Synthesis of folate derivatives in aerated storage tissue disks

Microbiological assay of extracts prepared from carrot, potato, turnip and beet storage tissue disks revealed that folate derivatives were synthesized during a 48 hr aeration period in sterile distilled water. The composition of the folate pool in carrot was examined by DEAE—cellulose column chromatography, γ-glutamylcarboxypeptidase treatment and differential assay of individual derivatives using Lactobacillus casei and Streptococcus faecalis. The principal folates were polyglutamates of formyl and methyl tetrahydrofolate. Smaller quantities of the corresponding mono- and di-glutamates were also detected. The latter derivatives occurred in pools having a high degree of metabolic turnover. The specific activities of three enzymes catalyzing production of these derivatives from tetrahydrofolate increased during the first 12 hr of aeration. Amino acid analyses revealed that folate synthesis in carrot disks was accompanied by depletion of free serine and by net synthesis of free and protein methionine.     

Cation-Dependent Binding of Substrate to the Folate Transport Protein of Lactobacillus casei

Lactobacillus casei cells grown in the presence of limiting folate contained large amounts of a membrane-associated binding protein which mediates folate transport. Binding to this protein at 4°C was time and concentration dependent and at low levels (1 to 10 nM) of folate required 60 min to reach a steady state. The apparent dissociation constant (K_d) for folate was 1.2 nM at pH 7.5 in 100 mM K-phosphate buffer, and it varied by less than twofold when measured over a range of pH values (5.5 to 7.5) or in buffered salt solutions of differing ionic compositions. Conversely, removal of ions and their replacement with isotonic sucrose (pH 7.5) led to a 200-fold reduction in binding affinity for folate. Restoration of the high-affinity state of the binding protein could be achieved by the readdition of various cations to the sucrose medium. K_d measurements over a range of cation concentrations revealed that a half-maximal restoration of binding affinity was obtained with relatively low levels (10 to 50 μM) of divalent cations (e.g., Ca²⁺, Mg²⁺, and ethylenediammonium²⁺ ions). Monovalent cations (e.g., Na⁺, K⁺, and Tris⁺) were also effective, but only at concentrations in the millimolar range. The K_d for folate reached a minimum of 0.6 nM at pH 7.5 in the presence of excess CaCl₂. In cells suspended in sucrose, the affinity of the binding protein for folate increased 20-fold by decreasing the pH from 7.5 to 4.5, indicating that protons can partially fulfill the cation requirement. These results suggest that the folate transport protein of L. casei may contain both a substrate- and cation-binding site and that folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites would support the hypothesis that folate is transported across the cell membrane via a cation-folate symport mechanism.     

Transport of folate compounds into Lactobacillus Casei

Transport of folate, 5-methyl tetrahydrofolate, and amethopterin into Lactobacillus casei occurs against a concentration gradient, is pH and temperature dependent, requires glucose, exhibits saturation kinetics, is maximal when cells are harvested in late-log phase, and is repressed by excess folate in the growth medium. K_m values are 0.35, 0.90, and 0.21 μm for the influx of folate, 5-methyl tetrahydrofolate, and amethopterin, respectively. Dihydrofolate, tetrahydrofolate, 5-formyl tetrahydrofolate, 5-methyl tetrahydrofolate, aminopterin, and amethopterin are inhibitors of folate influx. Countertransport of 5-methyl tetrahydrofolate is enhanced by various other folate compounds. Uptake of folate, 5-methyl tetrahydrofolate, and amethopterin is inhibited to the same degree by increasing concentrations of iodoacetate. These results indicate that a single system is responsible for transport of a variety of folate compounds into L. casei.     

Irreversible inhibition of folate transport in Lactobacillus casei by covalent modification of the binding protein with carbodiimide-activated folate

Activated folate formed by reaction of folic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide irreversibly inhibits the folate transport system of Lactobacillus casei. Complete inhibition of both folate binding to the carrier protein and folate transport was achieved by pretreatment of the cells at low temperature (4 °C) and at neutral pH with 200 nm activated folate. Fifty percent inhibition of binding and transport occurred at 35 and 40 nm activated folate, respectively. Specificity was demonstrated by the fact that excess nonactivated folate added during the pretreatment step afforded complete protection of the binding protein against inhibition, and that activated folate had no effect on the binding or transport of thiamine. Rapid measurements at 4 °C were employed to show that, prior to the appearance of irreversible inhibition, activated folate (K_i = 15 nM) interacted reversibly with the binding site for folate (K_d = 0.8 nM). Cells treated with activated [³H]folate incorporated 1 mol of folate per mole of binding protein. Purification of the labeled protein followed by digestion with Pronase led to the isolation of a compound identified as ?-N-folyl lysine. The ?-amino group of a lysyl residue thus appears to be the nucleophilic group at the binding site that reacts with activated folate.     

