J Food Prot, 2000 Nov, 63(11), 1551 - 5 Formation of biogenic amines in raw milk Hispánico cheese manufactured with proteinases and different levels of starter culture; Fernandez-Garcia E et al.; Two proteinases, a neutral proteinase from Bacillus subtilis and a cysteine proteinase from Micrococcus sp., were used to accelerate the ripening process of raw cow's milk Hispanico cheese, a semihard variety . Two levels (0.1% and 1%) of a commercial starter culture containing Lactococcus lactis subsp . lactis and L . lactis subsp . cremoris were added for cheese manufacture . The influence of both factors, proteinase addition and level of starter culture, on the growth of amino acid-decarboxylating microorganisms and on the formation of biogenic amines during cheese ripening was investigated in duplicate experiments . The population of tyrosine decarboxylase-positive bacteria, which represented less than 1% of the total bacterial population in most cheese samples, and tyrosine decarboxylase-positive lactobacilli was not influenced by proteinase addition or level of starter culture . Tyramine was detected in all batches of cheese from day 30 . Its concentration was significantly (P < 0.05) influenced by proteinase addition but not by the level of starter culture and increased with cheese age . After 90 days of ripening, 103 to 191 mg/kg of tyramine was found in the different cheese batches . Histamine was not detected until day 60 in cheese with neutral proteinase and 1% starter culture and until day 90 in the rest of the cheeses . The concentration of this amine did not exceed 20 mg/kg in any of the batches investigated . Phenylethylamine and tryptamine were not found in any of the samples. Biochim Biophys Acta, 2001 Nov 15, 1524(1), 38 - 44 Acoustic field assisted enhanced demixing of aqueous two-phase systems; Srinivas ND et al.; Aqueous two-phase extraction has been recognized as a versatile downstream processing technique for the recovery of biomolecules . A major deterrent to its industrial exploitation is the slow demixing of the two aqueous phases after extraction, due to their similar physical properties . A method to decrease the demixing times of these systems, employing a travelling acoustic wave field, is reported . The effects of phase composition and microbial cells on demixing in a polyethylene glycol/potassium phosphate two-phase system are studied in detail . As phase composition increased, demixing time decreased gradually . Phase volume ratio was found to have a significant effect on demixing time at low phase compositions . However, at intermediate and high phase compositions, only a small effect on demixing time was observed . The effect of phase composition and volume ratio on demixing behavior was explained based on the droplet size of the dispersed phase, which is the resultant effect of the physical properties of the phases . At all the phase compositions studied, the acoustically assisted process decreased the demixing time by 17-60% when compared to demixing under gravity alone . Increasing the cell concentration increased the demixing time markedly in case of yeast cells . However, it remained practically constant in the case of Lactobacillus casei cells . Application of an acoustic field reduced the demixing times up to 60% and 40% in the case of yeast and L . casei cells, respectively . Visual observations indicated that ultrasonication caused mild circulation currents in the phase dispersion enhancing droplet-droplet interaction, which in turn enhanced the rate of coalescence, eventually resulting in an enhanced demixing rate. Plasmid, 2000 Nov, 44(3), 239 - 49 Nucleotide sequence and analysis of pBL1, a bacteriocin-producing plasmid from Lactococcus lactis IPLA 972; Sanchez C et al.; The complete sequence of the 10.9-kbp bacteriocinogenic plasmid pBL1 from Lactococcus lactis subsp . lactis IPLA 972 has been determined . Thirteen ORFs were encountered, of which 5 were incomplete . pBL1 proved to be a narrow-host-range plasmid which replicates neither in Bacilus subtilis nor in Lactobacillus spp . The structural organization of the pBL1 replication region was highly similar to other well-known theta-replicating plasmids of lactococci, at both the untranslated (the replication origin) and the translated (repB and orfX) sequences . As in other plasmids, the product of orfX was not necessary for plasmid replication . However, it was shown to be involved in plasmid stability . Three genes organized in an operon-like structure encompassed, most likely, the bacteriocin-encoding region . Upstream of the origin of replication a nicking site (oriT) was found . This oriT sequence proved to be functional by mobilization of plasmids wearing it . One complete and several partial IS elements were identified on pBL1 . J Biol Chem, 2001 Mar 9, 276(10), 6998 - 7003 Epub 2000 Nov 16. FemABX family members are novel nonribosomal peptidyltransferases and important pathogen-specific drug targets; Hegde SS et al.; Pathogen-specific antibiotics kill the offending species without inviting the patient's flora to help develop a resistance mechanism . The current scarcity of pathogen-specific antibiotics reflects the rarity of essential genes that are also not widely represented in and conserved among species . The FemX enzyme that initiates the synthesis of the interchain peptide of the peptidoglycan in a subset of bacterial species was purified from Lactobacillus viridescens . Subsequently, the encoding femX gene was cloned and sequenced using reverse genetics . The femX gene is a member of the femAB family, a large family of genes previously implicated in interchain peptide synthesis but with unknown specific functions . Mutagenesis of the femX gene identified the members of the extended FemABX family as novel nonribosomal peptidyltransferases . Determinants of FemX complex substrate recognition and a strong stimulator of FemX activity were also identified . The FemABX family members are ideal candidates for pathogen-specific antibiotic development. Nahrung, 2000 Oct, 44(5), 364 - 7 Inhibition of Bacillus cereus ATCC 14579 by plantaricin UG1 in vitro and in food; Enan G; The inhibition of Bacillus cereus ATCC 14579 viable growth by Lactobacillus plantarum UG1 bacteriocin (plantaricin UG1) in vitro and in food (pasteurized milk and minced meat) was studied . The inhibitory effect against B . cereus food-borne pathogen noticed in this study was due to plantaricin UG1, but not due to lactic acid produced by the L . plantarum UG1 culture . Plataricin UG1 negative clone did not affect viable growth of B . cereus in both broth and meat or pasteurized milk . The inhibitory effect of L . plantarum UG1 and its bacteriocion was apparently more in liquid systems (BHI broth & pasteurized milk) than in minced meat . The inhibitory effect of plantaricin UG1 against B . cereus was dependent on its concentration . The 22880 AU/ml concentration appeared to be an ideal preservative against B . cereus ATCC 14579 in liquid systems. J Bacteriol, 2000 Dec, 182(23), 6857 - 61 Expression of cbsA encoding the collagen-binding S-protein of Lactobacillus crispatus JCM5810 in Lactobacillus casei ATCC 393(T); Martinez B et al.; 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. J Bacteriol, 2000 Dec, 182(23), 6724 - 31 Inducible metabolism of phenolic acids in Pediococcus pentosaceus is encoded by an autoregulated operon which involves a new class of negative transcriptional regulator; Barthelmebs L et al.; Pediococcus pentosaceus displays a substrate-inducible phenolic acid decarboxylase (PAD) activity on p-coumaric acid . Based on DNA sequence homologies between the three PADs previously cloned, a DNA probe of the Lactobacillus plantarum pdc gene was used to screen a P . pentosaceus genomic library in order to clone the corresponding gene of this bacteria . One clone detected with this probe displayed a low PAD activity . Subcloning of this plasmid insertion allowed us to determine the part of the insert which contains a 534-bp open reading frame (ORF) coding for a 178-amino-acid protein presenting 81.5% of identity with L . plantarum PDC enzyme . This ORF was identified as the padA gene . A second ORF was located just downstream of the padA gene and displayed 37% identity with the product of the Bacillus subtilis yfiO gene . Subcloning, transcriptional analysis, and expression studies with Escherichia coli of these two genes under the padA gene promoter, demonstrated that the genes are organized in an autoregulated bicistronic operonic structure and that the gene located upstream of the padA gene encodes the transcriptional repressor of the padA gene . Transcription of this pad operon in P . pentosaceus is acid phenol dependent. J Med Microbiol, 2000 Nov, 49(11), 1023 - 30 Inhibition of chemotaxis by organic acids from anaerobes may prevent a purulent response in bacterial vaginosis; Al-Mushrif S et al.; It has been postulated that certain organic acids produced by the anaerobes associated with bacterial vaginosis (BV) could prevent a purulent response in this infection . Varying concentrations of pure succinic, acetic and lactic acids were incubated in vitro with a monocytic cell line (MonoMac 6) . High inhibition of chemotaxis was produced by succinic acid; lower inhibition and no inhibition was shown by acetic acid and lactic acid respectively . Succinic and acetic acids were detected in high concentrations in the vaginal fluid of women with BV and in culture supernates of Prevotella and Mobiluncus spp.; these acids impaired chemotaxis of MonoMac 6 cells in vitro . The vaginal fluids of normal women and the culture supernates of Lactobacillus spp . had no effect on chemotaxis. Eur J Pharm Biopharm, 2000 Nov, 50(3), 389 - 95 Technological and biological evaluation of tablets containing different strains of lactobacilli for vaginal administration; Maggi L et al.; Ten strains of lactobacilli were evaluated for the administration of viable microorganisms to restore the normal indigenous flora in the treatment of urogenital tract infections (UTI) in women . As the strains considered are facultative anaerobes, optimization of the production process was particularly critical to preserve bacterial viability . The microorganisms were formulated in single- and double-layer vaginal tablets . The two layers were characterized by different release properties: one is an effervescent composition that ensures a rapid and complete distribution of the active ingredient over the whole vaginal surface; while the second is a sustained release composition capable of releasing the lactobacilli over a longer period of time . Three different retarding polymers were tested, and all the formulations and tablets were evaluated in terms of technological processability, bacterial viability and stability, and cell adhesion properties of the microorganisms . From the results obtained, three out of ten strains appear particularly suitable for their application in the treatment of UTI . A larger batch of tablets made with a mixture of the three strains was then evaluated, confirming the feasibility of their industrial production and a good bacterial viability in the final dosage form. Kokubyo Gakkai Zasshi, 2000 Sep, 67(3), 240 - 50 {The changes of oral conditions caused by artificial crown contours in severely disabled patients}; Yoshida T; The purpose of this study was to investigate the relation between the artificial crown contour and plaque accumulation in severely disabled patients who have muscle hypoactivity in the oral region . This relation was statistically analyzed by the change in CFU numbers of Mutans Streptococci, Lactobacillus, and total anaerobic bacteria . The results were as follows: 1 . In the severely disabled group, there was no significant difference in the decrease of number of microorganisms between with and without tooth brushing . When the normal contour crown was exchanged for the under contour, however, the numbers of Mutans Streptococci and total anaerobic bacteria decreased significantly (p < 0.05) . 2 . In the normal control group, both the crown contour and tooth brushing were effective for decreasing the number of Mutans Streptococci and total anaerobic bacteria (p < 0.05) . 3 . In both groups, the number of Lactobacillus was decreased by changing the crown contours and by tooth brushing, with no statistical significance . 