Appl Environ Microbiol, 1997 May, 63(5), 1701 - 11 Sequence analysis and characterization of pOM1, a small cryptic plasmid from Butyrivibrio fibrisolvens, and its use in construction of a new family of cloning vectors for Butyrivibrios; Hefford MA et al.; As a preliminary step in the development of vector systems, we have isolated and begun to characterize small, cryptic plasmids from several strains of the rumen bacterium Butyrivibrio fibrisolvens . We present here the complete nucleotide sequence of Butyrivibrio plasmid pOM1, which was isolated from B . fibrisolvens Bu49 . While it is very similar in size to the previously characterized Butyrivibrio plasmids pRJF1 and pRJF2, pOM1 exhibits a restriction pattern which is quite distinct . Analysis of sequence data reveals that pOM1 contains only two open reading frames of significant length (ORF1 and ORF2), both of which are required for self-replication and maintenance . The protein encoded in ORF1 shows homologies with Pre (plasmid recombination enzyme) proteins encoded in plasmids from gram-positive organisms such as Staphylococcus aureus, Streptococcus agalactiae, Lactobacillus plantarum, and Bacillus thuringiensis . The putative translation product of ORF2, on the other hand, resembles Rep (replication) proteins of a different group of gram-positive plasmids, for which the Staphylococcus plasmid pSN2 is a prototype . Unlike the other characterized-Butyrivibrio plasmids, pOM1 appears to replicate via a rolling-circle mechanism . Experimental evidence showing the presence of a single-stranded replication intermediate consistent with this mechanism is presented . pOM1 has been used in the construction of a new Escherichia coli-B . fibrisolvens shuttle vector, pSMerm1, which has been successfully used to introduce a cloned gene into B . fibrisolvens harboring the pRJF1 plasmid. J Bacteriol, 1997 May, 179(9), 3039 - 42 Cloning and characterization of cspL and cspP, two cold-inducible genes from Lactobacillus plantarum; Mayo B et al.; Two cold shock genes, cspL and cspP, have been cloned from two Lactobacillus plantarum strains . These genes, which are nonallelic, were present in all strains tested . The genes encode 66-amino-acid polypeptides related to each other and to the cold shock Csp family . Transcription of cspP rendered a single mRNA, while two cspL mRNAs were found with common 5' ends . The amounts of these transcripts increased moderately upon exposure of the cultures to cold. Int J Food Microbiol, 1997 Apr 15, 35(3), 271 - 4 Microflora of Boza, a traditional fermented Turkish beverage; Hancioglu O et al.; Boza, a Turkish traditional beverage made by yeast and lactic acid fermentation of cooked maize, wheat and rice flours was prepared and the microbial characteristics were investigated . During the course of fermentation from 0 to 24 h, populations of lactic acid bacteria and yeast raised from 7.6 x 10(6) and 2.25 x 10(5) after inoculation to 4.6 x 10(8) and 8.1 x 10(6), respectively; pH dropped from 6.1 to 3.5; total titratable acidity by means of lactic acid increased from 0.02 to 0.27 mmol/g; alcohol content increased from 0.017% to 0.79% . Seventy seven isolates of lactic acid bacteria and 70 yeast isolates were identified . The lactic acid bacteria isolated during the fermentation included Leuconostoc paramesenteroides (25.6%), Lactobacillus sanfrancisco (21.9%), Leuconostoc mesenteroides subsp . mesenteroides (18.6%), Lactobacillus coryniformis (9.1%), L . confusus (7.8%), Leuconostoc mesenteroides subsp . dextranicum (7.3%), Lactobacillus fermentum (6.5%), Leuconostoc oenos (3.7%) . The yeasts isolated comprised Saccharomyces uvarum (83.0%) and S . cerevisiae (17.0%). Int J Food Microbiol, 1997 Apr 15, 35(3), 259 - 65 The effect of electron beam irradiation, combined with acetic acid, on the survival and recovery of Escherichia coli and Lactobacillus curvatus; Fielding LM et al.; The preservation of food by ionising radiation may lead to undesirable sensory changes within the food . These changes can be reduced by combining irradiation with other treatments, for example the addition of organic acids . Late exponential phase cultures of Escherichia coli and Lactobacillus curvatus were irradiated, in a liquid medium, at doses of 0-1.8 kilograys (kGy), in the presence of acetic acid (0-2%) at pH 4.6 . A synergistic effect occurred when E . coli was irradiated in the presence of acetic acid (0.02-1.0%) at all doses used (0.145-1.1 kGy) . There is evidence to suggest that membrane disruption occurred in the cells as a result of the combined treatments and this may account, to some extent, for the synergism observed . The addition of acetic acid up to a concentration of 2.0% had no effect upon the radiation survival or upon the subsequent growth of L . curvatus. Tidsskr Nor Laegeforen, 1997 Apr 10, 117(9), 1282 - 4 {Occurrence of bacterial vaginosis among abortion seekers}; Bjornerem A et al.; Bacterial vaginosis is the most common vaginal infection during the fertile period . The clinical diagnosis is based on three of Amsel's four criteria: thin, grey-white discharge, vaginal fluid pH above 4.5, a fishy odour on addition of 10% potassium hydroxide solution to the vaginal fluid, and the presence of clue cells on a saline wet mount . A probably more sensitive indicator of the diagnosis is based on Gram-stain, where the normal lactobacillus-dominated vaginal flora is changed to the lactobacillus deficient flora of bacterial vaginosis . The condition