scholarly journals Two Nucleoside Uptake Systems in Lactococcus lactis: Competition between Purine Nucleosides and Cytidine Allows for Modulation of Intracellular Nucleotide Pools

2003 ◽  
Vol 185 (5) ◽  
pp. 1503-1508 ◽  
Author(s):  
Jan Martinussen ◽  
Steen L. L. Wadskov-Hansen ◽  
Karin Hammer
Keyword(s):  
Growth Rate ◽  
Lactococcus Lactis ◽  
Laboratory Strain ◽  
Defined Medium ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Nucleoside Triphosphate ◽  
Buffer System ◽  
Uridine Uptake ◽  
Nucleotide Pools

ABSTRACT A method for measuring internal nucleoside triphosphate pools of lactococci was optimized and validated. This method is based on extraction of 33P-labeled nucleotides with formic acid and evaluation by two-dimensional chromatography with a phosphate buffer system for the first dimension and with an H3BO3-LiOH buffer for separation in the second dimension. We report here the sizes of the ribo- and deoxyribonucleotide pools in laboratory strain MG1363 during growth in a defined medium. We found that purine- and pyrimidine-requiring strains may be used to establish physiological conditions in batch fermentations with altered nucleotide pools and growth rates by addition of nucleosides in different combinations. Addition of cytidine together with inosine to a purine-requiring strain leads to a reduction in the internal purine nucleotide pools and a decreased growth rate. This effect was not seen if cytidine was replaced by uridine. A similar effect was observed if cytidine and inosine were added to a pyrimidine-requiring strain; the UTP pool size was significantly decreased, and the growth rate was reduced. To explain the observed inhibition, the nucleoside transport systems in Lactococcus lactis were investigated by measuring the uptake of radioactively labeled nucleosides. The Km for for inosine, cytidine, and uridine was determined to be in the micromolar range. Furthermore, it was found that cytidine and inosine are competitive inhibitors of each other, whereas no competition was found between uridine and either cytidine or inosine. These findings suggest that there are two different high-affinity nucleoside transporters, one system responsible for uridine uptake and another system responsible for the uptake of all purine nucleosides and cytidine.

scholarly journals Nucleoside uptake in rat liver parenchymal cells

1996 ◽  
Vol 317 (3) ◽  
pp. 835-842 ◽  
Author(s):  
Joan MERCADER ◽  
Mireia GOMEZ-ANGELATS ◽  
Belén del SANTO ◽  
Javier CASADO ◽  
Antonio F. FELIPE ◽  
...  
Keyword(s):  
Rat Liver ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Dependent Manner ◽  
Transport Activity ◽  
Liver Parenchymal ◽  
Uridine Uptake ◽  
High Concentrations ◽  
Parenchymal Cells ◽  
Dose Dependent

Rat liver parenchymal cells express Na+-dependent and Na+-independent nucleoside transport activity. The Na+-dependent component shows kinetic properties and substrate specificity similar to those reported for plasma membrane vesicles [Ruiz-Montasell, Casado, Felipe and Pastor-Anglada (1992) J. Membr. Biol. 128, 227–233]. This transport activity shows apparent Km values for uridine in the range 8–13 μM and a Vmax of 246 pmol of uridine per 3 min per 106 cells. Most nucleosides, including the analogue formycin B, cis-inhibit Na+-dependent uridine transport, although thymidine and cytidine are poor inhibitors. Inosine and adenosine inhibit Na+-dependent uridine uptake in a dose-dependent manner, reaching total inhibition. Guanosine also inhibits Na+-dependent uridine uptake, although there is some residual transport activity (35% of the control values) that is resistant to high concentrations of guanosine but may be inhibited by low concentrations of adenosine. The transport activity that is inhibited by high concentrations of thymidine is similar to the guanosine-resistant fraction. These observations are consistent with the presence of at least two Na+-dependent transport systems. Na+-dependent uridine uptake is sensitive to N-ethylmaleimide treatment, but Na+-independent transport is not. Nitrobenzylthioinosine (NBTI) stimulates Na+-dependent uridine uptake. The NBTI effect involves a change in Vmax, it is rapid, dose-dependent, does not need preincubation and can be abolished by depleting the Na+ transmembrane electrochemical gradient. Na+-independent uridine transport seems to be insensitive to NBTI. Under the same experimental conditions, NBTI effectively blocks most of the Na+-independent uridine uptake in hepatoma cells. Thus the stimulatory effect of NBTI on the concentrative nucleoside transporter of liver parenchymal cells cannot be explained by inhibition of nucleoside efflux.