Improved methods for the preparation of [3H]folate polyglutamates: biosynthesis with Lactobacillus casei and enzymatic synthesis with Escherichia coli folylpolyglutamate synthetase

Tritiated forms of polyglutamyl folates are not commercially available but are often needed for experimental uses in folate biochemistry. Thus, considerable interest exists in the preparation of polyglutamyl [³H]folates from the commercial monoglutamyl [³H]folates. However, refinement of established enzymatic and biological synthesis methods is still needed. To address this need we developed improved procedures for the conversion of monoglutamyl [³H]folates to various polyglutamyl forms. In the bacterial synthesis, Lactobacillus casei was grown in the presence of 1 ng/ml (2.27 nM) [³H]folic acid in Folic Acid Casei Medium. Washed cells were resuspended in 2% sodium ascorbate containing 10 mM β-mercaptoethanol and heated to release the folates. The extracted [³H]folates were purified on a folate-binding protein affinity column and then applied to a Sephadex G-10 column to separate the eluted poly- from the monoglutamyl folate species. High performance liquid chromatography with multichannel electrochemical detection indicated that the bacterial synthesis yielded predominantly polyglutamates of [³H]5-methyltetrahydrofolate and [³H]5-formyltetrahydrofolate (di- through heptaglutamates). The alternative method consisted of enzymatic polyglutamylation of [³H]folic acid catalyzed by recombinant Escherichia coli folylpolyglutamate synthetase. This enzymatic synthesis yielded predominantly tri-, tetra-, and pentaglutamyl species for the [³H]folate substrate.     

Differences between Lactobacillus casei subsp. casei 2206 and Citrate-Positive Lactococcus lactis subsp. lactis 3022 in the Characteristics of Diacetyl Production

Lactobacillus casei subsp. casei 2206 exhibited much lower levels of diacetyl reductase activity than Citr⁺Lactococcus lactis subsp. lactis 3022 but two-, three-, and more than eightfold-higher levels of diacetyl synthase, lactate dehydrogenase, and NADH oxidase activities, respectively. A requirement for metal ions by the diacetyl synthases in both species was observed. The extracts of strain 2206 but not strain 3022 produced more diacetyl from pyruvate when the reaction for diacetyl synthase was aerated than when it was conducted statically.     

Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells

Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10⁹ CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10⁹ CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain.     

Identification of genes encoding the folate- and thiamine-binding membrane proteins in Firmicutes

Genes encoding high-affinity folate- and thiamine-binding proteins (FolT, ThiT) were identified in the Lactobacillus casei genome, expressed in Lactococcus lactis, and functionally characterized. Similar genes occur in many Firmicutes, sometimes next to folate or thiamine salvage genes. Most thiT genes are preceded by a thiamine riboswitch.     

High-performance liquid chromatographic determination of the distribution of naturally occurring folic acid derivatives in rat liver

Procedures which allow extraction and quantitation of labile, reduced folic acid derivatives in rat liver have been developed. These procedures entail extraction of hepatic folates at 100°C in 2% ($\text{w}\text{v}$) sodium ascorbate, 0.2 m 2-mercaptoethanol, pH 7.85. The extract was treated with conjugase to hydrolyze folate polyglutamates and reverse-phase, ion-pair high-performance liquid chromatography was used to separate the resulting monoglutamates which were measured by microbiological assay using Lactobacillus casei. Experiments with HPLC-purified standard derivatives, so treated, showed excellent stability of tetrahydropteroylglutamic acid (H₄PteGlu), 10-formyl-H₄PteGlu, 5-formyl-H₄PteGlu, 5-methyl-H₄PteGlu, and pteroylglutamic acid (PteGlu). Under these conditions, approximately 56% of H₂PteGlu was recovered unchanged while about 27% was converted to PteGlu; 5,10-methylene-H₄PteGlu was quantitatively recovered as H₄PteGlu. These procedures were applied to the task of measuring the distribution of naturally occurring folate cofactors in rat liver. These results indicated that rat liver folates have the following compositions: 5-methyl-H₄PteGlu, 37.2%; H₄PteGlu, 32.7%; 10-formyl-H₄PteGlu, 22.6%; and 5-formyl-H₄PteGlu, 7.7%. Experiments with [³H]PteGlu injection showed that all hepatic folates had the same specific radioactivity as determined by radioassay and L. casei assay, indicating that L. casei exhibited the same growth response to all the folates detected in rat liver.     