is probably associated with higher risk of complications in connection with pregnancy and gynaecological surgery . A prospective study of bacterial vaginosis based on microscopy of Gram-stained smears was conducted among 168 women applying for first trimester abortion . The prevalence of bacterial vaginosis was 24% and of Chlamydia trachomatis 8.4% . Four patients (10.3%) in the vaginosis group were treated with antibiotics for certain or suspected postabortal endometritis, as against six patients (5.4%) in the group without bacterial vaginosis. J Mol Biol, 1997 Apr 4, 267(3), 640 - 60 Crystal structure of a ternary complex of D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei, NAD+ and 2-oxoisocaproate at 1.9 A resolution; Dengler U et al.; D-2-hydroxyisocaproate dehydrogenase (D-HicDH) from Lactobacillus casei is a homodimer with 333 amino acids and a molecular mass of 37 kDa per subunit . The enzyme belongs to the protein family of NAD+-dependent D-2-hydroxycarboxylate dehydrogenases and within this family to the subgroup of D-lactate dehydrogenases (D-LDHs) . Compared with other D-LDHs D-HicDH is characterized by a very low specificity regarding size and chemical constitution of the accepted D-2-hydroxycarboxylates . Hexagonal crystals of recombinant D-HicDH in the presence of NAD+ and 2-oxoisocaproate (4-methyl-2-oxopentanoate) were grown with ammonium sulphate as precipitating agent . The structure of these crystals was solved by molecular replacement and refined to a final R-factor of 19.6% for all measured X-ray reflections in the resolution range (infinity to 1.86 A) . Both NAD+ and 2-oxoisocaproate were identified in the electron density map; binding of the latter in the active site, however, competes with a sulphate ion, which is also defined by electron density . Additionally the final model contains 182 water molecules and a second sulphate ion . The binding of both an in vitro substrate and the natural cosubstrate in the active site provides substantial insight into the catalytic mechanism and allows us to assess previously published active site models for this enzyme family, in particular the two most controversial points, the role of the conserved Arg234 and substrate binding . Furthermore the overall topology and details of the D-HicDH structure are described, discussed against the background of homologous structures and compared with one closely and one distantly related protein. Oral Microbiol Immunol, 1997 Apr, 12(2), 91 - 7 Inhibition of purified enolases from oral bacteria by fluoride; Guha-Chowdhury N et al.; Enolase activity in strains of oral streptococci previously has been found to be inhibited by 50% (Ki) by fluoride concentrations ranging from 50 to 300 microM or more in the presence of 0.5 to 1.0 mM inorganic phosphate ions . In this study, enolase was extracted and partly purified by a two-step process from five oral bacterial species and the effect of fluoride on the kinetics of enolase examined . The molecular weight of the putative enolase proteins was 46-48 kDa . The Vmax values ranged from 20 to 323 IU/mg and K(m) for glycerate-2-phosphate from 0.22 to 0.74 mM . Enolase activity was inhibited competitively by fluoride, with Ki values ranging from 16 to 54 microM in the presence of 5 mM inorganic phosphate ions . Ki values for phosphate ranged from 2 to 8 mM . The enolase from Streptococcus sanguis ATCC 10556 was more sensitive to fluoride (Ki = 16 +/- 2) than was enolase from Streptococcus salivarius ATCC 10575 (Ki = 19 +/- 2) or Streptococcus mutans NCTC 10449 (Ki = 40 +/- 4) and all three streptococcal strains were more sensitive to fluoride than either Actinomyces naeslundii WVU 627 (Ki = 46 +/- 6) or Lactobacillus rhamnosus ATCC 7469 (Ki = 54 +/- 6) enolases . The levels of fluoride found to inhibit the streptococcal enolases in this study are much lower than previously reported and are likely to be present in plaque, especially during acidogenesis, and could exert an anti-glycolytic effect. J Public Health Dent, 1997 Spring, 57(2), 82 - 8 Do root lesions tend to develop in the same people who develop coronal lesions? Beck JD, Drake CW. OBJECTIVES: The three purposes of this study are to: (1) describe the relationship between the prevalence of coronal caries and root caries; (2) describe the relationship between the three-year incidence of coronal caries and root caries; and (3) if the two conditions are associated, develop a multiple regression model that identifies characteristics distinguishing people who had increments of both root caries and coronal caries from people who had increments of either coronal caries or root caries, or who had no new caries . METHODS: Dental examinations and interviews were conducted in the homes of a randomly selected, stratified sample of people over the age of 65 years in five North Carolina counties . The relationships between coronal and root D and DF were analyzed through contingency table analyses, and ordinal logistic regression was used to identify characteristics that differentiated people who had both coronal and root D over the three years from people who had either coronal or root D and people who had no new disease . RESULTS: Evidence of root and coronal caries in whites was much more likely to be in the form of fillings, while for blacks, it was more likely to be in the form of untreated decay . Prevalence rates of coronal and root D and DF were significantly associated for both blacks and whites . Incidence rates