylopyranoside nor alpha-glucosides were substrates. Glucose repressed the synthesis of alpha-xylosidase fivefold, and 80% of this repression was released in an L. pentosus delta ccpA mutant. The alpha-xylosidase gene was also expressed in the absence of xylose when xylR was disrupted.

Regulation of expression of the Lactobacillus pentosus xylAB operon.

The xylose cluster of Lactobacillus pentosus consists of five genes, two of which, xylAB, form an operon and code for the enzymes involved in the catabolism of xylose, while a third encodes a regulatory protein, XylR. By introduction of a multicopy plasmid carrying the xyl operator and by disruption of the chromosomal xylR gene, it was shown that L. pentosus xylR encodes a repressor. Constitutive expression of xylAB in the xylR mutant is repressed by glucose, indicating that glucose repression does not require XylR. The xylR mutant displayed a prolonged lag phase compared to wild-type bacteria when bacteria were shifted from glucose to xylose medium. Differences in the growth rate in xylose medium at different stages of growth are not correlated with differences in levels of xylAB transcription in L. pentosus wild-type or xylR mutant bacteria but are positively correlated in Lactobacillus casei with a plasmid containing xylAB. Glucose repression was further investigated with a ccpA mutant. An 875-bp internal fragment of the ccpA gene of L. pentosus was isolated by PCR and used to construct a ccpA knockout mutant. Transcription analysis of L. pentosus xylA showed that CcpA is involved in glucose repression. CcpA was also shown to be involved in glucose repression of the alpha-amylase promoter of Lactobacillus amylovorus by demonstrating that glucose repression of the chloramphenicol acetyltransferase gene under control of the alpha-amylase promoter is strongly reduced in the L. pentosus ccpA mutant strain.

Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus corniculatus L. plants triggers changes in ammonium assimilation and plant development.

A soybean cytosolic glutamine synthetase gene (GS15) was fused with the constitutive 35S cauliflower mosaic virus (CaMV) promoter in order to direct overexpression in Lotus corniculatus L. plants. Following transformation with Agrobacterium rhizogenes, eight independent Lotus transformants were obtained which synthesized additional cytosolic glutamine synthetase (GS) in the shoots. To eliminate any interference caused by the T-DNA from the Ri plasmid, three primary transformants were crossed with untransformed plants and progeny devoid of TL- and TR-DNA sequences were chosen for further analyses. These plants had a 50-80% increase in total leaf GS activity. Plants were grown under different nitrogen regimes (4 or 12 mM NH4+) and aspects of carbon and nitrogen metabolism were examined. In roots, an increase in free amino acids and ammonium was accompanied by a decrease in soluble carbohydrates in the transgenic plants cultivated with 12 mM NH4+ in comparison to the wild type grown under the same conditions. Labelling experiments using 15NH4+ were carried out in order to monitor the influx of ammonium and its subsequent incorporation into amino acids. This experiment showed that both ammonium uptake in the roots and the subsequent translocation of amino acids to the shoots was lower in plants overexpressing GS. It was concluded that the build up of ammonium and the increase in amino acid concentration in the roots was the result of shoot protein degradation. Moreover, following three weeks of hydroponic culture early floral development was observed in the transformed plants. As all these properties are characteristic of senescent plants, these findings suggest that expression of cytosolic GS in the shoots may accelerate plant development, leading to early senescence and premature flowering when plants are grown on an ammonium-rich medium.

Responses of soybean to ammonium and nitrate supplied in combination to the whole root system or separately in a split-root system.

To address the questions of whether allocation of carbohydrates to roots is influenced by ionic form of nitrogen absorbed and whether allocation of carbohydrates to roots in turn influences proportionality between NH4+ and NO3- uptake from mixed sources, NH4+ and NO3- were supplied separately to halves of a split-root hydroponic system and were supplied in combination to a whole-root system. Dry matter accumulation in the split-root system was 18% less in the NH4(+)-fed axis than in the NO3(-)-fed axis. This, however, does not indicate that partitioning of carbohydrate between the two axes was different. Most of the reduction in dry matter accumulation in the NH4(+)-fed axis can be accounted for by the retransport of CH2O equivalents from the root back to the shoot with amino acids produced by NH4+ assimilation. Uptake of NH4+ or NO3- by the respective halves of the split-root system was proportional to the estimated allocation of carbohydrate to that half. When NH4+ and NO3- were supplied to separate halves of the split-root system, the cumulative NH4+ to NO3- uptake ratio was 0.81. When supplied in combination to the whole-root system, the cumulative NH4+ to NO3- uptake ratio was 1.67. Thus, while the shoot may affect total nitrogen uptake through the export of carbohydrates to roots, the shoot (common for halves of the split-root system) apparently does not exert a direct effect on proportionality of NH4+ and NO3- uptake by roots. For whole roots supplied with both NH4+ and NO3-, the restriction in uptake of NO3- may involve a stimulation of NO3- efflux rather than an inhibition of NO3- influx. While only the net uptake of NH4+ and NO3- was measured by ion chromatography, monitoring at approximately hourly intervals during the first 3 days of treatment revealed irregularly occurring intervals of both depletion (net influx) and enrichment (net efflux) in solutions. In the case of NH4+, numbers of net efflux events were similar (21 to 24 out of 65 sequential sampling intervals) whether NH4+ was supplied with NO3- to whole-root systems or separately to an axis of the split-root system. In the case of NO3-, however, the number of net efflux events increased from 8 when NO3- was supplied to a separate axis of the split-root system to between 19 and 24 when NO3- was supplied with NH4+ to whole-root systems.