Preparation of extracts of culture liquids for gas-chromatographic determination of acidic fermentation products

An extraction method coupled with gas-chromatographic analysis has been devised for the determination of small amounts of C₁-C₇ branched and straight-chain saturated fatty acids as well as 18 mono-, di- and tricarboxylic acids and related compounds, the latter being analyzed either as their methylesters or as their trimethylsilyl-derivatives. Methods for extracting non-volatile acids or both volatile and non-volatile acids are described for large and small volumes of culture media. The proposed methods have been successfully applied to the determination of non-acidic fermentation products, known amounts of alcohols, and to volatile and non-volatile acids in bacterial fermentation solutions, namely a batch culture ofLactobacillus casei subsp.rhamnosus ATCC 7469. A carbon balance sheet of this organism's metabolic behaviour throughout growth is proposed.     

Characterization of the Cell Wall-Bound Proteinase of Lactobacillus casei HN14

Lactobacillus casei HN14, which was isolated from homemade cheese, produces an extracellular, cell wall-bound proteinase. The HN14 proteinase can be removed from the cell envelope by washing the cells in a Ca²⁺-free buffer. The activity of the crude proteinase extract is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the HN14 proteinase is similar to the lactococcal PI-type enzyme, since it hydrolyzes β-casein only. Lactobacillus casei HN14 appeared to be plasmid free, which suggests that the proteinase gene is chromosomally located. Chromosomal DNA of this strain hybridizes with DNA probes Q1 (which contains a fragment of the prtM gene) and Q6 and Q92 (which contain fragments of the prtP gene); all three probes originated from the proteinase gene region of Lactococcus lactis subsp. cremoris Wg2. A restriction enzyme map of the proteinase region of Lactobacillus casei HN14 was constructed on the basis of hybridization experiments. Comparison of the restriction enzyme maps of the Lactobacillus casei HN14 proteinase gene region and those of lactococcal proteinase gene regions studied so far indicates that they are highly similar.     

Multilocus Sequence Typing of Lactobacillus casei Reveals a Clonal Population Structure with Low Levels of Homologous Recombination

Robust genotyping methods for Lactobacillus casei are needed for strain tracking and collection management, as well as for population biology research. A collection of 52 strains initially labeled L. casei or Lactobacillus paracasei was first subjected to rplB gene sequencing together with reference strains of Lactobacillus zeae, Lactobacillus rhamnosus, and other species. Phylogenetic analysis showed that all 52 strains belonged to a single compact L. casei-L. paracasei sequence cluster, together with strain CIP107868 (= ATCC 334) but clearly distinct from L. rhamnosus and from a cluster with L. zeae and CIP103137^T (= ATCC 393^T). The strains were genotyped using amplified fragment length polymorphism, multilocus sequence typing based on internal portions of the seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA, and recG, and tandem repeat variation (multilocus variable-number tandem repeats analysis [MLVA] using nine loci). Very high concordance was found between the three methods. Although amounts of nucleotide variation were low for the seven genes (π ranging from 0.0038 to 0.0109), 3 to 12 alleles were distinguished, resulting in 31 sequence types. One sequence type (ST1) was frequent (17 strains), but most others were represented by a single strain. Attempts to subtype ST1 strains by MLVA, ribotyping, clustered regularly interspaced short palindromic repeat characterization, and single nucleotide repeat variation were unsuccessful. We found clear evidence for homologous recombination during the diversification of L. casei clones, including a putative intragenic import of DNA into one strain. Nucleotides were estimated to change four times more frequently by recombination than by mutation. However, statistical congruence between individual gene trees was retained, indicating that recombination is not frequent enough to disrupt the phylogenetic signal. The developed multilocus sequence typing scheme should be useful for future studies of L. casei strain diversity and evolution.     