based on DF indicated that root and coronal caries were not associated in whites, but were associated in blacks . People more likely to experience both types of caries had more gingival recession at baseline, greater average attachment loss over the three years, and lactobacilli at baseline . In addition, the presence of Porphymonas gingivalis at three years was important for whites . CONCLUSIONS: It appears that coronal and root caries do tend to appear together in the same individuals, but fillings attenuate that relationship . The impact of dental treatment on the epidemiology of dental caries appears to be considerable and calls into question whether the F component of the caries index is related to disease as defined by epidemiologic criteria. Community Dent Oral Epidemiol, 1997 Apr, 25(2), 165 - 9 On the pH-lowering potential of lactobacilli and mutans streptococci from dental plaque related to the prevalence of caries; Borgstrom MK et al.; The common method used today to identify persons at risk of dental caries is to estimate the numbers of cariogenic bacteria such as lactobacilli and mutans streptococci in saliva or plaque samples taken from the patient . However, the value of these bacterial counts for explaining and predicting individuals at risk of caries has not been powerful enough . Evaluating one virulence factor such as the acidogenicity of these bacteria might increase their explanatory values for caries . Sixty children aged 14-15 yrs participated in this study . Smooth surface caries and restorations were registered and total plaque samples collected . Counts of lactobacilli and mutans streptococci were estimated, and the pH-lowering potential of both bacteria was measured in an adapted glucose broth . The results showed a weak association between dental caries and lactobacilli, but in the subgroup with this bacterium the explanatory value increased to 14% and in the subgroup with a strong pH-lowering potential it was as high as 27% . For mutans streptococci the associations were weak in all groups. Acta Odontol Scand, 1997 Apr, 55(2), 111 - 5 Oral sugar clearance and other caries-related factors in patients with myotonic dystrophy; Engvall M et al.; The aim of the investigation was to try to explain why patients with myotonic dystrophy (MD) have a high caries prevalence . Seventeen MD patients, 15 of whom had been examined 8 years earlier, and 17 matched, healthy controls participated . In connection with this follow-up examination, the oral sugar clearance was evaluated after chewing a glucose tablet . A paraffin-stimulated whole saliva sample was collected for determination of secretion rate, buffer capacity, and numbers of mutans streptococci and lactobacilli . Dietary score, plaque index, oral muscular coordination, and self-cleaning ability were also recorded . For all factors, the MD patients showed less favorable mean values than the controls; the differences between the groups were statistically significant, except for the bacterial counts and the salivary buffer capacity . Thus, the high caries prevalence in MD patients may be explained by longer oral sugar clearance time, lower salivary secretion rate, higher intake frequency of sugar-containing products, higher plaque index, and less pronounced oral muscular coordination and self-cleaning ability than in healthy individuals. J Ind Microbiol Biotechnol, 1997 Apr, 18(4), 284 - 91 Use of virginiamycin to control the growth of lactic acid bacteria during alcohol fermentation; Hynes SH et al.; The antibiotic virginiamycin was investigated for its effects on growth and lactic acid production by seven strains of lactobacilli during the alcoholic fermentation of wheat mash by yeast . The lowest concentration of virginiamycin tested (0.5 mg Lactrol kg-1 mash), was effective against most of the lactic acid bacteria under study, but Lactobacillus plantarum was not significantly inhibited at this concentration . The use of virginiamycin prevented or reduced potential yield losses of up to 11% of the produced ethanol due to the growth and metabolism of lactobacilli . However, when the same concentration of virginiamycin was added to mash not inoculated with yeast, Lactobacillus rhamnosus and L . paracasei grew after an extensive lag of 48 h and L . plantarum grew after a similar lag even in the presence of 2 mg virginiamycin kg-1 mash . Results showed a variation in sensitivity to virginiamycin between the different strains tested and also a possible reduction in effectiveness of virginiamycin over prolonged incubation in wheat mash, especially in the absence of yeast. J Ind Microbiol Biotechnol, 1997 Apr, 18(4), 272 - 7 Caroteno-protein and exopolysaccharide production by co-cultures of Rhodotorula glutinis and Lactobacillus helveticus; Frengova G et al.; The lactose-negative yeast Rhodotorula glutinis 22P and the homofermentative lactic acid bacterium Lactobacillus helveticus 12A were cultured together in a cheese whey ultrafiltrate containing 42 g L-1 lactose . The chemical composition of the caroteno-protein has been determined . The carotenoid and protein contents are 248 micrograms g-1 dry cells and 48.2% dry weight . Carotenoids produced by Rhodotorula glutinis 22P have been identified as beta-carotene 15%, torulene 10%, and torularhodin 69% . After separating the cell mass from the microbial association, the exopolysaccharides synthesized by Rhodotorula glutinis 22P were isolated from the supernatant medium in a yield of 9.2 g L-1 . The monosaccharide composition of the synthesized biopolymer was predominantly D-mannose (57.5%). Eur J Oral Sci, 1997 Apr, 105(2), 162 - 9 Effects of frequent mouthrinses with palatinose and xylitol on dental plaque; Lingstrom P et al.; The aim was to evaluate the effects of frequent mouthrinses with palatinose, xylitol and a mixture of palatinose and xylitol on plaque pH, plaque formation and cariogenic microorganisms . 