4 . In view of the cariogenic potential of microorganisms, it is concluded that the under contour crown prevents plaque accumulation, and that this crown should be used on severely disabled patients, as it makes tooth brushing more effective. Can J Microbiol, 2000 Oct, 46(10), 938 - 45 Molecular analysis of mutated Lactobacillus acidophilus promoter-like sequence P15; Arsenijevic S et al.; The promoter-like sequence P15 that was previously cloned from the chromosome of Lactobacillus acidophilus ATCC 4356 is active in Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus acidophilus, and Escherichia coli, but not in Lactococcus lactis . N-methyl-N-nitroso-N-guanidine (MNNG) mutagenesis of P15 was used to select for a promoter active in L . lactis MG1363 . Molecular analysis of the mutated promoter (designated P16) revealed a 90 bp deletion and a T-->A transversion . This deletion, in combination with the addition to the transversion, created a promoter with putative -35 and -10 hexamers identical to the consensus promoter sequence found in E . coli and Bacillus subtilis vegetative promoters . The activity of P16 was measured by its ability to promote chloramphenicol resistance in different bacteria when inserted in the promoter-probe plasmid pBV5030 (designated pLA16) . The MIC of chloramphenicol in L . lactis, L . reuteri, L . plantarum, E . coli, and L . acidophilus harbouring pLA16 were 30, 170, 180, > 500, and 3 micrograms/mL, respectively . This represents an increase in promoter activity compared to P15 in L . reuteri of 3-fold, in L . plantarum of 9-fold, and in E . coli of at least 2.5-fold, but a decrease in L . acidophilus of 7-fold. Can J Microbiol, 2000 Oct, 46(10), 892 - 7 Improved growth and viability of lactobacilli in the presence of Bacillus subtilis (natto), catalase, or subtilisin; Hosoi T et al.; In an effort to demonstrate the potential usefulness of Bacillus subtilis (natto) as a probiotic, we examined the effect of this organism on the growth of three strains of lactobacilli co-cultured aerobically in vitro . Addition of B . subtilis (natto) to the culture medium resulted in an increase in the number of viable cells of all lactobacilli tested . Since B . subtilis (natto) can produce catalase, which has been reported to exhibit a similar growth-promoting effect on lactobacilli, we also examined the effect of bovine catalase on the growth of Lactobacillus reuteri JCM 1112 and L . acidophilus JCM 1132 . Both catalase and B . subtilis (natto) enhanced the growth of L . reuteri JCM 1112, whereas B . subtilis (natto) but not catalase enhanced the growth of L . acidophilus JCM 1132 . In a medium containing 0.1 mM hydrogen peroxide, its toxic effect on L . reuteri JCM 1112 was abolished by catalase or B . subtilis (natto) . In addition, a serine protease from B . licheniformis, subtilisin, improved the growth and viability of L . reuteri JCM 1112 and L . acidophilus JCM 1132 in the absence of hydrogen peroxide . These results indicate that B . subtilis (natto) enhances the growth and (or) viability of lactobacilli, possibly through production of catalase and subtilisin. J Anim Sci, 2000 Nov, 78(11), 2980 - 9 Intake, digestibility, and composition of orchardgrass and alfalfa silages treated with cellulase, inoculant, and formic acid fed to lambs; Nadeau EM et al.; The objectives of this study were to determine the effect of a cellulase (from Trichoderma longibrachiatum) alone or combined with a bacterial inoculant (Lactobacillus plantarum and Pediococcus cerevisiae) or formic acid on composition, intake, and digestibility of orchardgrass (Dactylis glomerata L.) and alfalfa (Medicago sativa L.) silages . Orchardgrass and alfalfa were harvested at the early heading stage and at the early bloom stage of maturity and wilted to approximately 22 and 32% DM, respectively . Forages were then ensiled in 100-L sealed barrels for at least 60 d before they were fed to lambs . Silage treated with cellulase had lower (P < .001) pH and lower (P < .001) acetic acid and NH3 N concentrations than untreated silage of both plant species and a higher (P = .004) lactic acid concentration than the control treatment of alfalfa silage . Fermentation characteristics of cellulase-treated silages, especially of alfalfa, were further enhanced by use of inoculant . Formic acid addition increased (P < .001), reducing sugar concentration of cellulase-treated orchardgrass and alfalfa silage by 90 and 154%, respectively, and decreased (P < .001) NH3 N concentration of cellulase-treated alfalfa silage by 19% . Averaged across plant species, cellulase, combined with inoculant or formic acid, resulted in 8 and 13% greater (P = .03) DMI, respectively, than the control silage . Extensive enzymatic cell-wall degradation during ensiling decreased (P = .003) NDF intake of cellulase-treated orchardgrass silage by 25% and decreased (P = .001) cellulose intake by 23%, when averaged across plant species . Addition of formic acid increased (P = .003) NDF intake of cellulase-treated orchardgrass silage by 19% . Averaged across species, cellulase application decreased (P < .05) silage NDF digestibility by 18% . Greater sugar and lower acetic acid, NH3 N, and NDF concentrations resulted in greater DMI of cellulase-treated silage than of control silage, when cellulase was combined with formic acid or inoculant. New Microbiol, 2000 Oct, 23(4), 423 - 31 Conservation in probiotic preparations of Lactobacillus with inhibitory capacity on other species; Paraje MG et al.; Strains of Lactobacillus isolated from dairy products and genital tract competed with Candida albicans through a membrane of 12000 dalton cut-off . This inhibition was due to hydrogen peroxide and was trypsin-stable, heat-sensitive and antagonized by catalase . Lactobacillus coming from "starters" showed antimicrobial activity against fungus isolated in a yogurt factory . Penicillium, Alternaria, Phialophora, Microsporum and Candida spp . were inhibited when 10(2) spores were inoculated in the assay . No inhibition was observed with 10(5) spores . Besides, one of 21 Lactobacillus strains isolated from the vaginas of healthy women inhibited pathogenic bacteria by means a bacteriocin trypsin-sensitive, heat-stable and retained by dialysis membrane . Tablets for future probiotic use were prepared and the viability of bacteria was assayed using media with different compositions . Pharmaceutical preparations with polyethyleneglycol was the best formulation for the Lactobacillus viability, the counts remained between 10(7) and 10(6) cfu/tablet for up to 1 year. Eur J Gastroenterol Hepatol, 2000 Oct, 12(10), 1077 - 88 Micro-organisms administered in the benefit of the host: myths and facts; Marchand J et al.; OBJECTIVE: To evaluate the published literature on the potential benefit of micro-organisms on the general well being of the host . STUDY DESIGN: All published prospective, randomized, placebo-controlled trials with micro-organisms to improve the health of the host were critically reviewed . RESULTS: According to published data, there is evidence suggesting that Lactobacillus rhamnosus strain GG or Lactobacillus casei strain GG and Saccharomyces boulardii may be of possible benefit for the treatment of several medical conditions . However, published data on the therapeutic effect of other micro-organisms are almost non-existent . CONCLUSION: Better designed prospective, randomized, and placebo-controlled studies are needed . Most of the present strains have not been selected in a rational way, but apparently represent rather randomly picked isolates . Although the theoretical advantages of micro-organisms administered to the benefit of the host are extremely interesting and promising, results of clinical trials are disappointing. Appl Environ Microbiol, 2000 Nov, 66(11), 5030 - 4 Colonization of the stratified squamous epithelium of the nonsecreting area of horse stomach by lactobacilli; Yuki N et al.; Selective adhesion to only certain epithelia is particularly common among the bacterial members of the indigenous microflora of mammals . We have found that the stratified squamous epithelium of the nonsecreting area of horse stomach is colonized by gram-positive rods . The microscopic features of a dense layer of these bacteria on the epithelium were found to be similar to those reported in mice, rats, and swine . Adhering microorganisms were isolated and identified as Lactobacillus salivarius, L . crispatus, L . reuteri, and L . agilis by DNA-DNA hybridization and 16S rRNA gene sequencing techniques . These lactobacilli associated with the horse, except for L . reuteri, were found to adhere to horse epithelial cells in vitro but not to those of rats . A symbiotic relationship of these lactobacilli with the horse is suggested. Appl Environ Microbiol, 2000 Nov, 66(11), 4822 - 8 Integrative food-grade expression system based on the lactose regulon of Lactobacillus casei; Gosalbes MJ et al.; The lactose operon from Lactobacillus casei is regulated by very tight glucose repression and substrate induction mechanisms, which made it a tempting candidate system for the expression of foreign genes or metabolic engineering . An integrative vector was constructed, allowing stable gene insertion in the chromosomal lactose operon of L . casei . This vector was based on the nonreplicative plasmid pRV300 and contained two DNA fragments corresponding to the 3' end of lacG and the complete lacF gene . Four unique restriction sites were created, as well as a ribosome binding site that would allow the cloning and expression of new genes between these two fragments . Then, integration of the cloned genes into the lactose operon of L . casei could be achieved via homologous recombination in a process that involved two selection steps, which yielded highly stable food-grade mutants . This procedure has been successfully used for the expression of the E . coli gusA gene and the L . lactis ilvBN genes in L . casei . Following the same expression pattern as that for the lactose genes, beta-glucuronidase activity and diacetyl production were repressed by glucose and induced by lactose . This integrative vector represents a useful tool for strain improvement in L . casei that could be applied to engineering fermentation processes or used for expression of genes for clinical and veterinary uses. Biosci Biotechnol Biochem, 2000 Sep, 64(9), 1868 - 73 Preventive effect of lactobacillus delbrueckii subsp . bulgaricus on the oxidation of LDL; Terahara M et al.; Lactobacillus delbrueckii subsp . bulgaricus 2038 was examined for its activity to prevent the oxidation of the erythrocyte membrane in vitro, and the oxidation of LDL in vivo . Strain 2038 produced radical scavengers that reacted with 1,1-diphenyl-2-picrylhydrazl (DPPH) during cultivation . Moreover, the ethereal extract from the supernatant of the culture prevented the oxidation of the erythrocyte membrane in vitro . As an in vivo study, male F344 rats were fed on diets containing 20% fresh soybean oil (or 13% oxidized oil and 7% fresh oil) with 10% freeze-dried powder of the 2038 culture (or with skim milk powder) for 4 weeks . The level of thiobarbituric acid-reactive substances was lower in the low-density lipoproteins (per milligram of cholesterol) from rats fed on the oxidized oil with freeze-dried powder of the 2038 culture than without it . The level of vitamin E in the plasma was higher in the rats fed on the oxidized oil with the freeze-dried powder than without it. Biosci Biotechnol Biochem, 2000 Sep, 64(9), 1836 - 41 Preparative 2'-reduction of ATP catalyzed by ribonucleotide reductase purified by liquid-liquid extraction; Brunella A et al.; Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate reductase (RTPR, E.C.1.17.4.2) constitutively expressed by E . coli HB101 pSQUIRE has been purified from sonicated cell material in a one-step procedure by PEG 4000 (16% (w/w))/phosphate (7% (w/w)) liquid-liquid extraction . A high yield of 75.1% RTPR in the top phase and a partitioning of 8.5:1 between total RTPR activity in top and bottom phase were obtained in this preparative system . The RTPR-containing top phase was used to reduce ATP in the 2'-position on a gram scale with high final conversion and yield proving the ribonucleotide reductase approach feasible for the preparative synthesis of 2'-deoxyribonucleotides . High concentrations of sodium acetate in the reaction served to substitute for allosteric effectors of RTPR . 