scholarly journals Transcriptional Pattern of Genes Coding for the Proteolytic System of Lactococcus lactis and Evidence for Coordinated Regulation of Key Enzymes by Peptide Supply

2001 ◽  
Vol 183 (12) ◽  
pp. 3614-3622 ◽  
Author(s):  
Eric Guédon ◽  
Pierre Renault ◽  
S. Dusko Ehrlich ◽  
Christine Delorme
Keyword(s):  
Lactococcus Lactis ◽  
Defined Medium ◽  
Transport Systems ◽  
Luciferase Reporter ◽  
Catabolic Repression ◽  
Proteolytic System ◽  
Transcriptional Pattern ◽  
Genes Encoding ◽  
Highly Expressed Genes ◽  
Responsive Element

ABSTRACT The transcription of 16 genes encoding 12 peptidases (pepC, pepN, pepX, pepP, pepA, pepF2, pepDA1, pepDA2, pepQ, pepT, pepM, and pepO1), PI and PIII proteinases (prtP1 and prtP3), and three transport systems (dtpT, dtpP, and opp-pepO1) ofLactococcus lactis MG1363 was analyzed in response to different environmental factors. Promoter fusions with luciferase reporter genes and/or mRNA analysis were used to study the effects of sugar sources, growth at 37°C, and peptide supply on the transcription of these genes. Only transcription of thepepP gene is modulated by the source of sugar. The presence of potential catabolite-responsive element (CRE) boxes in its promoter region suggests that expression of this gene is directly controlled by catabolic repression. Elevated temperature had no significant effect on the level of transcription of these genes. prtP1, prtP3, pepC, pepN, pepX, and the opp-pepO1 operon are the most highly expressed genes in chemically defined medium, and their expression is repressed 5- to 150-fold by addition of peptide sources such as Casitone in the medium. Moreover, the transcription ofprtP1, prtP3, pepC, pepN, and the opp-pepO1operon is repressed two- to eight-fold by the dipeptides leucylproline and prolylleucine. The transcription of pepDA2 might also be repressed by the peptide sources, but this effect is not observed on the regulation of dtpT, pepP, pepA, pepF2, pepDA1, pepQ, pepT, pepM, and the dtpP operon. The significance of these results with respect to the functions of different components of the proteolytic system in L. lactis are discussed.

Characterization of nucleoside transport systems in cultured rat epididymal epithelium

2001 ◽  
Vol 280 (5) ◽  
pp. C1076-C1082 ◽  
Author(s):  
George P. H. Leung ◽  
Jeffrey L. Ward ◽  
Patrick Y. D. Wong ◽  
Chung-Ming Tse
Keyword(s):  
Nucleoside Transport ◽  
Transport Systems ◽  
Thymidine Uptake ◽  
Nucleoside Transporter ◽  
Equilibrative Nucleoside Transporter ◽  
Functional Studies ◽  
Uridine Uptake ◽  
Concentrative Nucleoside Transporter ◽  
Nucleoside Uptake

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na+ dependent, while 30% was Na+ independent. The Na+-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na+-dependent [3H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na+-dependent [3H]guanosine uptake was inhibited by thymidine by 20% and Na+-dependent [3H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na+-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.

scholarly journals Twofold Reduction of Phosphofructokinase Activity in Lactococcus lactis Results in Strong Decreases in Growth Rate and in Glycolytic Flux

2001 ◽  
Vol 183 (11) ◽  
pp. 3458-3467 ◽  
Author(s):  
Heidi W. Andersen ◽  
Christian Solem ◽  
Karin Hammer ◽  
Peter R. Jensen
Keyword(s):  
Growth Rate ◽  
Lactococcus Lactis ◽  
Defined Medium ◽  
Glycolytic Flux ◽  
Wild Type ◽  
Synthetic Promoters ◽  
Phosphofructokinase Activity ◽  
Sugar Phosphates ◽  
Strong Control ◽  
Reduced Activity

ABSTRACT Two mutant strains of Lactococcus lactis in which the promoter of the las operon, harboring pfk, pyk, and ldh, were replaced by synthetic promoters were constructed. These las mutants had an approximately twofold decrease in the activity of phosphofructokinase, whereas the activities of pyruvate kinase and lactate dehydrogenase remained closer to the wild-type level. In defined medium supplemented with glucose, the growth rate of the mutants was reduced to 57 to 70% of wild-type levels and the glycolytic flux was reduced to 62 to 76% of wild-type levels. In complex medium growth was even further reduced. Surprisingly, the mutants still showed homolactic fermentation, which indicated that the limitation was different from standard glucose-limited conditions. One explanation could be that the reduced activity of phosphofructokinase resulted in the accumulation of sugar-phosphates. Indeed, when one of the mutants was starved for glucose in glucose-limited chemostat, the growth rate could gradually be increased to 195% of the growth rate observed in glucose-saturated batch culture, suggesting that phosphofructokinase does affect the concentration of upstream metabolites. The pools of glucose-6-phosphate and fructose-6-phosphate were subsequently found to be increased two- to fourfold in the las mutants, which indicates that phosphofructokinase exerts strong control over the concentration of these metabolites.