15 subjects refrained from toothbrushing during 3 test periods and rinsed 15 x daily for 4 d with 10 ml of: (1) 50% palatinose, (2) 37.5% palatinose + 12.5% xylitol, or (3) 50% xylitol . A contrast period with no mouthrinses was also carried out . The 4 periods were carried out in a randomized order with a cross-over design . After the 4-day periods, 3 parameters were measured: (1) plaque pH during the first 30 min after a mouthrinse with palatinose, a mixture of palatinose and xylitol or xylitol alone, directly followed by a 2nd rinse with 10% sucrose; (2) number of mutans streptococci and lactobacilli in plaque and saliva; (3) plaque index . The most pronounced pH drop for the sugar substitutes was found when rinsing with 50% palatinose after the palatinose period, and the least pH drop with 50% xylitol after the xylitol period . The sucrose rinse gave similar pH fall after all 4 periods . The microbial data showed no differences between the 4 periods, but the mutans streptococcus counts in saliva decreased after the xylitol period in contrast to the 3 other periods . Regarding the plaque index, xylitol gave lower scores compared to the other 3 periods. J Pediatr Gastroenterol Nutr, 1997 Apr, 24(4), 399 - 404 Lactobacillus reuteri as a therapeutic agent in acute diarrhea in young children; Shornikova AV et al.; BACKGROUND: Certain strains of lactobacilli may promote recovery from acute diarrhea . Lactobacillus reuteri is of human origin and is a natural colonizer of gastrointestinal tract . In this trial, exogenously administered L . reuteri was studied as a therapeutic agent in acute diarrhea . METHODS: Forty patients between 6 and 36 months of age hospitalized with acute diarrhea (75% rotavirus) were studied . After parental consent, the patients were randomized to one of two treatment groups to receive either 10(10) to 10(11) colony-forming units of L . reuteri or a matching placebo daily for the length of hospitalization or up to 5 days . The clinical outcome of diarrhea and colonization of L . reuteri were evaluated . RESULTS: The mean (SD) duration of watery diarrhea after treatment was 1.7 (1.6) days in the L . reuteri group and 2.9 (2.3) days in the placebo group (p = 0.07) . On the second day of treatment only 26% of patients receiving L . reuteri had watery diarrhea, compared with 81% of those receiving placebo (p = 0.0005) . Cultures of lactobacilli from stool samples demonstrated that administration of L . reuteri resulted in colonization of the gastrointestinal tract . Lactobacillus reuteri accounted for > 75% of the total lactobacilli found in children fed with this product . CONCLUSIONS: Lactobacillus reuteri is effective as a therapeutic agent in acute rotavirus diarrhea in children . Further studies are warranted to confirm the present finding and to explore the full therapeutic potential of L . reuteri in acute viral diarrhea. Clin Microbiol Rev, 1997 Apr, 10(2), 345 - 57 Management of listeriosis; Hof H et al.; Determination of the MIC in vitro is often used as the basis for predicting the clinical efficacy of antibiotics . Listeriae are uniformly susceptible in vitro to most common antibiotics except cephalosporins and fosfomycin . However, the clinical outcome is poor . This is partially because listeriae are refractory to the bactericidal mechanisms of many antibiotics, especially to ampicillin-amoxicillin, which still is regarded as the drug of choice . A true synergism can be achieved by adding gentamicin . Another point is that listeriae are able to reside and multiply within host cells, e.g., macrophages, hepatocytes, and neurons, where they are protected from antibiotics in the extracellular fluid . Only a few agents penetrate, accumulate, and reach the cytosol of host cells, where the listeriae are found . Furthermore, certain host cells may exclude antibiotics from any intracellular compartment . Thus, determination of the antibacterial efficacy of a drug against listeriae in cell cultures may be a better approximation of potential therapeutic value . Certain host cells may have acquired the property of excluding certain antibiotics, for example macrolides, from intracellular spaces, which might explain therapeutic failures of antibiotic therapy in spite of low MICs . Animal models do not completely imitate human listeriosis, which is characterized by meningitis, encephalitis, soft tissue and parenchymal infections, and bacteremia . Meningitis produced in rabbits is a hyperacute disease, whereby most listeriae lie extracellularly, fairly accessible to antibiotics that can cross the blood-cerebrospinal fluid barrier . In the murine model of systemic infection, Listeria monocytogenes is located mainly within macrophages and parenchymal cells of the spleen and liver, hardly accessible to certain drugs, such as ampicillin and gentimicin . The therapeutic efficacy of drugs clearly depends on the model used . Thus, for example, the combination of ampicillin with gentamicin acts synergistically in the rabbit meningitis model but not in the mouse model . Since