1,4-Dithio-DL-threitol was used as an artificial reducing agent for RTPR. J Appl Microbiol, 2000 Oct, 89(4), 678 - 86 Extrusion of wheat or sorghum and/or addition of exogenous enzymes to pig diets influences the large intestinal microbiota but does not prevent development of swine dysentery following experimental challenge; Durmic Z et al.; A study was made of dietary influences on the large intestinal microbiota of pigs and on the incidence of swine dysentery (SD) after experimental infection with Brachyspira hyodysenteriae, the aetiological agent of SD . Animals were fed diets based either on wheat (expts 1 and 2) or sorghum (expt 2) . Grains were ground and fed either raw or after high temperature and pressure extrusion and/or after addition of exogenous enzymes to the whole diet to reduce the starch and soluble non-starch polysaccharides available for fermentation in the large intestine . Limiting fermentation creates conditions that apparently reduce the incidence of SD after infection with B . hyodysenteriae . The diets were fed to weaned pigs for 4-6 weeks, then half the animals on each diet were killed and gut samples collected for microbiology . The treatments had little effect on bacterial numbers . In expt 1, dietary extrusion of wheat reduced lactobacilli in the large intestine . Addition of enzymes to extruded wheat-based diets in expt 2 reduced facultative anaerobes and increased non-sporing anaerobes . Addition of enzymes to a raw sorghum diet in expt 3 decreased numbers of facultative anaerobes, while extrusion of sorghum increased total anaerobes . Bacteroides spp . and Fusobacterium spp., which act in synergy with B . hyodysenteriae in SD, were isolated at a higher percentage in pigs fed the untreated wheat diet than in pigs fed the treated wheat diets . Following experimental infection the incidence of SD amongst pigs fed treated wheat diets was slightly lower than those fed the untreated diet, but with sorghum-based diets the opposite was found . Overall, the different dietary treatments used did not significantly reduce SD. J Appl Microbiol, 2000 Oct, 89(4), 553 - 63 Isolation and characterization of a Lactobacillus amylovorus mutant depleted in conjugated bile salt hydrolase activity: relation between activity and bile salt resistance; Grill JP et al.; Growth experiments were conducted on Lactobacillus amylovorus DN-112 053 in batch culture, with or without pH regulation . Conjugated bile salt hydrolase (CBSH) activity was examined as a function of culture growth . The CBSH activity increased during growth but its course depended on bile salts type and culture conditions . A Lact . amylovorus mutant was isolated from the wild-type strain of Lact . amylovorus DN-112 053 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine . An agar plate assay was used to detect mutants without CBSH activity . In resting cell experiments, the strain showed reduced activity . Differences between growth parameters determined for wild-type and mutant strains were not detected . Comparative native gel electrophoresis followed by CBSH activity staining demonstrated the loss of proteins harbouring this activity in the mutant . Four protein bands corresponding to CBSH were observed in the wild-type strain but only one was detected in the mutant . The specific growth rate of the mutant strain was affected more by bile salts than the wild-type strain . Nevertheless, bile was more toxic for the wild-type strain . In viability studies in the presence of nutrients, it was demonstrated that glycodeoxycholic acid exerted a higher toxicity than taurodeoxycholic acid in a pH-dependent manner . No difference was apparent between the two strains . In the absence of nutrients, the wild-type strain died after 2 h whereas no effect was observed for the mutant . The de-energization experiments performed using the ionophores nigericin and valinomycin suggested that the chemical potential of protons (ZDeltapH) was involved in Lactobacillus bile salt resistance. J Bacteriol, 2000 Nov, 182(22), 6525 - 8 Cholic acid is accumulated spontaneously, driven by membrane deltapH, in many lactobacilli; Kurdi P et al.; Many lactobacilli from various origins were found to apparently lack cholic acid extrusion activity . Cholic acid was accumulated spontaneously, driven by the transmembrane proton gradient . Accumulation is a newly identified kind of interaction between intestinal microbes and unconjugated bile acids and is different from extrusion and modification, which have been described previously. J Bacteriol, 2000 Nov, 182(22), 6440 - 50 Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus; Sillanpaa J et al.; The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli . The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein . The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens . Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced . In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion . Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule . The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA . Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA . RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions . S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L . crispatus. J Med Chem, 2000 Oct 19, 43(21), 3837 - 51 Design, synthesis, and X-ray crystal structure of a potent dual inhibitor of thymidylate synthase and dihydrofolate reductase as an antitumor agent; Gangjee A et al.; A novel N- inverted question mark2-amino-4-methyl{(pyrrolo{2, 3-d}pyrimidin-5-yl)ethyl}benzoyl inverted question mark-L-glutamic acid (3a) was designed and synthesized as a potent dual inhibitor of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) and as an antitumor agent . Compound 3b, the N7-benzylated analogue of 3a, was also synthesized as an antitumor agent . The synthesis of 3a was accomplished via a 12-step sequence which involved the synthesis of 2-amino-4-methylpyrrolo{2,3-d}pyrimidine (10) in 5 steps from 2-acetylbutyrolactone . Protection of the 2-amino group of 10 and regioselective iodination at the 5-position followed by palladium-catalyzed coupling afforded intermediate 14 which was converted to 3a by reduction and saponification . Similar synthetic methodology was used for 3b . X-ray crystal structure of the ternary complex of 3a, DHFR, and NADPH showed that the pyrrolo{2, 3-d}pyrimidine ring binds in a "2,4-diamino mode" in which the pyrrole nitrogen mimics the 4-amino moiety of 2,4-diaminopyrimidines . This is the first example of a classical pyrrolo{2,3-d}pyrimidine antifolate shown to have this alternate mode of binding to DHFR . Compounds 3a and 3b were more inhibitory than LY231514 against TS from Lactobacillus casei and Escherichia coli . Analogue 3a was also more inhibitory against DHFR from human, Toxoplasma gondii, and Pneumocystis carinii . Evaluation of 3a against methotrexate (MTX)-resistant cell lines with defined mechanisms indicated that cross-resistance of 3a was much lower than that of MTX . Metabolite protection studies and folylpoly-gamma-glutamate synthetase studies suggest that the antitumor activity of 3a against the growth of tumor cells in culture is a result of dual inhibition of TS and DHFR . Compound 3a inhibited the growth of CCRF-CEM and FaDu cells in culture at ED(50) values of 12.5 and 7.0 nM, respectively, and was more active against FaDu cells than MTX . In contrast, compound 3b was inactive against both cell lines . Compound 3a was evaluated in the National Cancer Institute in vitro preclinical antitumor screening program and afforded IG(50) values in the nanomolar range against a number of tumor cell lines. Arch Latinoam Nutr, 2000 Jun, 50(2), 157 - 63 {Obtaining a fermented chickpea extract (Cicer arietinum L.) and its use as a milk extensor}; Morales de Leon J et al.; Chickpea (Cicer arietinum L) is cultivated in the North part of Mexico and it is considered a good source of vegetal protein of low cost (20% average), nevertheless, the 80% used for the exportation and only the 20% less was used for animal feeding . The main objective in this study is to obtain a fermented chickpea extract for using in dairy extensor . Chickpea water absorbtion kinetics were carried out in e temperature conditions:while the conditions were established, chickpea was grounded and fermented in different amounts with its natural flora, L . casei, L . plantarum and a mixture culture of both microorganism in logarithmic phase . The results showed that the presence of microorganism of chickpea natural flora interferes during the fermentation, so before the inoculation it was necessary treat the chickpea extract (CE) terminally in a dilution 1:4 during 20 min at 7.7 kg/cm2 of pressure . The use of a mixture culture of 5% of L . casei and 5% L . plantarum inoculated in MRS broth was used to decrease fermentation time . Its addition in logarithmic phase to the sterile chickpea extract increased the lactic acid production and decreased the pH value in 6 h which was less time that one obtained with each of lactobacillus . The fermented extract obtained finally, presented similar sensory characteristics to the ones of dairy products . Therefore, chickpea is a good alternative as a extensor for this kind of products. J Pediatr Gastroenterol Nutr, 2000 Oct, 31(4), 453 - 7 Is lactobacillus GG helpful in children with Crohn's disease? Results of a preliminary, open-label study; Gupta P et al.; BACKGROUND: Lactobacillus GG is a safe probiotic bacterium known to transiently colonize the human intestine . It has been found to be useful in treatment of several gastrointestinal conditions characterized by increased gut permeability . In the current study, the efficacy of Lactobacillus GG was investigated in children with Crohn's disease . METHODS: In this open-label pilot evaluation viewed as a necessary preliminary step for a possible subsequent randomized placebo-controlled trial, four children with mildly to moderately active Crohn's disease were given Lactobacillus GG (10(10) colony-forming units {CFU}) in enterocoated tablets twice a day for 6 months . Changes in intestinal permeability were measured by a double sugar permeability test . Clinical activity was determined by measuring the pediatric Crohn's disease activity index . RESULTS: There was a significant improvement in clinical activity 1 week after starting Lactobacillus GG, which was sustained throughout the study period . Median pediatric Crohn's disease activity index scores at 4 weeks were 73% lower than baseline . Intestinal permeability improved in an almost parallel fashion . CONCLUSIONS: Findings in this pilot study show that Lactobacillus GG may improve gut barrier function and clinical status in children with mildly to moderately active, stable Crohn's disease . Randomized, double-blind, placebo-controlled trials are warranted for a final assessment of the efficacy of Lactobacillus GG in Crohn's disease. J Food Prot, 2000 Oct, 63(10), 1338 - 46 Surface application of lysozyme, nisin, and EDTA to inhibit spoilage and pathogenic bacteria on ham and bologna; Gill AO et al.; A study was conducted to determine if the effectiveness of an antimicrobial treatment for cooked ham and bologna would be increased or maintained when applied in a surface coating . Cooked 10-g disks of ham and bologna sausage received one of three treatments: no coating (control), coating with 0.2 g of 7% (wt/vol) gelatin gel (gel-control), or coating with 0.2 g of 7% gelatin gel containing 25.5 g/liter of lysozyme-nisin (1:3) plus 25.5 g/liter of EDTA (gel-treated) . The samples were then inoculated with one of six test organisms: Brochothrix thermosphacta, Escherichia coli O157:H7, Lactobacillus sakei, Leuconostoc mesenteroides, Listeria monocytogenes, or Salmonella Typhimurium . Inoculated samples were vacuum packed and stored at 8 degrees C for 4 weeks . The antimicrobial gel treatment had an immediate bactericidal effect up to 4 log CFU/cm2 on the four gram-positive organisms tested (B . thermosphacta, Lactobacillus sakei, Leuconostoc mesenteroides, and Listeria monocytogenes) and inhibited the growth of these organisms during the 4 weeks of storage . The antimicrobial gel treatment also had a bactericidal effect on the growth of Salmonella Typhimurium during storage . The numbers of E . coli O157:H7 on ham were reduced by 2 log CFU/cm2 following treatment with both antimicrobial-containing and non-antimicrobial-containing gels during the 4-week storage period . No effect was observed on the growth of E . coli O157:H7 on bologna. Adv Biochem Eng Biotechnol, 2000, 68, 21 - 60 Antimicrobial peptides of lactic acid bacteria: mode of action, genetics and biosynthesis; Sablon E et al.; A survey is given of the main classes of bacteriocins, produced by lactic acid bacteria: I . lantibiotics II . small heat-stable non-lanthionine containing membrane-active peptides and III . large heat-labile proteins . First, their mode of action is detailed, with emphasis on pore formation in the cytoplasmatic membrane . Subsequently, the molecular genetics of several classes of bacteriocins are described in detail, with special attention to nisin as the most prominent example of the lantibiotic-class . Of the small non-lanthionine bacteriocin class, the Lactococcus lactococcins, and the Lactobacillus sakacin A and plantaricin A-bacteriocins are discussed . The principles and mechanisms of immunity and resistance towards bacteriocins are also briefly reported . The biosynthesis of bacteriocins is treated in depth with emphasis on response regulation, post-translational modification, secretion and proteolytic activation of bacteriocin precursors . To conclude, the role of the leader peptides is outlined and a conceptual model for bacteriocin maturation is proposed. Curr Microbiol, 2000 Mar, 40(3), 181 - 4 Purification and characterization of invertase from Lactobacillus reuteri CRL 1100; Cuezzo de Gines S et al.; The invertase of Lactobacillus reuteri CRL 1100 is a glycoprotein composed by a single subunit with a molecular weight of 58 kDa . The enzyme was stable below 45 degrees C over a wide pH range (4.5-7.0) with maximum activity at pH 6.0 and 37 degrees C . The invertase activity was significantly inhibited by bivalent metal ions (Ca(++), Cu(++), Cd(++), and Hg(++)), beta-mercaptoethanol, and dithiothreitol and partially improved by ethylenediaminetetraacetic acid . The enzyme was purified 32 times over the crude extract by gel filtration and ion-exchange chromatography with a recovery of 17% . The K(m) and V(max) values for sucrose were 6.66 mM and 0.028 micromol/min, respectively . An invertase is purified and characterized for the first time in Lactobacillus, and it proved to be a beta-fructofuranosidase. Int J Syst Evol Microbiol, 2000 Sep, 50 Pt 5, 1803 - 9 Lactobacillus arizonensis sp . nov., isolated from jojoba meal; Swezey JL et al.; Five strains of