Ammonium and nitrate uptake by soybean during recovery from nitrogen deprivation.

Soybean [Glycine max (L.) Merrill] plants that had been subjected to 15 d of nitrogen deprivation were resupplied for 10 d with 1.0 mol m-3 nitrogen provided as NO3-, NH4+, or NH4(+) + NO3- in flowing hydroponic culture. Plants in a fourth hydroponic system received 1.0 mol m-3 NO3- during both stress and resupply periods. Concentrations of soluble carbohydrates and organic acids in roots increased 210 and 370%, respectively, during stress. For the first day of resupply, however, specific uptake rates of nitrogen, determined by ion chromatography as depletion from solution, were lower for stressed than for non-stressed plants by 43% for NO3- resupply, by 32% for NH4(+) + NO3- resupply, and 86% for NH4+ resupply. When specific uptake of nitrogen for stressed plants recovered to rates for non-stressed plants at 6 to 8 d after nitrogen resupply, carbohydrates and organic acids in their roots had declined to concentrations lower than those of non-stressed plants. Recovery of nitrogen uptake capacity of roots thus does not appear to be regulated simply by the content of soluble carbon compounds within roots. Solution concentrations of NH4+ and NO3- were monitored at 62.5 min intervals during the first 3 d of resupply. Intermittent 'hourly' intervals of net influx and net efflux occurred. Rates of uptake during influx intervals were greater for the NH4(+)-resupplied than for the NO3(-)-resupplied plants. For NH4(+)-resupplied plants, however, the hourly intervals of efflux were more numerous than for NO3(-)-resupplied plants. It thus is possible that, instead of repressing NH4+ influx, increased accumulation of amino acids and NH4+ in NH4(+)-resupplied plants inhibited net uptake by stimulation of efflux on NH4+ absorbed in excess of availability of carbon skeletons for assimilation. Entry of NH4+ into root cytoplasm appeared to be less restricted than translocation of amino acids from the cytoplasm into the xylem.

Expression of characteristics of ammonium nutrition as affected by pH of the root medium.

To study the effect of root-zone pH on characteristic responses of NH4+ -fed plants, soybeans (Glycine max¿L.¿ Merr. cv. Ransom) were grown in flowing solution culture for 21 d on four sources of N (1.0 mol m-3 NO3-, 0.67 mol m-3 NO3- plus 0.33 mol m-3 NH4+, 0.33 mol m-3 NO3- plus 0.67 mol m-3 NH4+, and 1.0 mol m-3 NH4+) with nutrient solutions maintained at pH 6.0, 5.5, 5.0, and 4.5. Amino acid concentration increased in plants grown with NH4+ as the sole source of N at all pH levels. Total amino acid concentration in the roots of NH4+ -fed plants was 8 to 10 times higher than in NO3(-)-fed plants, with asparagine accounting for more than 70% of the total in the roots of these plants. The concentration of soluble carbohydrates in the leaves of NH4+ -fed plants was greater than that of NO3(-)-fed plants, but was lower in roots of NH4+ -fed plants, regardless of pH. Starch concentration was only slightly affected by N source or root-zone pH. At all levels of pH tested, organic acid concentration in leaves was much lower when NH4+ was the sole N source than when all or part of the N was supplied as NO3-. Plants grown with mixed NO3- plus NH4+ N sources were generally intermediate between NO3(-)- and NH4+ -fed plants. Thus, changes in tissue composition characteristic of NH4+ nutrition when root-zone pH was maintained at 4.5 and growth was reduced, still occurred when pH was maintained at 5.0 or above, where growth was not affected. The changes were slightly greater at pH 4.5 than at higher pH levels.

Cyclic variations in nitrogen uptake rate of soybean plants: effects of pH and mixed nitrogen sources.

To determine if the daily pattern of NO3- and NH4+ uptake is affected by acidity or NO3- : NH4+ ratio of the nutrient solution, non-nodulated soybean plants (Glycine max) were exposed for 21 days to replenished, complete nutrient solutions at pH 6.0, 5.5, 5.0, and 4.5 which contained either 1.0 mM NH4+, 1.0 mM NO3- [correction of NO3+], 0.67 mM NH4+ plus 0.33 mM NO3- (2:1 NH4+ : NO3-) [correction of (2:1 NH3+ : NO4-)], or 0.33 mM NH4+ plus 0.67 mM NO3- (1:2 NH4+ : NO3-). Net uptake rates of NH4+ and NO3- were measured daily by ion chromatography as depletion from the replenished solutions. When NH4+ and NO3- were supplied together, cumulative uptake of total nitrogen was not affected by pH or solution NH4+ : NO3- ratio. The cumulative proportion of nitrogen absorbed as NH4+ decreased with increasing acidity; however, the proportional uptake of NH4+ and NO3- was not constant, but varied day-to-day. This day-to-day variation in relative proportions of NH4+ and NO3- absorbed when NH4+ : NO3- ratio and pH of solution were constant indicates that the regulatory mechanism is not directly competitive. Regardless of the effect of pH on cumulative uptake of NH4+, the specific nitrogen uptake rates from mixed and from individual NH4+ and NO3- sources oscillated between maxima and minima at each pH with average periodicities similar to the expected interval of leaf emergence.