conventional antimicrobial therapy with antibiotics is not satisfactory, particularly in the immunocompromised host (about 30% of patients with listeriosis die in spite of a rational choice of antibiotics), other possibilities must be considered for therapy as well as prevention . Indeed, listeriae are highly susceptible to several endogenous antibiotics, such as defensins . Bacteriocins produced by related bacterial species, e.g., lactobacilli and enterococci, are rapidly bactericidal . However, unfortunately, the use of such alternative measures along with immunization and immunmodulation is not yet feasible. Sex Transm Dis, 1997 Apr, 24(4), 236 - 9 The use of sequential self-obtained vaginal smears for detecting changes in the vaginal flora; Schwebke JR et al.; BACKGROUND AND OBJECTIVES: The ability to study daily changes in the vaginal flora may provide insight into the pathogenesis of bacterial vaginosis . Because culture of the vaginal fluid is tedious and expensive, the utility of self-obtained vaginal smears for documenting changes in the flora was evaluated . GOALS: To validate the adequacy of self-collected vaginal fluid Gram stains and use them to monitor vaginal flora . STUDY DESIGN: Ten asymptomatic premenopausal women collected daily vaginal smears for 30 days . The smears were Gram stained and interpreted using a standardized scoring system (Nugent criteria) . In addition, results from self- and clinician-obtained vaginal smears from 18 women were compared to validate the adequacy of self-obtained smears . RESULTS: Two women had asymptomatic bacterial vaginosis . One woman, who was postpartum, had intermediate flora that toward the end of the collection period changed to Lactobacillus predominant . The remaining seven women exhibited two patterns . One was Lactobacillus morphotypes only; the second consisted of Lactobacillus-predominant days interspersed with days with moderate to high numbers of Gardnerella/Bacteroides morphotypes . There was a significant correlation of the point of change in the flora of this group with menses . CONCLUSIONS: The adequacy of self-collected vaginal fluid Gram's stains was validated . Changes in vaginal flora were demonstrated over a 30-day period by use of this methodology. J Bacteriol, 1997 Apr, 179(8), 2697 - 706 Arginine biosynthesis and regulation in Lactobacillus plantarum: the carA gene and the argCJBDF cluster are divergently transcribed; Bringel F et al.; A cluster of citrulline biosynthetic genes has been cloned and sequenced from a fragment of Lactobacillus plantarum CCM 1904 (ATCC 8014) DNA isolated as complementing a Bacillus subtilis argF mutation . The gene order was carA-argCJBDF, with carA transcribed divergently from the arg cluster . Although other gram-positive bacteria show similar arg clusters, this arrangement for carA is thus far unprecedented . Downstream from the arg cluster, two open reading frames (ORF7 and ORF8) having unknown functions were found . Sequence analysis of the end of a 10.5-kb cloned DNA fragment showed that argF was 3.5 kb from the ldhL gene coding for L-(+)-lactate dehydrogenase . A tree representation of amino acid sequence clustering relationships of 31 ornithine carbamoyltransferases (OTCases) from various organisms revealed two prokaryotic groups: one with ArgF of L . plantarum and one with ArgF of B . subtilis, which are paralogous . This divergence was not observed in vivo because an L . plantarum argF mutant (AM 1215) harboring no OTCase activity was complemented by the argF genes of L . plantarum and B . subtilis . No OTCase activity was detectable when L . plantarum was grown in the presence of saturating amounts of arginine or citrulline . Arginine may repress the citrulline biosynthetic genes in L . plantarum by using 11 identified DNA motifs which resemble the Escherichia coli ARG box consensus and which are in most cases separated by multiples of 11 bp, corresponding to a DNA helical turn . The carA and argCJBDF genes are divergently transcribed . Their putative promoters are 6 bp apart and are partially overlapped by putative ARG boxes, suggesting concerted transcription regulation. J Bacteriol, 1997 Apr, 179(8), 2472 - 8 Structure and regulation of expression of the Bacillus subtilis valyl-tRNA synthetase gene; Luo D et al.; We have sequenced the valyl-tRNA synthetase gene (valS) of Bacillus subtilis and found an open reading frame coding for a protein of 880 amino acids with a molar mass of 101,749 . The predicted amino acid sequence shares strong similarity with the valyl-tRNA synthetases from Bacillus stearothermophilus, Lactobacillus casei, and Escherichia coli . Extracts of B . subtilis strains overexpressing the valS gene on a plasmid have increased valyl-tRNA aminoacylation activity . Northern analysis shows that valS is cotranscribed with the folC gene (encoding folyl-polyglutamate synthetase) lying downstream . The 300-bp 5' noncoding region of the gene contains the characteristic regulatory elements, T box, "specifier codon" (GUC), and rho-independant transcription terminator of a gene family in gram-positive bacteria that encodes many aminoacyl-tRNA synthetases and some amino acid biosynthetic enzymes and that is regulated by tRNA-mediated antitermination . We have shown that valS expression is induced by valine limitation and that the specificity of induction can be switched to threonine by changing the GUC (Val) specifier triplet to ACC (Thr) . Overexpression of valS from a recombinant plasmid