simmondsin-degrading, lactic-acid-producing bacteria were isolated from fermented jojoba meal . These isolates were facultatively anaerobic, gram-positive, non-motile, non-spore-forming, homofermentative, rod-shaped organisms . They grew singly and in short chains, produced lactic acid but no gas from glucose, and did not exhibit catalase activity . Growth occurred at 15 and 45 degrees C . All strains fermented cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannitol, D-mannose, melibiose, D-ribose, salicin, D-sorbitol, sucrose and trehalose . Some strains fermented L-(-)-arabinose and L-rhamnose . D-Xylose was not fermented and starch was not hydrolysed . The mean G+C content of the DNA was 48 mol% . Phylogenetic analyses of 16S rDNA established that the isolates were members of the genus Lactobacillus . DNA reassociation of 45% or less was obtained between the new isolates and the reference strains of species with G+C contents of about 48 mol% . The isolates were differentiated from other homofermentative Lactobacillus spp . on the basis of 16S rDNA sequence divergence, DNA relatedness, stereoisomerism of the lactic acid produced, growth temperature and carbohydrate fermentation . The data support the conclusion that these organisms represent strains of a new species, for which the name Lactobacillus arizonensis is proposed . The type strain of L . arizonensis is NRRL B-14768T (= DSM 13273T). Int J Syst Evol Microbiol, 2000 Sep, 50 Pt 5, 1789 - 95 Lactobacillus kimchii sp . nov., a new species from kimchi; Yoon JH et al.; A bacteriocin-producing lactic acid bacterium, which was isolated from the Korean fermented-vegetable food kimchi, was subjected to a polyphasic taxonomic study using phenotypic characterization and phylogenetic and genetic methods . This organism (MT-1077T) has phenotypic properties that are consistent with the description characterizing the genus Lactobacillus . Phylogenetic analysis based on 16S rDNA sequences showed clearly that strain MT-1077T is a member of the genus Lactobacillus . The closest phylogenetic relatives are Lactobacillus alimentarius KCTC 3593T and Lactobacillus farciminis LMG 9200T, with levels of 16S rDNA similarity of 98.4 and 98.2%, respectively . Levels of 16S rDNA similarity between strain MT-1077T and other Lactobacillus species were less than 93.0% . Differences in some phenotypic characteristics and DNA-DNA relatedness data indicated that strain MT-1077T should be distinguished from L . alimentarius KCTC 3593T and L . farciminis LMG 9200T . On the basis of the data presented, it is proposed that strain MT-1077T should be placed in the genus Lactobacillus as a new species, Lactobacillus kimchii sp . nov . The type strain of the new species is strain MT-1077T (= KCTC 8903PT = JCM 10707T). Microbios, 2000, 103(404), 31 - 41 The minimum inhibitory concentration of oral antibacterial agents against cariogenic organisms; Botelho MG; The minimum inhibitory concentrations (MIC) of eight common dental antibacterial agents against three genera of bacteria which have been implicated in dentine caries, namely streptococci, lactobacilli and actinomycetes were investigated . The ultimate aim was to determine the most appropriate antibacterial agent which could be added to dental restorative materials for filling cavities where there was residual dentine caries . The antibacterial agents tested were chlorhexidine diacetate, chlorhexidine dihydrochloride, chlorhexidine gluconate, benzalkonium chloride, cetrimide, cetylpyridinium chloride, thymol and sodium hypochlorite . Thymol and sodium hypochlorite did not inhibit microbial growth at any of the concentrations tested . For the active antibacterial agents tested the MIC values against lactobacilli and streptococci were 0.25 microg/ml to 8.0 microg/ml and for actinomycetes 0.125 to 8.0 microg/ml . These results illustrate the wide spectrum of sensitivity of caries associated bacteria against dental antibacterial agents . From the MIC values alone, it is difficult to recommend which of the active antibacterial agents would be most effective in eliminating cariogenic organisms. FEMS Microbiol Lett, 2000 Sep 15, 190(2), 335 - 9 A Lactobacillus helveticus plasmid detects restriction fragment length polymorphism in different bacterial species; Gancheva AG et al.; A small cryptic Lactobacillus helveticus plasmid, pLBL4, was able to reveal restriction fragment length polymorphism in different bacterial species including Lactobacillus species, Bacillus species, and Escherichia coli when used as a DNA probe . The observed polymorphism was a result of the combined hybridization of several microsatellite sequences . The 6-bp sequence (TTGTTT) was repeated 12 times, seven of which were concentrated within the region between 1791 and 1997 bp of the plasmid sequence . The polymorphic patterns generated with pLBL4 differed from those obtained with M13 DNA in the larger number of bands observed . The results presented here open the possibility of using pLBL4 as a new broad-spectrum polymorphic DNA probe for fingerprint analysis. FEMS Microbiol Lett, 2000 Sep 15, 190(2), 305 - 8 Production kinetics of acidophilin 801, a bacteriocin produced by Lactobacillus acidophilus IBB 801; Zamfir M et al.; Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801 . Studying the relationship between growth and bacteriocin biosynthesis revealed primary metabolite kinetics of bacteriocin production with a peak activity at the end of the exponential growth phase followed by a decrease during the stationary phase . Both microbial growth and bacteriocin production was inhibited by lactic acid . Whereas volumetric bacteriocin production (activity units (AU) ml(-1)) was favoured under pH-controlled conditions, bacteriocin titres rapidly decreased because of strong adsorption of the bacteriocin molecules to the producing cells under less acidic conditions. Cleft Palate Craniofac J, 2000 Sep, 37(5), 447 - 52 Dental health indices and caries associated microflora in children with unilateral cleft lip and palate; Lucas VS et al.; OBJECTIVE: To investigate the dental health and caries related microflora of children with unilateral cleft lip and palate . STUDY GROUP: Sixty children with unilateral cleft lip and palate and matched controls . OUTCOME MEASURES: The decayed, missing, and filled teeth and surfaces in both the deciduous and permanent dentitions . The presence of developmental defects and plaque and gingivitis scores were also recorded . Plaque was collected from 25 of the children and their matched controls from three different sites, which were (1) the first approximal site distal to the cleft, (2) a contralateral anterior site, and (3) a remote site . It was cultured for Streptococcus mutans and lactobacilli . Plaque was collected from two sites in the matched controls . RESULTS: There was no significant difference in the caries, plaque, and gingivitis scores between the children with cleft palate and the controls . A greater number of enamel opacities were recorded in the control group, and there was a higher prevalence of enamel discoloration in the children with cleft lip and palate . There was no significant difference in the proportion of S . mutans or lactobacilli at the cleft site, compared with the unaffected site in the study group, although there was an anterior-posterior gradient in the proportion of S . mutans . There was no significant association between the stagnation area at the cleft site and the bacteria associated with dental caries. Appl Microbiol Biotechnol, 2000 Sep, 54(3), 311 - 8 Intestinal receptors for adhesive fimbriae of enterotoxigenic Escherichia coli (ETEC) K88 in swine--a review; Jin LZ et al.; Determining the structure of the intestinal receptor for enterotoxigenic Escherichia coli (ETEC) K88 fimbriae will make it possible to develop new strategies to prevent K88+ ETEC-induced disease in pigs . Putative K88 adhesin receptors have been identified in both intestinal brush border and mucus preparations as either glycoproteins or glycolipids . Proteins with sizes of 25, 35, 40-42, 60, and 80 kDa in the intestinal mucus and 16, 23, 35, 40-70, 74, 210, and 240 kDa in brush border membranes were reported to bind specifically to K88ab and K88ac fimbriae . The factors accounting for these variable results may include the variants of K88, ages, breeds, and phenotypes of pigs, and even the sampling sites in the small intestine . Of the reported K88 receptors, only three brush border receptors, i.e., a pair of mucin-type sialoglycoproteins (210 kDa or 240 kDa), an intestinal neutral glycosphingolipid (IGLad), and a 74-kDa transferrin glycoprotein (GP74), have fulfilled the criteria as phenotype-specific K88 fimbrial receptors . Inhibiting the attachment of ETEC to intestine by modifying the receptor attachment sites has been the key for developing novel approaches to preventing ETEC-induced diarrhea in pigs . These include: (1) receptor analogs from a variety of biological sources, (2) an enteric protected protease, (3) chicken egg-yolk containing anti-K88 fimbrial antibodies, and (4) some Lactobacillus isolates producing proteinaceous components or carbohydrates interacting with mucus components . Future studies should be directed to further characterize the carbohydrate and protein moieties of receptors recognized by the K88 adhesin variants and to identify the genes responsible for susceptibility to K88+ infections. Prim, Care Update Ob Gyns . 2000 Sep 1, 7(5), 181 - 185 Bacterial vaginosis; Wang J; Bacterial vaginosis is the most common cause of vaginitis, affecting over 3 million women in the United States annually . Depopulation of lactobacilli from the normal vaginal flora and overgrowth of Gardnerella vaginalis and other anaerobic species are the presumed etiology . To date, no scientific evidence shows that bacterial vaginosis is a sexually transmitted disease . Malodorous vaginal discharge is the most common symptom . Differential diagnoses include trichomoniasis, moniliasis, and allergic or chemical dermatitis . The diagnosis is confirmed when at least three of the following four findings are present (Amsel's criteria): 1) thin, homogenous discharge, 2) pH greater than 4.5, 3) positive amine test, and 4) presence of clue cells . The sensitivity and positive predictive value are both 90% . Vaginal Gram stain is also reliable and allows for permanent record . Cultures are nonspecific because G . vaginalis resides in normal vaginal flora as well . Papanicolaou smears are not particularly sensitive, but their positive predictive value is very high . The Centers for Disease Control and Prevention recommend three treatment regimens in nonpregnant patients: oral metronidazole (500 mg twice daily for 7 days), intravaginal 2% clindamycin cream (one applicatorful at bedtime for 7 days), or intravaginal metronidazole gel (one to two applicatorfuls per day for 5 days) . Alternative regimens include a single 2-g oral dose of metronidazole or a 7-day course of oral clindamycin, 300 mg twice daily . The association between bacterial vaginosis and adverse pregnancy outcomes has satisfied many criteria for a causal inference . Treatment of bacterial vaginosis in women with previous history of preterm labor results in fewer preterm deliveries than in untreated women from the same population. J Appl Microbiol, 2000 Sep, 89(3), 547 - 52 Kinetics of the arginine metabolism of malolactic wine lactic acid bacteria Lactobacillus buchneri CUC-3 and Oenococcus oeni Lo111; Mira de Orduna R et al.; The excretion of citrulline, a precursor of carcinogenic ethyl carbamate, formed from arginine degradation by malolactic bacteria in wine is of toxicological concern . The arginine metabolism of resting cells of Lactobacillus buchneri CUC-3 and Oenococcus oeni Lo1l1 was examined . The citrulline excretion rate was found to be linearly correlated to the arginine degradation rate . It was possible to calculate an arginine to citrulline conversion ratio which could be used to predict the amount of citrulline expected after the degradation of a known quantity of arginine . The conversion ratios determined in this study were similar to data calculated from other authors for fermentations in wine and ranged between 4.0% and 7.7% . Ribose, fructose and glucose inhibited the degradation of arginine in Lact . buchneri CUC-3, and inhibition of arginine degradation by glucose correlated with higher arginine to citrulline conversion ratios . The work presents new results of arginine metabolism in malolactic bacteria and gives starting points for investigations in wine. J Appl Microbiol, 2000 Sep, 89(3), 511 - 6 Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex; Kullen MJ et al.; The Lactobacillus acidophilus complex includes Lact . acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii . The objective of this work was to develop a rapid and definitive DNA sequence-based identification system for unknown isolates of the Lact . acidophilus complex . A approximately = 500 bp region of the 16S rRNA gene, which contained the V1 and V2 variable regions, was amplified from the isolates by the polymerase chain reaction . The sequence of this region of the 16S rRNA gene from the type strains of the Lact . acidophilus complex was sufficiently