Varying influence of the autolysin, N-acetyl muramidase, and the cell envelope proteinase on the rate of autolysis of six commercial Lactococcus lactis cheese starter bacteria grown in milk

2000 ◽  
Vol 67 (4) ◽  
pp. 585-596 ◽  
Author(s):  
SELVARANI GOVINDASAMY-LUCEY ◽  
PRAMOD K. GOPAL ◽  
PATRICK A. SULLIVAN ◽  
CHRISTOPHER J. PILLIDGE
Keyword(s):  
Lactococcus Lactis ◽  
Cell Walls ◽  
Cell Envelope ◽  
Laboratory Strain ◽  
Proteolytic Cleavage ◽  
Zymogram Analysis ◽  
Sds Page ◽  
Derivatives Of ◽  
Free Strains

The autolysin, N-acetyl muramidase (AcmA), of six commercial Lactococcus lactis subsp. cremoris starter strains and eight Lc. lactis subsp. cremoris derivatives or plasmid-free strains was shown by renaturing SDS-PAGE (zymogram analysis) to be degraded by the cell envelope proteinase (lactocepin; EC 3.4.21.96) after growth of strains in milk at 30 °C for 72 h. Degradation of AcmA was less in starter strains and derivatives producing lactocepin I/III (intermediate specificity) than in strains producing lactocepin I. This supports previous observations on AcmA degradation in derivatives of the laboratory strain Lc. lactis subsp. cremoris MG1363 (Buist et al. Journal of Bacteriology180 5947–5953 1998). In contrast to the MG1363 derivatives, however, the extent of autolysis in milk of the commercial Lc. lactis subsp. cremoris starter strains in this study did not always correlate with lactocepin specificity and AcmA degradation. The distribution of autolysins within the cell envelope of Lc. lactis subsp. cremoris starter strains and derivatives harvested during growth in milk was compared by zymogram analysis. AcmA was found associated with cell membranes as well as cell walls and some cleavage of AcmA occurred independently of lactocepin activity. An AcmA product intermediate in size between precursor (46 kDa) and mature (41 kDa) forms of AcmA was clearly visible on zymograms, even in the absence of lactocepin I activity. These results show that autolysis of commercial Lc. lactis subsp. cremoris starter strains is not primarily determined by AcmA activity in relation to lactocepin specificity and that proteolytic cleavage of AcmA in vivo is not fully defined.

scholarly journals Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex

1989 ◽  
Vol 264 (1) ◽  
pp. 223-231 ◽  
Author(s):  
T C Williams ◽  
A J Doherty ◽  
D A Griffith ◽  
S M Jarvis
Keyword(s):  
Brush Border ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Facilitated Diffusion ◽  
Basolateral Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Outer Cortex ◽  
Overshoot Phenomenon

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.

scholarly journals Characterization of the Role of para-Aminobenzoic Acid Biosynthesis in Folate Production by Lactococcus lactis

2007 ◽  
Vol 73 (8) ◽  
pp. 2673-2681 ◽  
Author(s):  
Arno Wegkamp ◽  
Wietske van Oorschot ◽  
Willem M. de Vos ◽  
Eddy J. Smid
Keyword(s):  
Gene Cluster ◽  
Lactococcus Lactis ◽  
Gene Clusters ◽  
Defined Medium ◽  
Biosynthesis Pathway ◽  
Chemically Defined Medium ◽  
Aminobenzoic Acid ◽  
Biosynthesis Gene Cluster ◽  
Gene Coding ◽  
Aba Biosynthesis