leads to autorepression of a valS-lacZ transcriptional fusion . Like induction by valine starvation, autoregulation of valS depends on the presence of the GUC specifier codon . Disruption of the valS gene was not lethal, suggesting the existence of a second gene, as is the case for both the thrS and the tyrS genes. Appl Environ Microbiol, 1997 Apr, 63(4), 1284 - 7 Characterization of an intracellular oligopeptidase from Lactobacillus paracasei; Tobiassen RO et al.; An intracellular oligopeptidase from Lactobacillus paracasei Lc-01 has been purified to homogeneity by Fast Flow Q Sepharose, hydroxyapatite, and Mono Q chromatography . The molecular mass of the enzyme was determined to be 140 kDa by gel filtration and approximately 30 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and SDS-capillary electrophoresis . The pI of the enzyme was at pH 4.5 . The enzyme expressed maximum activity at pH 8.0 and 40 degrees C . Oligopeptidase activity on bradykinin was inhibited strongly by 1,10-phenantroline and EDTA and partly by p-chloromercuribenzoic acid but not by phosphoramidon or phenylmethylsulfonyl fluoride . Marked inhibition by beta-casein fragment 58 to 72 was demonstrated . The enzyme showed neither general aminopeptidase nor caseinolytic activity, and it degraded only oligopeptides between 8 and 13 amino acids . The enzyme readily hydrolyzed the Phe-Ser and Pro-Phe bonds of bradykinin; the Phe-His bond of angiotensin I; the Pro-Gln, Gln-Phe, and Phe-Gly bonds of substance P; and the Pro-Tyr bond of neurotensin . Weak activity toward the Ala-Tyr and Pro-Ser bonds of alpha(s1)-casein fragment 157 to 164, was observed . The N-terminal amino acid sequence of the oligopeptidase showed a high degree of homology to the lactacin B inducer from Lactobacillus acidophilus. J Bacteriol, 1997 Apr, 179(7), 2459 - 63 Identification and characterization of IS1381, a new insertion sequence in Streptococcus pneumoniae; Sanchez-Beato AR et al.; A new insertion sequence (IS1381) was identified in the genome of Streptococcus pneumoniae R6 as an 846-bp segment containing 20-bp terminal inverted repeats and flanked by 7-bp direct repeats . The three sequenced copies of this element have two overlapping open reading frame (ORF) genes named orfA and orfB . However, significant variations between individual copies were found, suggesting that inactivating mutations have occurred in an original single ORF . Accordingly, the consensus IS1381 element derived from the comparison of the three available copies should contain a single ORF sufficient to encode a basic protein of 267 amino acids which exhibited high similarity to the putative transposases of ISL2 from Lactobacillus helveticus and of IS702 from the cyanobacterium Calothrix sp . strain PCC 7601 . A minimum of five to seven copies were detected by hybridization experiments in the R6 genome . In remarkable contrast with the two previously reported pneumococcal insertion sequences, several copies of IS1381 have been detected in all of the clinical isolates tested so far . Interestingly, Streptococcus oralis NCTC 11427 (type strain), a close relative of pneumococcus, does not contain this element, but its occurrence in the type strain of Streptococcus mitis (NCTC 12261) suggests that this species has exchanged DNA with S . pneumoniae directly or through an intermediate species. Int J Food Microbiol, 1997 Mar 18, 35(1), 83 - 90 Processing and storage of lye-treated carrots fermented by a mixed starter culture; Montano A et al.; A mixed culture of Lactobacillus plantarum and Saccharomyces cerevisiae was compared with a single culture of L . plantarum as starter for the fermentation of lye-treated carrots . Using the mixed culture, more than 95% of glucose, fructose and malic acid was consumed after 7 days of fermentation in a brine containing 2.5% w/v NaCl and 0.7% acetic acid, but only 54% of sucrose was degraded . The fermentation products quantified were lactic acid, ethanol and acetic acid and carbon recovery was 88% . Using the single culture of L . plantarum, substrate consumption was lower, and carbon recovery almost 100% . In uninoculated lye-treated carrots, heterofermentative bacteria grew, with the production of considerable amounts of mannitol . In all cases, the stability, sensorial characteristics and carotenoid content of the packed product were studied during 9 months of storage at 30 degrees C, and two different preservation systems were compared: addition of preservatives, approximately 500 and 1000 mg/kg of sorbic and benzoic acid, respectively, and pasteurization at 80 degrees C for 10 min . The pasteurized samples were microbiologically stable during the storage period, while lactic acid bacteria grew in the samples with preservatives . Storage time significantly (P < 0.05) affected the texture and colour parameters (L*, a*, b*) of the carrots, but not the amounts of alpha- and beta-carotene . The type of fermentation had no significant effect on texture, colour parameters or carotenoid content . The preservation method had no significant effect on texture or carotenoid content, but did affect colour (parameter a*) . The flavour of carrots fermented by the mixed culture was significantly (P < 0.05) preferred to that of those fermented with a single culture of L . plantarum. FEBS Lett, 1997 Mar 17, 405(1), 16 - 20 Correlated bond rotations in interactions of arginine residues with ligand carboxylate groups in protein ligand complexes; Nieto PM et al.; The 