variable to allow for clear differentiation amongst each of the strains . As an initial step in the characterization of potentially probiotic strains, this technique was successfully used to identify a variety of unknown human intestinal isolates . The approach described here represents a rapid and definitive method for the identification of Lact . acidophilus complex members. J Appl Microbiol, 2000 Sep, 89(3), 442 - 51 Effects of lactic acid bacteria in inoculants on changes in amino acid composition during ensilage of sterile and non-sterile ryegrass; Winters AL et al.; A study was carried out on the changes occurring in the amino acid fraction of a hybrid ryegrass during ensilage in laboratory-scale silos to help to establish the relative roles of plant and microbial proteases on protein degradation in the silo . Herbage treatments included (i) normal grass without treatment (ii) lambda-irradiated grass (sterile) without treatment (iii) sterile, inoculated with a strain of Lactobacillus plantarum and (iv) sterile, inoculated with a strain of Lactobacillus paracasei subsp . paracasei . These treatments had a significant effect on silage amino acid profiles . Concentrations of free amino acids and the extent of amino acid catabolism varied with treatment . However, levels were notably higher in control silages after 90 days (free amino acid nitrogen constituting 54% of total amino acid nitrogen compared with 37, 32 and 22% for treatments i, ii and iv, respectively) . These results indicate that the extent of protein hydrolysis during ensilage is influenced by factors other than rate of pH decline and plant protease activity, and that microbial proteases play a role. Curr Opin Pediatr, 2000 Oct, 12(5), 477 - 81 Probiotics in pediatric gastrointestinal disorders; Davidson GP et al.; Probiotics have been defined most recently as living microorganisms which, upon ingestion in certain numbers, exact health benefits beyond inherent general nutrition . They have been a part of human nutrition for centuries, but in recent years they have been more closely studied for their potential to improve health and treat disease . This review of probiotics is not extensive, highlighting the most recent reviews and well controlled clinical studies in both animals and humans . The safety issues are also discussed as well as potential mechanisms of action . The importance of studying each probiotic bacterium individually in each condition where a health benefit is claimed is highlighted by Lactobacillus GG, the most widely studied probiotic which has proven benefit in reducing the severity and duration of viral diarrhea but no benefit against bacterial diarrhea. Int J Food Microbiol, 2000 Sep 10, 59(3), 241 - 7 Study of the cryotolerance of Lactobacillus acidophilus: effect of culture and freezing conditions on the viability and cellular protein levels; Baati L et al.; Slow cooling rate and pre-freezing stress brings about a high increase in the cell resistance and preservation of their physiological characteristics . A brutal decrease in temperature (from 37 degrees C to - 80 degrees C) causes a considerable loss of cell viability, in contrast a slow one preserves a survival rate of 75% . Pre-incubation of cells at low temperature (22 degrees C) during 6 h led to the development of cryotolerance indicated by an enhanced capacity to survive after a freezing treatment of 24 h at - 80 degrees C . Exposure of the cells to low pH (5.5) caused a large decrease in cell resistance but did not lead to any significant decrease of survival rate after freezing treatment . However, an increase of 15 +/- 3% in protein level compared to cells cultivated at regulated pH was observed. Int J Food Microbiol, 2000 Sep 10, 59(3), 185 - 209 Structural model requirements to describe microbial inactivation during a mild heat treatment; Geeraerd AH et al.; The classical concept of D and z values, established for sterilisation processes, is unable to deal with the typical non-loglinear behaviour of survivor curves occurring during the mild heat treatment of sous vide or cook-chill food products . Structural model requirements are formulated, eliminating immediately some candidate model types . Promising modelling approaches are thoroughly analysed and, if applicable, adapted to the specific needs: two models developed by Casolari (1988), the inactivation model of Sapru et al . (1992), the model of Whiting (1993), the Baranyi and Roberts growth model (1994), the model of Chiruta et al . (1997), the model of Daughtry et al . (1997) and the model of Xiong et al . (1999) . A range of experimental data of Bacillus cereus, Yersinia enterocolitica, Escherichia coli O157:H7, Listeria monocytogenes and Lactobacillus sake are used to illustrate the different models' performances . Moreover, a novel modelling approach is developed, fulfilling all formulated structural model requirements, and based on a careful analysis of literature knowledge of the shoulder and tailing phenomenon . Although a thorough insight in the occurrence of shoulders and tails is still lacking from a biochemical point of view, this newly developed model incorporates the possibility of a straightforward interpretation within this framework. Cryobiology, 2000 Aug, 41(1), 17 - 24 Stabilization and preservation of Lactobacillus acidophilus in saccharide matrices; Conrad PB et al.; Lyophilization and vacuum- or spray-drying are some of the most useful techniques for preserving foods, agricultural products, and pharmaceuticals . Biological materials, however, can be irreversibly damaged during these treatments . Therefore, it is essential to design protective agents to preserve protein activity and cell viability . In this paper we examine the use of alpha, alpha-trehalose-borate systems as protectants for Lactobacillus acidophilus during freeze- and vacuum-drying . Trehalose was found to be an effective protectant for freeze-dried and vacuum-dried samples, and it is equivalent to a protective formulation which is in current industrial use . It is known from our previous work on enzymes that the presence of borate can dramatically enhance the protective ability of trehalose . In this work, the addition of trehalose-borate to bacterial concentrate greatly improves the recovery of viable cells after storage . This improvement was seen in freeze-dried samples stored at 37 degrees C as well as for vacuum-dried samples held at room temperature . A tailored buffering strategy was tested to counteract the high pH resulting from the addition of borate to the mixture . Use of citric or lactic acids in combination with ammonium hydroxide gave a protectant solution with high pH (resulting in effective crosslinking between trehalose and borate) but a dry product with reduced pH upon rehydration (conducive to cell survival) . These results raise exciting possibilities for protection of more labile prokaryotic species as well as simple eukaryotes . Cryobiology, 2000 Aug, 41(1), 10 - 6 Changes in the surface potential of Lactobacillus acidophilus under freeze-thawing stress; Fernndez Murga ML et al.; The zeta potential of Lactobacillus acidophilus CRL 640, a measure of the net distribution of electrical charges on the bacterial surface, is a function of the glucose concentration in the growing media . With 2% glucose, cells in the stationary phase showed a zeta potential of -45 +/- 2 mV . With these cells, the zeta potential after freezing and thawing decreased to -32 +/- 2 mV and there was a decrease in viability . The changes in the surface potential correlated with damage to the cell surface as shown by electron microscopy . Freeze-thawed cells incubated in a rich medium recovered a zeta potential of -38 +/- 2 mV without cell growth . L . acidophilus CRL 640 showed the same value of surface potential as control cells when they were frozen and thawed in 2 M glycerol . Antonie Van Leeuwenhoek, 2000 Jul, 78(1), 73 - 85 Numerical phenetic study of the genus Carnobacterium; Lai S et al.; Eighty-nine strains representing the genus Carnobacterium, Enterococcus durans, Vagacoccus salmoninarum and atypical Lactobacillus strains MT12 and MT13 were examined for 92 unit characters . Computer analysis of the data resulted in the recovery of four major, five minor and thirteen single membered clusters . Three cluster-groups contained seventy-four of the Carnobacterium strains, Enterococcus durans NCFB 596T and Lactobacillus maltaromicus NCFB 2382T . Cluster-group A was equated with Carnobacterium piscicola and cluster-group B with Carnobacterium divergens . Lactobacillus maltaromicus NCFB 2382T shared many properties in common with the C . piscicola strains . The recovery of several Carnobacterium strains as single membered clusters suggests that the genus Carnobacterium is underspeciated . Further work is also required to determine the subspecific structure of Carnobacterium divergens and Carnobacterium piscicola. Int J Food Microbiol, 2000 Sep 15, 60(1), 91 - 7 Cluster analysis, richness and biodiversity indexes derived from denaturing gradient gel electrophoresis fingerprints of bacterial communities demonstrate that traditional maize fermentations are driven by the transformation process; Amp F et al.; The bacterial communities of maize fermented foods (pozol, poto-poto and ogi) from Mexico, Congo and Benin was compared using a culture-independent approach {denaturing gradient gel electrophoresis (DGGE) analysis of total DNA} . Foods produced following the same flow chart (i) grouped in distinct clusters, (ii) shared similar richness and biodiversity indexes and (iii) exhibited a high intra-specific variability . Structural biodiversity was higher in pozol samples, probably due to oxic conditions and higher initial pH . DGGE bands found in foods of different origins suggest that Lactobacillus plantarum, Lb . delbrueckii and Lb . fermentum are particularly well adapted to the fermentation of maize. Int J Food Microbiol, 2000 Sep 15, 60(1), 75 - 81 Chemical, physical and enzymatic pre-treatments of probiotic lactobacilli alter their adhesion to human intestinal mucus glycoproteins; Tuomola EM et al.; Intestinal mucus glycoproteins extracted from faeces of healthy adult subjects were used as a substratum for bacterial adhesion to investigate the effects of physical, chemical and enzymatic pre-treatments of the bacteria on their adhesion . The strains studied were Lactobacillus acidophilus 1 (LCI, Nestle), L . rhamnosus strain GG (ATCC 53103), L . rhamnosus LC-705, and L . casei strain Shirota (Yakult, Yakult Ltd) . Hereafter the strains are referred to as LA1, LGG, LC-705, and Shirota, respectively . Strains LA1 and LGG adhered greatly whereas the adhesion of strains LC-705 and Shirota to intestinal mucus glycoproteins was low . Adhesion of LA1 and LGG was reduced by boiling, autoclaving and by pepsin and trypsin treatments suggesting that the bacterial protein structures are essential for their adhesion . Treatment in ethanol and in propanol prior to adhesion significantly increased the adhesion of LA1 and LC-705, respectively . Adhesion of Shirota strain was not altered by any of the treatments. Int J Food Microbiol, 2000 Sep 15, 60(1), 25 - 32 Action of lysozyme and nisin mixtures against lactic acid bacteria; Chun W et al.; Lysozyme was formulated together with nisin for usage against food spoilage lactobacilli . The mixtures demonstrated improved minimal inhibitory concentrations (MIC), compared to the parent compounds, for many of the bacteria and media tested, including high salt media in which lysozyme lost virtually all of its activity . Synergy was also observed through measurement of the kinetics of bacterial killing of L . curvatus 845, in which strain synergy had been observed in MIC assays . The combination of lysozyme and nisin caused more severe cell damage as viewed by scanning electron microscopy, and a consequent change in optical density at 600 nm, compared to the parent compounds, effects that were presumed to reflect the action of lysozyme . In addition, the combination caused more rapid permeabilization (depolarization) of the cytoplasmic membranes of Staphylococcus aureus, an effect that reflected the mechanism of action of nisin . Thus, nisin and lysozyme appear to demonstrate synergy against gram-positive bacteria because they reinforce each others mechanisms of bacterial killing. Appl Environ Microbiol, 2000 Oct, 66(10), 4427 - 32 Adaptation of the nisin-controlled expression system in Lactobacillus plantarum: a tool to study in vivo biological effects; Pavan S et al.; The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today . For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus . We therefore implemented in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by Lactococcus lactis . The original two-plasmid NICE system turned out to be poorly suited to L . plantarum . In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (beta-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L . plantarum NCIMB8826 . Moreover, recombinant L . plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice . The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli. Appl Environ Microbiol, 2000 Oct, 66(10), 4396 - 400 Identification of collagen-binding proteins