ABSTRACT The pab genes for para-aminobenzoic acid (pABA) biosynthesis in Lactococcus lactis were identified and characterized. In L. lactis NZ9000, only two of the three genes needed for pABA production were initially found. No gene coding for 4-amino-4-deoxychorismate lyase (pabC) was initially annotated, but detailed analysis revealed that pabC was fused with the 3′ end of the gene coding for chorismate synthetase component II (pabB). Therefore, we hypothesize that all three enzyme activities needed for pABA production are present in L. lactis, allowing for the production of pABA. Indeed, the overexpression of the pABA gene cluster in L. lactis resulted in elevated pABA pools, demonstrating that the genes are involved in the biosynthesis of pABA. Moreover, a pABA knockout (KO) strain lacking pabA and pabB C was constructed and shown to be unable to produce folate when cultivated in the absence of pABA. This KO strain was unable to grow in chemically defined medium lacking glycine, serine, nucleobases/nucleosides, and pABA. The addition of the purine guanine, adenine, xanthine, or inosine restored growth but not the production of folate. This suggests that, in the presence of purines, folate is not essential for the growth of L. lactis. It also shows that folate is not strictly required for the pyrimidine biosynthesis pathway. L. lactis strain NZ7024, overexpressing both the folate and pABA gene clusters, was found to produce 2.7 mg of folate/liter per optical density unit at 600 nm when the strain was grown on chemically defined medium without pABA. This is in sharp contrast to L. lactis strains overexpressing only one of the two gene clusters. Therefore, we conclude that elevated folate levels can be obtained only by the overexpression of folate combined with the overexpression of the pABA biosynthesis gene cluster, suggesting the need for a balanced carbon flux through the folate and pABA biosynthesis pathway in the wild-type strain.

scholarly journals Effect of serum proteins on sodium-linked transport processes in cultured kidney cells.

1995 ◽  
Vol 5 (11) ◽  
pp. 1964-1970
Author(s):  
S S Blumenthal ◽  
D L Lewand ◽  
P A Tipnis ◽  
J G Kleinman
Keyword(s):  
Amino Acid ◽  
Dextran Sulfate ◽  
Cultured Cells ◽  
Serum Proteins ◽  
Fluid Phase ◽  
Defined Medium ◽  
Transport Processes ◽  
Transport Systems ◽  
Heat Treated ◽  
Fluid Phase Endocytosis

The mechanism for increased Na+ retention in the nephrotic syndrome is unknown. To determine if Na+ transport systems in the proximal tubule might be affected by filtered proteins, mouse cortical tubule cells grown in defined medium were exposed to concentrations of bovine serum albumin (BSA) ranging from 0.01 to 0.5%. Activity of the Na(+)-glucose cotransporter, measured as Na(+)-dependent uptake of alpha-methylglucoside, increased progressively to a maximum of 2.3-fold above baseline (P < 0.001; N = 10). The increase in transporter activity was due to an increased Vmax, and the magnitude of the increase was inversely related to the basal cotransporter activity of the cultures. Increased cotransporter activity was detectable 6 h after exposure, was sustained for 24 h after cells were removed from an albumin-free medium, and was prevented by cycloheximide. Heat-treated BSA, fatty-acid and globulin-free BSA, and gamma-globulins were as effective at increasing Na(+)-glucose cotransporter activity as untreated Fraction V BSA. Dextran, dextran-sulfate, and amino acid supplements were ineffective. Neither protease inhibitors nor chloroquine added to an albumin-containing medium prevented increased alpha-methylglucoside uptake. Albumin did not change the rate of fluid-phase endocytosis in the cultured cells. Na(+)-amino acid cotransport and Na(+)-H+ exchange were either decreased or unchanged after BSA exposure. Exposing apical surfaces of cells grown on permeable membranes to BSA led to a greater increase in activity of the Na(+)-glucose cotransporter relative to controls than did exposing the basolateral surface (145 versus 89%; P < 0.05; N = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid uptake systems in Bacteroides ruminicola

1979 ◽  
Vol 25 (10) ◽  
pp. 1161-1168 ◽  
Author(s):  
Roselynn M. W. Stevenson
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Nitrogen Sources ◽  
Amino Acid Uptake ◽  
Defined Medium ◽  
High Rate ◽  
Transport Systems ◽  
Acid Uptake ◽  
Chemical Structures ◽  
Kinetic Inhibition

Uptake of amino acids by Bacteroides ruminicola was observed in cells grown in a complete defined medium, containing ammonia as the nitrogen source. A high rate of uptake occurred only in fresh medium, as an inhibitory substance, possibly acetate, apparently accumulated during growth. All amino acids except proline were taken up and incorporated into cold trichloroacetic acid precipitable material. Different patterns of incorporation and different responses to 2,4-dinitrophenol and potassium ferricyanide indicated multiple uptake systems were involved. Kinetic inhibition patterns suggested six distinct systems were present for amino acid uptake, with specificities related to the chemical structures of the amino acids. Thus, the failure of free amino acids to act as sole nitrogen sources for growth of B. ruminicola is not due to the absence of transport systems for these compounds.

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