1H/15N HSQC NMR spectra of complexes of Lactobacillus casei dihydrofolate reductase containing methotrexate recorded at 1 degree C show four resolved signals for the four NH(eta) protons of the Arg57 residue . This is consistent with hindered rotation in the guanidino group resulting from interactions with the alpha-carboxylate of methotrexate . Increasing the temperature causes exchange line-broadening and coalescence of signals . Rotation rates for the N(epsilon)C(zeta) and C(zeta)N(eta) bonds have been calculated from lineshape analysis and from zz-HSQC exchange experiments . The interactions between the methotrexate alpha-carboxylate group and the Arg57 guanidino group decrease the rotation rates for the N(epsilon)C(zeta) bond by about a factor of 10 and those for the C(zeta)N(eta) bonds by more than a factor of 100 with respect to their values in free arginine . Furthermore, the relative rates of rotation about these two bonds are reversed in the protein complexes compared with their values in free arginine indicating that there are concerted rotations about the N(epsilon)C(zeta) bond of the Arg57 guanidino group and the C'C(alpha) bond of the glutamate alpha-carboxylate group of methotrexate. FEMS Microbiol Lett, 1997 Mar 15, 148(2), 223 - 6 Identification of the replication region of the Lactobacillus acidophilus plasmid pLA106; Sano K et al.; The complete nucleotide sequence of plasmid pLA106 (2862 bp) from Lactobacillus acidophilus TK8912 was determined . Based on this sequence, the location of the genes for mobilization-plasmid recombination protein (mob), replication origin (ori), transcriptional repressor protein (repA) and replication initiation protein (repB) were predicted . Deletion analysis showed that the 1.4-kb PstI-EcoRV fragment carrying the ori, repA and repB genes is able to replicate in Lactobacillus and Escherichia coli cells . The plasmid pLA106 appears to have most features of the pLS1/pE194 plasmid family. J Biol Chem, 1997 Mar 14, 272(11), 6890 - 7 cis-Active Ras G2-like sequence implicated in the heterotropic activation of the deoxyadenosine kinase of Lactobacillus acidophilus R-26; Guo S et al.; Deoxyadenosine kinase (dAK) forms a heterodimer with either deoxyguanosine kinase (dGK) or deoxycytidine kinase (dCK), and is heterotropically activated 3-5 times by dGuo or dCyd . Expressed alone, dAK is inactive and exhibits no response to dGuo or dCyd; activity and heterotropic response are fully restored upon reassociation with dGK or dCK . However, turnover of independently expressed dGK or dCK is nearly maximal, being further activated only 50-100% upon reassociation with dAK . In neither case is the heterotropic activation due to ligand-induced heterodimer formation . A proline/alanine substitution within a dAK segment homologous to loop G2 of Ras proteins yielded a heterodimer with dAK permanently cis-activated 2-fold, with a corresponding reduction in heterotropic activation by dGuo . A chimeric dAK, with 25% of its C terminus substituted by the homologous sequence from dGK, was inactive alone, and its characteristics were unchanged in the reconstituted heterodimer . Superimposing the Pro/Ala substitution on this chimera also reduced heterotropic activation by half . Cross-linking the dimer by 1,5-difluoro-2,4-dinitrobenzene was inhibited by ATP, dATP, dGTP, and dAdo, suggesting the proximity of the active site(s) to the interface . These data suggest that dAK depends on dGK or dCK in a manner resembling the reliance of Ras upon GTPase activating protein. Gene, 1997 Mar 10, 187(1), 45 - 53 Genome structure of the Lactobacillus temperate phage phi g1e: the whole genome sequence and the putative promoter/repressor system; Kodaira KI et al.; The complete genome sequence of a Lactobacillus temperate phage phi g1e was established . The double-stranded DNA is composed of 42,259 bp, and encodes for sixty-two possible open reading frames (ORF) as well as several potential regulatory sequences . Based on comparative analysis with other related proteins of the Lactobacillus and Lactococcus phages as well as the Escherichia coli phages (such as lambda), functions were putatively assigned to several phi g1e ORFs: cng and cpg (encoding for repressors), hel (helicase), ntp (NTPase), and several ORFs (e.g., minor capsid proteins) . An about 1000-bp DNA region of phi g1e containing cpg and cng was inferred to function as a promoter/repressor system for the phi g1e lysogenic and lytic pathway. J Mol Biol, 1997 Mar 7, 266(4), 776 - 96 Domain motions in dihydrofolate reductase: a molecular dynamics study; Verma CS et al.; Molecular dynamics simulations have been carried out on the enzyme dihydrofolate reductase from Lactobacillus casei complexed with methotrexate, NADPH and 264 crystallographic water molecules . Analysis of correlations in atomic fluctuations reveal the presence of highly correlated motion (correlation coefficient > 0.6) in the region between residues 30 to 35 and 85 to 90 leading to the identification of two domains, an "adenosine-binding domain" and a "large domain", which rotate by 3 to 4 degrees with respect to each other . The strongest correlation (> 0.6) within the large domain involves a coupling between the motions of the "teen-loop", and the spatially contiguous loops linking beta 6-beta 7 and beta 7-beta 8 . Moreover, there is a significant correlation (approximately 0.5) between the adenosine fragment of NADPH and the pteridine and p-aminobenzoyl fragments of methotrexate, which