in Lactobacillus spp . with surface-enhanced laser desorption/ionization-time of flight ProteinChip technology; Howard JC et al.; Biosurfactants produced by Lactobacillus fermentum RC-14, L . rhamnosus GR-1 and 36, and L . casei Shirota were found to contain proteins that bind to both collagen types III and VI, as determined by surface-enhanced laser desorption/ionization (SELDI)-time of flight mass spectrometry . Both collagen types III and VI immobilized on SELDI preactivated ProteinChip arrays detected several different sizes (2 to 48 kDa) of collagen-binding proteins . Overall, the RC-14-produced biosurfactant contained the greatest number of collagen-binding proteins (RC-14 > GR-1 > 36 > Shirota), including the mature form of a previously cloned 29-kDa collagen-binding protein (referred to in its mature 26-kDa form) . Although biosurfactants isolated from L . casei Shirota and L . rhamnosus 36 and GR-1 also contain several collagen-binding proteins, they do not contain the 26-kDa collagen-binding protein . Together, these results demonstrate the utility of the SELDI system as a means of rapidly characterizing clinically important but complex biosurfactant solutions. Appl Environ Microbiol, 2000 Oct, 66(10), 4272 - 8 Development of genetic tools for Lactobacillus sakei: disruption of the beta-galactosidase gene and use of lacZ as a reporter gene To study regulation of the putative copper ATPase, AtkB; Stentz R et al.; Downstream from the ptsHI operon of Lactobacillus sakei, the genes atkY and atkB, organized in an operon, were observed . The two putative proteins, AtkB and AtkY, show sequence similarity to the Enterococcus hirae copper P-type ATPase, responsible for copper efflux, and its negative regulator . Characterization of AtkB as a copper P-type ATPase could not be demonstrated since an atkB mutant did not show any phenotype . Thus, another strategy was followed in order to investigate the transcriptional regulation of the atkYB locus, leading to the development of new genetic tools for L . sakei . A plasmid was constructed, the use of which allowed gene replacement at the lacLM locus in L . sakei by two successive crossovers . A strain deleted of the lacLM operon encoding the beta-galactosidase of L . sakei was constructed by this method, and the Escherichia coli lacZ gene could then be used as a reporter gene to investigate the regulation of atkYB . Results show that the atkYB operon is induced by small concentrations of CuSO(4) (30 to 40 microM) but not when CuSO(4) is omitted or added at higher concentrations. Appl Environ Microbiol, 2000 Oct, 66(10), 4230 - 6 Biochemical and genetic characterization of propionicin T1, a new bacteriocin from Propionibacterium thoenii; Faye T et al.; A collection of propionibacteria was screened for bacteriocin production . A new bacteriocin named propionicin T1 was isolated from two strains of Propionibacterium thoenii . This bacteriocin shows no sequence similarity to other bacteriocins . Propionicin T1 was active against all strains of Propionibacterium acidipropionici, Propionibacterium thoenii, and Propionibacterium jensenii tested and also against Lactobacillus sake NCDO 2714 but showed no activity against Propionibacterium freudenreichii . The bacteriocin was purified, and the N-terminal part of the peptide was determined with amino acid sequencing . The corresponding gene pctA was sequenced, and this revealed that propionicin T1 is produced as a prebacteriocin of 96 amino acids with a typical sec leader, which is processed to give a mature bacteriocin of 65 amino acids . An open reading frame encoding a protein of 424 amino acids was found 68 nucleotides downstream the stop codon of pctA . The N-terminal part of this putative protein shows strong similarity with the ATP-binding cassette of prokaryotic and eukaryotic ABC transporters, and this protein may be involved in self-protection against propionicin T1 . Propionicin T1 is the first bacteriocin from propionibacteria that has been isolated and further characterized at the molecular level. Appl Environ Microbiol, 2000 Oct, 66(10), 4187 - 92 Urea hydrogen peroxide reduces the numbers of lactobacilli, nourishes yeast, and leaves no residues in the ethanol fermentation; Narendranath NV et al.; Urea hydrogen peroxide (UHP) at a concentration of 30 to 32 mmol/liter reduced the numbers of five Lactobacillus spp . (Lactobacillus plantarum, L . paracasei, Lactobacillus sp . strain 3, L . rhamnosus, and L . fermentum) from approximately 10(7) to approximately 10(2) CFU/ml in a 2-h preincubation at 30 degrees C of normal-gravity wheat mash at approximately 21 g of dissolved solids per ml containing normal levels of suspended grain particles . Fermentation was completed 36 h after inoculation of Saccharomyces cerevisiae in the presence of UHP, even when wheat mash was deliberately contaminated (infected) with L . paracasei at approximately 10(7) CFU/ml . There were no significant differences in the maximum ethanol produced between treatments when urea hydrogen peroxide was used to kill the bacteria and controls (in which no bacteria were added) . However, the presence of L . paracasei at approximately 10(7) CFU/ml without added agent resulted in a 5.84% reduction in the maximum ethanol produced compared to the control . The bactericidal activity of UHP is greatly affected by the presence of particulate matter . In fact, only 2 mmol of urea hydrogen peroxide per liter was required for disinfection when mashes had little or no particulate matter present . No significant differences were observed in the decomposition of hydrogen peroxide in normal-gravity wheat mash at 30 degrees C whether the bactericidal agent was added as H(2)O(2) or as urea hydrogen peroxide . NADH peroxidase activity (involved in degrading H(2)O(2)) increased significantly (P = 0.05) in the presence of 0.75 mM hydrogen peroxide (sublethal level) in all five strains of lactobacilli tested but did not persist in cells regrown in the absence of H(2)O(2) . H(2)O(2)-resistant mutants were not expected or found when lethal levels of H(2)O(2) or UHP were used . Contaminating lactobacilli can be effectively managed by UHP, a compound which when used at ca . 30 mmol/liter happens to provide near-optimum levels of assimilable nitrogen and oxygen that aid in vigorous fermentation performance by yeast. Mikrobiologiia, 2000 Jul-Aug, 69(4), 471 - 7 {Manganese-dependent ribonucleotide reductase from Propionibacterium freudenreichii subsp . Shermanii: partial purification, characteristics and role in DNA biosynthesis}; Iordan EP et al.; Like Lactobacillus leichmanii, Rhizobium meliloti, and Euglena gracilis, P . freudenreichii implicates cobalamin in DNA anabolism via adenosylcobalamin-dependent ribonucleotide reductase . However, in the absence of corrinoids, P . freudenreichii is able to synthesize DNA with the involvement of an alternative ribonucleotide reductase, which is independent of adenosylcobalamin . This enzyme is localized in both the cytoplasm (80% of activity) and the cytoplasmic membrane (20% of activity), being loosely bound to the latter . Experiments with crude ribonucleotide reductase isolated from extracts of corrinoid-deficient cells showed that manganese specifically stimulates this enzyme and that it is composed of two protein subunits, a feature that is typical of all metal-containing reductases activated by molecular oxygen . Low concentrations of manganese ions enhanced DNA synthesis in corrinoid-deficient manganese-limited cells . This effect was prevented by the addition of 80 mM hydroxyurea, a specific inhibitor of metal-containing aerobic ribonucleotide reductases . It was concluded that, in adenosylcobalamin-deficient P . freudenreichii cells, DNA synthesis is provided with deoxyribosyl precursors through the functioning of manganese-dependent aerobic ribonucleotide reductase composed of two subunits. FEMS Microbiol Lett, 2000 Oct 1, 191(1), 51 - 5 Acetaldehyde metabolism by wine lactic acid bacteria; Osborne JP et al.; Acetaldehyde is a volatile flavor compound present in many fermented foods and is important in the production of red and white wines . Nine strains of the genera Lactobacillus and Oenococcus were able to metabolize acetaldehyde in a resting cell system, whereas two Pediococcus strains were not . Acetic acid and ethanol were produced from its degradation . A Lactobacillus and an Oenococcus were able to degrade SO(2)-bound acetaldehyde, as well . A coincubation of resting cells of Saccharomyces bayanus Premiere Cuvee and Oenococcus oeni Lo111 showed that strain Lo111 metabolized acetaldehyde produced by the yeast . The ability of malolactic bacteria to degrade free and SO(2)-bound acetaldehyde has implications for sensory and color qualities and the use of SO(2) in wine. Arch Virol, 2000, 145(8), 1521 - 34 Cloning, sequence analysis, and expression of Lactobacillus casei phage PL-1 lysis genes; Kashige N et al.; The genes encoding the host cell wall-lytic proteins were searched in the genome DNA of phage PL-1 active against Lactobacillus casei ATCC 27092 by comparing the amino acid sequences with those of others using a computer software of the DDBJ data base . The gene regions found were cloned into E . coli by inserting PCR-amplified DNA fragments into the EcoRI site of pUC 19, and the nucleotide sequences were determined . One of the ORFs (hol) consisted of 270 bp encoding 90 amino acids . The hol product (holin) possessed a putative secretion signal, two putative transmembrane helices, and a highly charged C-terminus . Another ORF (lys) consisted of 1050 bp encoding an N-acetylmuramoyl-L-alanine amidase of 350 amino acids . The gene lys was expressed in E . coli using pCALn expression vector, and the purified gene product hydrolysed the amide linkage in the peptidoglycans of L . casei . The amino acid sequence of PL-1 amidase showed a high homology to those of Lactococcus lactis phage rlt and Listeria monocytogenes phage A511 . It was suggested that the N-terminal region was involved in enzyme activity and the C-terminal region in binding the enzyme to the cell wall substrate, respectively. Oral Dis, 2000 Sep, 6(5), 297 - 302 Coptidis rhizoma inhibits growth and proteases of oral bacteria; Hu JP et al.; OBJECTIVE: The aim of this study was to investigate the antibacterial effect of Coptidis Rhizoma (CR), a traditional medicinal plant, on oral bacteria . MATERIALS AND METHODS: CR extract was prepared by boiling CR in water for 2 h . Alkaloids contained in CR extract were assayed by high performance liquid chromatography (HPLC) . Antibacterial activity of CR extract was estimated from the lowest concentration that did not permit bacterial growth (minimum inhibitory concentration, MIC) and the concentrations that inhibited 50% of bacterial proteolytic activity (IC50) . RESULTS: CR extract inhibited the growth of Actinomyces naeslundii, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Actinobacillus actinomycetemcomitans at MIC of 0.031-0.25 mg ml(-1), whereas it had less inhibitory effect (MIC: 0.5-2 mg ml(-1)) on the growth of Streptococcus and Lactobacillus . The major active component of CR extract was berberine (Ber), an alkaloid, and its inhibiting specificity to bacterial growth was similar to that of CR extract . CR extract and Ber were bacteriostatic at the MICs against most of the bacteria, and bacteriocidal at the concentrations higher than the MICs . Ber inhibited the activities of collagenase from P . gingivalis and A . actinomycetemcomitans . CONCLUSION: CR extract and Ber had an inhibitory effect on periodontopathogenic bacteria . These results suggest the possibility of their clinical application for the treatment of periodontal diseases. Chem Biol Interact, 2000 Aug 15, 128(1), 39 - 49 Factors affecting the sequestration of aflatoxin by Lactobacillus rhamnosus strain GG; Haskard C et al.; The interaction of a potent carcinogen, aflatoxin B(1) (AFB(1)), with a probiotic strain of lactic acid bacteria, Lactobacillus rhamnosus strain GG (GG), has been investigated . The binding of AFB(1) to GG in the late exponential-early stationary phase was studied for viable, heat-killed and acid-killed bacteria . In general, viable, heat-killed and acid-killed GG responded in a similar manner . The effects of pronase E, lipase and m-periodate on AFB(1) binding and release were consistent with AFB(1) binding predominantly to carbohydrate components of the bacteria . The effect of urea suggested hydrophobic interactions play a major role in binding . Increasing concentration (0.01-1 M) of NaCl or CaCl(2) had minor effects on AFB(1) binding suggesting some involvement of electrostatic interactions . An increase in pH from 2.5 to 8.5 had no effect on AFB(1) binding but decreased binding of AFB(2a), possibly due to hydrogen bonding interactions. Antibiot Khimioter, 2000, 45(7), 17 - 21 {The effect of netilmicin, amikacin, ceftazidime and cefotaxime on the adhesive properties of microorganisms isolated from newborn infants}; Kushnareva MV; The effect of netilmycin, amikacin, ceftazidime and cefotaxime on adhesion of Lactobacillus spp . (14 strains), Escherichia coli (21 strains), Klebsiella pneumonia (15 strains), Enterococcus sp . (18 strains), Candida albicans (15 