Ca2+-Citrate Uptake and Metabolism in Lactobacillus casei ATCC 334

The putative citrate metabolic pathway in Lactobacillus casei ATCC 334 consists of the transporter CitH, a proton symporter of the citrate-divalent metal ion family of transporters CitMHS, citrate lyase, and the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Resting cells of Lactobacillus casei ATCC 334 metabolized citrate in complex with Ca²⁺ and not as free citrate or the Mg²⁺-citrate complex, thereby identifying Ca²⁺-citrate as the substrate of the transporter CitH. The pathway was induced in the presence of Ca²⁺ and citrate during growth and repressed by the presence of glucose and of galactose, most likely by a carbon catabolite repression mechanism. The end products of Ca²⁺-citrate metabolism by resting cells of Lb. casei were pyruvate, acetate, and acetoin, demonstrating the activity of the membrane-bound oxaloacetate decarboxylase complex OAD-ABDH. Following pyruvate, the pathway splits into two branches. One branch is the classical citrate fermentation pathway producing acetoin by α-acetolactate synthase and α-acetolactate decarboxylase. The other branch yields acetate, for which the route is still obscure. Ca²⁺-citrate metabolism in a modified MRS medium lacking a carbohydrate did not significantly affect the growth characteristics, and generation of metabolic energy in the form of proton motive force (PMF) was not observed in resting cells. In contrast, carbohydrate/Ca²⁺-citrate cometabolism resulted in a higher biomass yield in batch culture. However, also with these cells, no generation of PMF was associated with Ca²⁺-citrate metabolism. It is concluded that citrate metabolism in Lb. casei is beneficial when it counteracts acidification by carbohydrate metabolism in later growth stages.     

Attenuation of Colitis by Lactobacillus casei BL23 Is Dependent on the Dairy Delivery Matrix

The role of the food delivery matrix in probiotic performance in the intestine is not well understood. Because probiotics are often provided to consumers in dairy products, we investigated the contributions of milk to the health-benefiting performance of Lactobacillus casei BL23 in a dextran sulfate sodium (DSS)-induced murine model of ulcerative colitis. L. casei BL23 protected against the development of colitis when ingested in milk but not in a nutrient-free buffer simulating consumption as a nutritional supplement. Consumption of (acidified) milk alone also provided some protection against weight loss and intestinal inflammation but was not as effective as L. casei and milk in combination. In contrast, L. casei mutants deficient in DltD (lipoteichoic acid d-alanine transfer protein) or RecA (recombinase A) were unable to protect against DSS-induced colitis, even when consumed in the presence of milk. Mice fed either L. casei or milk contained reduced quantities of colonic proinflammatory cytokines, indicating that the L. casei DltD⁻ and RecA⁻ mutants as well as L. casei BL23 in nutrient-free buffer were effective at modulating immune responses. However, there was not a direct correlation between colitis and quantities of these cytokines at the time of sacrifice. Identification of the cecal microbiota by 16S rRNA gene sequencing showed that L. casei in milk enriched for Comamonadaceae and Bifidobacteriaceae; however, the consumption of neither L. casei nor milk resulted in the restoration of the microbiota to resemble that of healthy animals. These findings strongly indicate that probiotic strain efficacy can be influenced by the food/supplement delivery matrix.     

Expression of cbsA encoding the collagen-binding S-protein of Lactobacillus crispatus JCM5810 in Lactobacillus casei ATCC 393(T)

The cbsA gene encoding the collagen-binding S-layer protein of Lactobacillus crispatus JCM5810 was expressed in L. casei ATCC 393^T. The S-protein was not retained on the surface of the recombinant bacteria but was secreted into the medium. By translational fusion of CbsA to the cell wall sorting signal of the proteinase, PrtP, of L. casei, CbsA was presented at the surface, rendering the transformants able to bind to immobilized collagens.     

Carbon Source Requirements for Exopolysaccharide Production by Lactobacillus casei CG11 and Partial Structure Analysis of the Polymer

Exopolysaccharide production by Lactobacillus casei CG11 was studied in basal minimum medium containing various carbon sources (galactose, glucose, lactose, sucrose, maltose, melibiose) at concentrations of 2, 5, 10, and 20 g/liter. L. casei CG11 produced exopolysaccharides in basal minimum medium containing each of the sugars tested; lactose and galactose were the poorest carbon sources, and glucose was by far the most efficient carbon source. Sugar concentrations had a marked effect on polymer yield. Plasmid-cured Muc^- derivatives grew better in the presence of glucose and attained slightly higher populations than the wild-type strain. The values obtained with lactose were considerably lower for both growth and exopolysaccharide yield. The level of specific polymer production per cell obtained with glucose was distinctively lower for Muc^- derivatives than for the Muc⁺ strain. The polymer produced by L. casei CG11 in the presence of glucose was different from that formed in the presence of lactose. The polysaccharide produced by L. casei CG11 in basal minimum medium containing 20 g of glucose per liter had an intrinsic viscosity of 1.13 dl/g. It was rich in glucose (76%), which was present mostly as 2- or 3-linked residues along with some 2,3 doubly substituted glucose units, and in rhamnose (21%), which was present as 2-linked or terminal rhamnose; traces of mannose and galactose were also present.     