strains) was investigated . The strains were isolated from respiratory tract and feces of the newborns . Antibiotics were used in the following subtherapeutic and therapeutic concentrations: netilmycin--1.2 and 12.0 micrograms/ml, amikacin--1.8 and 18 micrograms/ml, ceftazidime--7.5 and 75 micrograms/ml, cefotaxime--6.5 and 75 micrograms/ml . Adhesion of C . albicans was investigated with buccal epithelium cells, adhesion of other microorganisms--on formalinized human erythrocytes (1(0)Rh(+)) . It was shown that antibiotics in subtherapeutic and therapeutic concentrations inhibited adhesion of the most strains . Cefalosporins demonstrated maximum inhibitory activity . The number of the strains inhibited by cefalosporins and by aminoglycosides enhanced along with antibiotics concentrations enhancement from subtherapeutic to therapeutic concentrations. J Dairy Sci, 2000 Aug, 83(8), 1684 - 91 Ripening of cheddar cheese with added attenuated adjunct cultures of lactobacilli; Madkor SA et al.; We made Milled curd Cheddar cheese with Lactococcus starter and an adjunct culture of Lactobacillus helveticus I or Lactobacillus casei T subjected to different attenuation treatments: freeze shocking (FS), heat shocking (HS), or spray drying (SD) . Proteolysis during cheese ripening (0 to 6 mo), measured by urea-PAGE and water-soluble nitrogen, indicated only minor differences between control and most adjunct-treated cheeses . However, there were significant differences in the effect of Lactobacillus adjuncts on the level of free amino nitrogen in cheese . Cheeses made with FS or HS Lb . helveticus adjunct exhibited significantly greatest rates of free amino group formation . Lipolysis as measured by total free fatty acids was consistently highest in adjunct-treated cheeses, and FS Lb . casei-treated cheeses showed the highest rate of free fatty acid formation followed by FS Lb . helveticus treated cheeses . Mean flavor and aroma scores were significantly higher for cheeses made with Lb . helveticus strain . Freeze-shocked Lb . helveticus-treated cheeses obtained the highest flavor and aroma scores . Sensory evaluation indicated that most of the adjunct-treated cheeses promoted better texture and body quality. Indian J Biochem Biophys, 2000 Apr, 37(2), 121 - 9 Purification and characterization of dihydrofolate reductase from Lactobacillus leichmannii; Rao KN; Dihydrofolate reductase (DHFR) (5,6,7,8-THF: NADDP+ oxidoreductase, EC 1.5.1.3) was purified 205-fold to apparent homogeneity from the crude extracts of Lactobacillus leichmannii . It has UV absorption maxima at 280 nm, M(r) of 20,000, Stokes radius of 0.34 nm and a S20.w value of 0.12 S . The preparation showed the presence of 168 amino acid residues with threonine and lysine as the NH2- and COOH- terminal end-groups respectively and a single reactive sulfhydryl group . pCMB inhibited the enzyme activity (IC50 = 2 microM) . The enzyme has a pH optimum of 7.4 and is thermally inactivated at > 35 degrees C . It is activated by 0.1 M KCl and KI and 2 M urea . 3-4 M urea completely inactivated the enzyme . Enzyme has Km values of 3.5 microM and 6.2 microM for NADPH and DHF respectively, and a Ki value of 7 nM for MTX, the inhibition being competitive. Microbiol Immunol, 2000, 44(7), 551 - 6 Development of a chromosome-plasmid balanced lethal system for Lactobacillus acidophilus with thyA gene as selective marker; Fu X et al.; A chromosome-plasmid balanced lethal gene delivery system for Lactobacillus acidophilus based on the thyA gene was developed . The selected L . acidophilus DOM La strain carries a mutated thyA gene and has an obligate requirement for thymidine . This strain can be used as a host for the constructed shuttle vector pFXL03, lacking antibiotic-resistant markers but having the wild-type thyA gene from L . casei which complements the thyA chromosomal mutation . The vector also contains the replicon region from plasmid pUC19 and that of the Lactococcus plasmid pWV01, which allows the transfer between Escherichia coli, L . casei and L . acidophilus . Eight unique restriction sites (i.e., PstI, HindIII, SphI, SalI, AccI, XbaI, KpnI and SacI) are available for cloning . After 40-time transfers in modified MRS medium, no plasmid loss was observed . The vector pFXL03 is potentially useful as a food-grade vaccine delivery system for L . acidophilus. Biochemistry, 2000 Sep 5, 39(35), 10747 - 54 Mechanism of elementary catalytic steps of pyruvate oxidase from Lactobacillus plantarum; Tittmann K et al.; Single steps in the catalytic cycle of pyruvate oxidase from Lactobacillus plantarum have been characterized kinetically and mechanistically by stopped-flow in combination with kinetic solvent isotope effect studies . Reversible substrate binding of pyruvate occurs with an on-rate of 6.5 x 10(4) M(-1) s(-1) and an off-rate of pyruvate of 20 s(-1) . Decarboxylation of the intermediate lactyl-ThDP and the reduction of FAD which consists of two consecutive single electron-transfer steps from HEThDP to FAD occur with rates of about k(dec) = 112 s(-1) and k(red) = 422 s(-1) . Flavin radical intermediates are not observed during reduction, and kinetic solvent isotope effects are absent, indicating that electron transfer and protonation processes are not rate limiting in the overall reduction process . Reoxidation of FADH(2) by O(2) to yield H(2)O(2) takes place at a pseudo-first-order rate of about 35 s(-1) in air-saturated buffer . A comparable value of about 35 s(-1) was estimated for the phosphorolysis of the acetyl-ThDP intermediate at phosphate saturation . In competition with phosphorolysis, enzyme-bound acetyl-ThDP is hydrolyzed with a rate k = 0.03 s(-1) . This is the first report in which the reaction of enzyme-bound acetyl-ThDP with phosphate and OH(-) is monitored directly by FAD absorbance changes using the sequential stopped-flow technique. Microbiology, 2000 Sep, 146 ( Pt 9), 2155 - 60 The synthesis of the bacteriocin sakacin A is a temperature-sensitive process regulated by a pheromone peptide through a three-component regulatory system; Diep DB et al.; Sakacin A is a bacteriocin produced by Lactobacillus sakei Lb706 . The gene cluster (sap) encompasses a regulatory unit composed of three consecutive genes, orf4 and sapKR . sapKR encode a histidine protein kinase and a response regulator, while orf4 encodes the putative precursor of a 23-amino-acid cationic peptide (termed Sap-Ph) . The authors show that Sap-Ph serves as a pheromone regulating bacteriocin production . Lb706 produced bacteriocin when the growth temperature was kept at 25 or 30 degrees C, but production was reduced or absent at higher temperatures (33.5-35 degrees C) . Production was restored by lowering the growth temperature to 30 degrees C, but at temperatures of 33-34 degrees C also by adding exogenous Sap-Ph to the growth medium . A knock-out mutation in orf4 abolished sakacin A production . Exogenously added Sap-Ph complemented this mutation, unambiguously showing the essential role of this peptide for bacteriocin production . Another sakacin A producer, Lactobacillus curvatus LTH1174, had a similar response to temperature and exogenously added Sap-Ph. Lett Appl Microbiol, 2000 Sep, 31(3), 193 - 7 Solvent extraction of bacteriocins from liquid cultures; Burianek LL et al.; A solvent extraction method was developed to concentrate lacidin from the culture of Lactobacillus acidophilus OSU133 . The new method concentrates the bacteriocin at the interface between chloroform and the aqueous culture of the producing bacterium . Compared with other extraction procedures, the new method effectively recovers higher bacteriocin yield and results in relatively clean preparations . Recovery of lacidin by the chloroform extraction procedure, compared with ammonium sulphate precipitation and cell acidification methods, was >10-fold and about 100-fold greater, respectively . The new extraction procedure saves time and is easy to perform . This method is also effective in recovering subtilin, bacillicin, pediocin and nisin from cultures of Bacillus subtilis ATCC 6633, B . subtilis OSY1115/C, Pediococcus acidilactici PO2 and Lactococcus lactis ATCC 11454, respectively. Lett Appl Microbiol, 2000 Aug, 31(2), 129 - 33 The use of multiplex PCR reactions to characterize populations of lactic acid bacteria associated with meat spoilage; Yost CK et al.; A rapid, systematic and reliable approach for identifying lactic acid bacteria associated with meat was developed, allowing for detection of Carnobacterium spp., Lactobacillus curvatus, Lact . sakei and Leuconostoc spp . Polymerase chain reaction primers specific for Carnobacterium and Leuconostoc were created from 16S rRNA oligonucleotide probes and used in combination with species-specific primers for the 16S/23S rRNA spacer region of Lact . curvatus and Lact . sakei in multiplex PCR reactions . The method was used successfully to characterize lactic acid bacteria isolated from a vacuum-packaged pork loin stored at 2 degrees C . Seventy isolates were selected for identification and 52 were determined to be Lact . sakei, while the remaining 18 isolates were identified as Leuconostoc spp. J Mol Biol, 2000 Sep 15, 302(2), 427 - 40 Structural and functional similarities in the ADP-forming amide bond ligase superfamily: implications for a substrate-induced conformational change in folylpolyglutamate synthetase; Sheng Y et al.; Comparison of the three-dimensional structures of folylpolyglutamate synthetase (FPGS) and the bacterial cell wall ligase UDP-N-acetylmuramoyl-l-alanine:d-glutamate ligase (MurD) reveals that these two enzymes have a remarkable structural similarity despite a low level of sequence identity . Both enzymes have a modular, multi-domain structure and catalyse a similar ATP-dependent reaction involving the addition of a glutamate residue to a carboxylate-containing substrate, tetrahydrofolate in the case of FPGS, and UDP-N-acetylmuramoyl-l-alanine in the case of MurD . Site-directed mutations of selected residues in the active site of Lactobacillus casei FPGS (P74A, E143A, E143D, E143Q, K185A, D313A, H316A, G411A and S412A) showed that most of these changes resulted in an almost complete loss of activity . Several of these amino acid residues in FPGS are found in structurally equivalent positions to active-site residues in MurD . Some insights into the function of these residues in FPGS activity are proposed, based on the roles surmised from the structures of two MurD . UDP-N-acetylmuramoyl-l-alanine.ADP complexes and a MurD . UDP-N-acetylmuramoyl-l-alanine-d-glutamate complex . Furthermore, the comparison has led us to propose that conformational changes induced by substrate binding in the reaction mechanism of FPGS result in a movement of the domains towards each other to more closely resemble the orientation of the corresponding domains in MurD . This relative domain movement may be a key feature of this new family of ADP-forming amide bond ligases . Appl Microbiol Biotechnol, 2000 Aug, 54(2), 243 - 7 Bacterial response to acetate challenge: a comparison of tolerance among species; Lasko DR et al.; Although acetate formation and tolerance are important criteria for various aspects of biotechnological process development, available studies on acetate tolerance in different species are disparate . We evaluate the response of eight bacterial strains, including two variants of Escherichia coli, two variants of Staphylococcus capitis, and one each of Acetobacter aceti, Gluconobacter suboxydans, Lactobacillus acetotolerans, and L . bulgaricus, to acetate challenges under identical conditions . Our findings were: (1) wild-type organisms of species that are considered tolerant of acetate perform only slightly better than E . coli in unadapted shaker cultures; (2) the ability to tolerate acetate is strongly dependent on the carbon source, and is, especially for E . coli, much greater on glycerol than on glucose; (3) respiration is not as important to acetate tolerance in E . coli and S . capitis as has been reported for the acetic acid bacteria; (4) S . capitis was the least affected by acetate under all conditions and grew at up to 44 g/l acetate without any preconditioning; and (5) qualitative high-throughput screening of growth characteristics can be achieved with relatively inexpensive multiwell plate readers. J Environ Sci Health B, 2000 Sep, 35(5), 599 - 610 Salmonella enteritidis hilA gene fusion response after incubation in spent media from either S . enteritidis or a poultry Lactobacillus strain; Durant JA et al.; The purpose of this study was to determine if growth of a poultry probiotic lactobacilli strain can influence S . enteritidis virulence expression by measuring the response of a hilA-lacZY transcriptional fusion . beta-galactosidase activity was not detected when S . enteritidis was incubated in Lactobacillus-spent medium (24 h growth, pH 4.1, 50.4 mM lactate) but was detectable in spent medium from 4 h growth cultures of Lactobacillus sp . (final OD of 0.213, pH 5.7, 12 mM lactate) when pH and lactate were adjusted to that of the 24 h-pH 4 spent media levels . Adjusting the pH of the 24 h spent medium from 4 to 6, resulted in a measurable beta-galactosidase activity that was significantly higher than expression in LB broth . When S . enteritidis was grown in Salmonella-spent media (24 h growth, pH 4.2, 78 mM acetate), hilA expression was increased 4-fold over expression in the LB broth. Infect Dis Obstet Gynecol, 2000, 8(3-4), 184 - 90 Gynecologic conditions and bacterial vaginosis: implications for the non-pregnant patient; Sweet RL; Bacterial vaginosis is characterized by a shift from the predominant lactobacillus vaginal flora to an overgrowth of anaerobic bacteria . Bacterial vaginosis is associated with an increased risk of gynecologic complications, including pelvic inflammatory disease, postoperative infection, cervicitis, human immunodeficiency virus (HIV), and possibly cervical intraepithelial neoplasia (CIN) . The obstetrical risks associated with bacterial vaginosis include premature rupture of membranes, preterm labor and delivery, chorioamnionitis and postpartum endometritis . Despite the health risks associated with bacterial vaginosis and its high prevalence in women of childbearing age, bacterial vaginosis continues to be largely ignored by clinicians, particularly in asymptomatic women. FEMS Immunol Med Microbiol, 2000 Sep, 29(1), 47 - 52 Modulation of humoral immune response through probiotic intake; Fang H et al.; Thirty healthy volunteers were randomised into three different treatment groups and consumed Lactobacillus GG, Lactococcus lactis or placebo (ethyl cellulose) for 7 days . On days 1, 3 and 5, an attenuated Salmonella typhi Ty21a oral vaccine was given to all subjects to mimic an enteropathogenic infection . All subjects responded well to the vaccine, but no significant differences were observed in numbers of IgA-, IgG- and IgM-secreting cells among the different groups . There was a trend towards a greater increase in specific IgA among the subjects receiving the vaccine in combination with Lactobacillus GG . Those receiving L . lactis with their vaccine evinced significantly higher CR3 receptor expression on neutrophils than those receiving either the placebo or Lactobacillus GG . These results indicate that probiotics may influence differently the immune response to oral S . typhi vaccine and that the immunomodulatory effect of probiotics is strain-dependent. Appl Environ Microbiol, 2000 Sep, 66(9), 4084 - 90 Purification and characterization of novel antifungal compounds from the sourdough Lactobacillus plantarum strain 21B; Lavermicocca P et al.; Sourdough lactic acid bacteria were selected for antifungal activity by a conidial germination assay . The 10-fold-concentrated culture filtrate of Lactobacillus plantarum 21B grown in wheat flour hydrolysate almost completely inhibited Eurotium repens IBT18000, Eurotium rubrum FTDC3228, Penicillium corylophilum IBT6978, Penicillium roqueforti IBT18687, Penicillium expansum IDM/FS2, Endomyces fibuliger IBT605 and IDM3812, Aspergillus niger FTDC3227 and IDM1, Aspergillus flavus FTDC3226, Monilia sitophila IDM/FS5, and Fusarium graminearum IDM623 . The nonconcentrated culture filtrate of L . plantarum 21B grown in whole wheat flour hydrolysate had similar inhibitory activity . The activity was fungicidal . Calcium propionate at 3 mg ml(-1) was not effective under the same assay conditions, while sodium benzoate caused inhibition similar to L . plantarum 21B . After extraction with ethyl acetate, preparative silica gel thin-layer chromatography, and chromatographic and spectroscopic analyses, novel antifungal compounds such as phenyllactic and 4-hydroxy-phenyllactic acids were identified in the culture filtrate of L . plantarum 21B . Phenyllactic acid was contained at the highest concentration in the bacterial culture filtrate and had the highest activity . It inhibited all the fungi tested at a concentration of 50 mg ml(-1) except for P . roqueforti IBT18687 and P . corylophilum IBT6978 (inhibitory concentration, 166 mg ml(-1)) . L . plantarum 20B, which showed high antimold activity, was also selected . Preliminary studies showed that phenyllactic and 4-hydroxy-phenyllactic acids were also contained in the bacterial culture filtrate of strain 20B . Growth of A . niger FTDC3227 occurred after 2 days in breads started with Saccharomyces cerevisiae 141 alone or with S . cerevisiae and Lactobacillus brevis 1D, an unselected but acidifying lactic acid bacterium, while the onset of fungal growth was delayed for 7 days in bread started with S . cerevisiae and selected L . plantarum 21B. Appl Environ Microbiol, 2000 Sep, 66(9), 3974 - 80 Dissolution of xylose metabolism in Lactococcus lactis; Erlandson KA et al.; Xylose metabolism, a variable phenotype in strains of Lactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyl operon . The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci . Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L . lactis subsp . lactis B-4449 (formerly Lactobacillus xylosus), L . lactis subsp . lactis IO-1, and L . lactis subsp . lactis 210, respectively . The two environmental isolates, L . lactis B-4449 and L . lactis IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose . L . lactis 210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl(-) . Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl(+) strains . Sequence comparisons revealed a number of point mutations in the xylA, xylB, and xylR genes in L . lactis 210, IO-1, and B-4449 . None of these mutations, with the exception of a premature stop codon in xylB, are obviously lethal, since they lie outside of regions recognized as critical for activity . Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of L . lactis unable to metabolize xylose. Appl Environ Microbiol, 2000 Sep, 66(9), 3966 - 73 Effects of high pressure on survival and metabolic activity of Lactobacillus plantarum TMW1.460; Ulmer HM et al.; The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death . The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa . Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds . HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10(6) cells . During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture . Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability . Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation . An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells . Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions. Appl Environ Microbiol, 2000 Sep, 66(9), 3898 - 904 Production of angiotensin-I-converting-enzyme-inhibitory peptides in fermented milks started by Lactobacillus delbrueckii subsp . bulgaricus SS1 and Lactococcus lactis subsp . cremoris FT4; Gobbetti M et al.; Two fermented milks containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus delbrueckii subsp . bulgaricus SS1 and L . lactis subsp . cremoris FT4 . The pH 4.6-soluble nitrogen fraction of the two fermented milks was fractionated by reversed-phase fast-protein liquid chromatography . The fractions which showed the highest ACE-inhibitory indexes were further purified, and the related peptides were sequenced by tandem fast atom bombardment-mass spectrometry . The most inhibitory fractions of the milk fermented by L . delbrueckii subsp . bulgaricus SS1 contained the sequences of beta-casein (beta-CN) fragment 6-14 (f6-14), f7-14, f73-82, f74-82, and f75-82 . Those from the milk fermented by L . lactis subsp . cremoris FT4 contained the sequences of beta-CN f7-14, f47-52, and f169-175 and kappa-CN f155-160 and f152-160 . Most of these sequences had features in common with other ACE-inhibitory peptides reported in the literature . In particular, the beta-CN f47-52 sequence had high homology with that of angiotensin-II . Some of these peptides were chemically synthesized . The 50% inhibitory concentrations (IC(50)s) of the crude purified fractions containing the peptide mixture were very low (8.0 to 11.2 mg/liter) . When the synthesized peptides were used individually, the ACE-inhibitory activity was confirmed but the IC(50)s increased considerably . A strengthened inhibitory effect of the peptide mixtures with respect to the activity of individual peptides was presumed . Once generated, the inhibitory peptides were resistant to further proteolysis either during dairy processing or by trypsin and chymotrypsin. Appl Environ Microbiol, 2000 Sep, 66(9), 3835 - 41 Metabolic engineering of Lactobacillus helveticus CNRZ32 for production of pure L-(+)-lactic acid; Kyla-Nikkila K et al.; Expression of D-(-)-lactate dehydrogenase (D-LDH) and L-(+)-LDH genes (ldhD and ldhL, respectively) and production of D-(-)- and L-(+)-lactic acid were studied in Lactobacillus helveticus CNRZ32 . In order to develop a host for production of pure L-(+)-isomer of lactic acid, two ldhD-negative L . helveticus CNRZ32 strains were constructed using gene replacement . One of the strains was constructed by deleting the promoter region of the ldhD gene, and the other was constructed by replacing the structural gene of ldhD with an additional copy of the structural gene (ldhL) of L-LDH of the same species . The resulting strains were designated GRL86 and GRL89, respectively . In strain GRL89, the second copy of the ldhL structural gene was expressed under the ldhD promoter . The two D-LDH-negative strains produced only L-(+)-lactic acid in an amount equal to the total lactate produced by the wild type . The maximum L-LDH activity was found to be 53 and 93% higher in GRL86 and GRL89, respectively, than in the wild-type strain . Furthermore, process variables for L-(+)-lactic acid production by GRL89 were optimized using statistical experimental design and response surface methodology . The temperature and pH optima were 41 degrees C and pH 5.9 . At low pH, when the growth and lactic acid production are uncoupled, strain GRL89 produced approximately 20% more lactic acid than GRL86. Appl Environ Microbiol, 2000 Sep, 66(9), 3764 - 72 Characterization of Leuconostoc gasicomitatum sp . nov., associated with spoiled raw tomato-marinated broiler meat strips packaged under modified-atmosphere conditions; Bjorkroth KJ et al.; Lactic acid bacteria (LAB) associated with gaseous spoilage of modified-atmosphere-packaged, raw, tomato-marinated broiler meat strips were identified on the basis of a restriction fragment length polymorphism (RFLP) (ribotyping) database containing DNAs coding for 16S and 23S rRNAs (rDNAs) . A mixed LAB population dominated by a Leuconostoc species resembling Leuconostoc gelidum caused the spoilage of the product . Lactobacillus sakei, Lactobacillus curvatus, and a gram-positive rod phenotypically similar to heterofermentative Lactobacillus species were the other main organisms detected . An increase in pH together with the extreme bulging of packages suggested a rare LAB spoilage type called "protein swell." This spoilage is characterized by excessive production of gas due to amino acid decarboxylation, and the rise in pH is attributed to the subsequent deamination of amino acids . Protein swell has not previously been associated with any kind of meat product . A polyphasic approach, including classical phenotyping, whole-cell protein electrophoresis, 16 and 23S rDNA RFLP, 16S rDNA sequence analysis, and DNA-DNA reassociation analysis, was used for the identification of the dominant Leuconostoc species . In addition to the RFLP analysis, phenotyping, whole-cell protein analysis, and 16S rDNA sequence homology indicated that L . gelidum was most similar to the spoilage-associated species . The two spoilage strains studied possessed 98.8 and 99.0% 16S rDNA sequence homology with the L . gelidum type strain . DNA-DNA reassociation, however, clearly distinguished the two species . The same strains showed only 22 and 34% hybridization with the L . gelidum type strain . These results warrant a separate species status, and we propose the name Leuconostoc gasicomitatum sp . nov . for this spoilage-associated Leuconostoc species. Protein Eng, 2000 Aug, 13(8), 557 - 63 Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase; Kawase S et al.; Arginines R23, R178, R179 and R218 in thymidylate synthase (TS, EC 2 . 1.1.45) are hydrogen bond donors to the phosphate moiety of the substrate, dUMP . In order to investigate how these arginines contribute to enzyme function, we prepared complete replacement sets of mutants at each of the four sites in Lactobacillus casei TS . Mutations of R23 increase K:(m) for dUMP 2-20-fold, increase K:(m) for cofactor 8-40-fold and decrease k(cat) 9-20-fold, reflecting the direct role of the R23 side chain in binding and orienting the cofactor in ternary complexes of the enzyme . Mutations of R178 increase K:(m) for dUMP 40-2000-fold, increase K:(m) for cofactor 3-20-fold and do not significantly affect k(cat) . These results are consistent with the fact that this residue is an integral part of the dUMP-binding wall and contributes to the orientation and ordering of several other dUMP binding residues . Kinetic parameters for all R179 mutations except R179P were not significantly different from wild-type values, reflecting the fact that this external arginine does not directly contact the cofactor or other ligand-binding residues . R218 is essential for the structure of the catalytic site and all mutations of this arginine except R218K were inactive. Biol Pharm Bull, 2000 Aug, 23(8), 973 - 8