Distribution Dynamics of Recombinant Lactobacillus in the Gastrointestinal Tract of Neonatal Rats

One approach to deliver therapeutic agents, especially proteins, to the gastro-intestinal (GI) tract is to use commensal bacteria as a carrier. Genus Lactobacillus is an attractive candidate for use in this approach. However, a system for expressing exogenous proteins at a high level has been lacking in Lactobacillus. Moreover, it will be necessary to introduce the recombinant Lactobacillus into the GI tract, ideally by oral administration. Whether orally administered Lactobacillus can reach and reside in the GI tract has not been explored in neonates. In this study, we have examined these issues in neonatal rats. To achieve a high level of protein expression in Lactobacillus, we tested the impact of three promoters and two backbones on protein expression levels using mRFP1, a red fluorescent protein, as a reporter. We found that a combination of an L-lactate dehydrogenase (ldhL) promoter of Lactobacillus sakei with a backbone from pLEM415 yielded the highest level of reporter expression. When this construct was used to transform Lactobacillus casei, Lactobacillus delbrueckii and Lactobacillus acidophilus, high levels of mRFP1 were detected in all these species and colonies of transformed Lactobacillus appeared pink under visible light. To test whether orally administered Lactobacillus can be retained in the GI tract of neonates, we fed the recombinant Lactobacillus casei to neonatal rats. We found that about 3% of the bacteria were retained in the GI tract of the rats at 24 h after oral feeding with more recombinant Lactobacillus in the stomach and small intestine than in the cecum and colon. No mortality was observed throughout this study with Lactobacillus. In contrast, all neonatal rats died within 24 hours after fed with transformed E. coli. Taken together, our results indicate that Lactobacillus has the potential to be used as a vehicle for the delivery of therapeutic agents to neonates.     

On the nature of folate derivatives in neurospora crassa and the effect of sulphanilamide

Summary The folic acid complex of Neurospora crassa has been separated by chromatography on DEAE-cellulose column and the nature of the individual derivatives ascertained from microbiological response. The functional groups of the polyglutamyl folates were ascertained.Major portion of Neurospora folates is composed of the polyglutamyl derivatives, as seen from the increase in activities for Streptococcus faecalis R, Pediococcus cerevisiae, and Lactobacillus casei after conjugase treatment.The presence of N¹⁰ and N⁵-formyl and N⁵-methyl tetrahydrofolic acid, N⁵-methyl diglutamyl tetrahydrofolic acid, two formylated derivatives of polyglutamates, and two N⁵-methyl polyglutamyl folates was ascertained in the folate complex of Neurospora crassa.Sulphanilamide growth inhibition results in the lowering of all the Neurospora folate derivatives.     

Isolation and biochemical characterization of folate deficient mutants of Chinese hamster cells

This article describes the selection of auxotrophic mutants of Chinese Hamster Ovary (CHO) cells and the genetic and biochemical characterization of two mutant lines. AUXB1 is auxotrophic for glycine, adenosine, and thymidine (GAT-), whereas AUXB3 requires only glycine and adenosine (GA-). These mutants do not complement since hybrid cells formed between them are also auxotrophic. Experiments concerned with the reversion of AUXB1 to prototrophy suggest that a single genetic lesion is responsible for the multiple auxotrophy. Biochemical analysis indicates that the multiple auxotrophy of both AUXB1 and AUXB3 is a result of low levels of intracellular folates in mutant cells. Phenotypic reversion to complete or partial prototrophy can be accomplished by growing these cells in high concentrations of folic or folinic acids. However, neither the folate transport nor the dihydrofolate reductase are defective in mutant cells. Chromatographic analysis of intracellular folate derivatives indicates that while folates extracted from wild type cells exist almost exclusively as polyglutamyl derivates (primarily pentaglutamates), AUXB1 cells contain primarily folate derivates in monoglutamyl form and AUXB3 cells contain mono-, di-, and perhaps some triglutamates. This observation suggests that the enzyme responsible for linking glutamate residues onto intracellular folate derivates is the site of the biochemical lesion in the mutant cells. Our results also suggest that a possible function of polyglutamyl residues is to aid cellular retention of folates.     

Freezing-Out Technique Applied to the Concentration of Biologically Active Materials

When applied to a dilute solution of folic acid and glucose, a freezing-out (with agitation) technique was shown to be an effective method of achieving a 20-fold reduction in volume with a loss of 10% of the active material being concentrated. Concentration of a stimulatory factor for Lactobacillus casei produced by Candida albicans in a complex medium was limited by the total solute concentration. Salts in the medium were concentrated to levels inhibitory for L. casei. The process is not selective and all solutes are concentrated.