are separated by approximately 17 A, and is lost on removal of "rigid-body" motion from the original trajectory . This provides support for the idea that the relative motion of the two domains is a means by which the occupation of the binding site for the adenosine end of the coenzyme can affect methotrexate binding and vice versa . Quasiharmonic vibrational analysis of the trajectory reveals that the overall dynamics of the system are governed by domain motions whose contributions are dominant at low frequencies . In addition, different low-frequency modes are responsible for separately coupling the adenosine-binding site and parts of methotrexate. Int J Food Microbiol, 1997 Mar 3, 34(3), 249 - 58 Munkoyo beverage, a traditional Zambian fermented maize gruel using Rhynchosia root as amylase source; Zulu RM et al.; A typical munkoyo beverage was made by fermenting Rhynchosia heterophylla root extract-cooked maize meal mixture with Lactobacillus confusus LZI and Saccharomyces cerevisiae YZ20 . The fermented munkoyo beverage had a pH of 3.3, lactic acid content of 60 mmol/l, ethanol 320-410 mmol/l and a characteristic 'munkoyo' aroma . L . confusus, used alone, produced a beverage with a faint munkoyo flavour note whilst beverage produced with S . cerevisiae alone seemed not to have a typical munkoyo note . R . heterophylla root extract converted 75% of the starch in sterile cooked maize meat to maltose (80% of total sugars), maltotriose (17%) and glucose (3%) in I h at 45 degrees C . During fermentation by the mixed culture or the yeast alone most of the maltose was utilised but little or none of the maltotriose . The ratio of yeast to lactic acid bacteria in the starter culture affected the rate of production of ethanol but had no effect on the growth or acid production by the bacterium . To obtain a munkoyo beverage with the desired low alcohol concentration (< 100 mmol/l), the ratio of yeast concentration (cfu/ml) to Lactobacillus concentration in the starter culture should be 1:1000 or less and the beverage should be fermented for 24 h only. Protein Eng, 1997 Mar, 10(3), 255 - 62 Modified substrate specificity of L-hydroxyisocaproate dehydrogenase derived from structure-based protein engineering; Feil IK et al.; L-2-Hydroxyisocaproate dehydrogenase (L-HicDH) is characterized by a broad substrate specificity and utilizes a wide range of 2-oxo acids branched at the C4 atom . Modifications have been made to the sequence of the NAD(H)-dependent L-HicDH from Lactobacillus confusus in order to define and alter the region of substrate specificity towards various 2-oxocarbonic acids . All variations were based on a 3D-structure model of the enzyme using the X-ray coordinates of the functionally related L-lactate dehydrogenase (L-LDH) from dogfish as a template . This protein displays only 23% sequence identity to L-HicDH . The active site of L-HicDH was modelled by homology to the L-LDH based on the conservation of catalytically essential residues . Substitutions of the active site residues Gly234, Gly235, Phe236, Leu239 and Thr245 were made in order to identify their unique participation in substrate recognition and orientation . The kinetic properties of the L239A, L239M, L236V and T245A enzyme variants confirmed the structural model of the active site of L-HicDH . The substrates 2-oxocaproate, 2-oxoisocaproate, phenylpyruvate, phenylglyoxylate, keto-tert-leucine and pyruvate were fitted into the active site of the subsequently refined model . In order to design dehydrogenases with an improved substrate specificity towards keto acids branched at C3 or C4, amino acid substitutions at positions Leu239, Phe236 and Thr245 were introduced and resulted in mutant enzymes with completely different substrate specificities . The substitution T245A resulted in a relative shift of substrate specificity for keto-tert-leucine of more than 17000 compared with the 2-oxocaproate (kcat/KM) . For the substrates branched at C4 a relative shift of up to 500 was obtained for several enzyme variants . A total of nine mutations were introduced and the kinetic data for the set of six substrates were determined for each of the resulting mutant enzymes . These were compared with those of the wild-type enzyme and rationalized by the active site model of L-HicDH . An analysis of the enzyme variants provided new insight into the residues involved in substrate binding and residues of importance for the differences between LDHs and HicDH . After the protein design project was complete the X-ray structure of the enzyme was solved in our group . A comparison between the model and the experimental 3D structure proved the quality of the model . All the variants were designed, expressed and tested before the 3D structure became available. J Acquir Immune Defic Syndr Hum Retrovirol, 1997 Mar 1, 14(3), 213 - 8 A placebo-controlled, double-blind prospective study in healthy female volunteers of dextrin sulphate gel: a novel potential intravaginal virucide; Stafford MK et al.; A double-blind, placebo-controlled study was designed to evaluate the safety and tolerability of intravaginal dextrin sulphate (D2S) gel to assess its preliminary suitability as a potential vaginal virucide . Tolerability was assessed by questionnaire and patient interview . Colposcopy with vaginal biopsy was performed to assess the macroscopic and microscopic evidence of inflammation . The potential impact of the gel on normal vaginal flora was examined by quantitative lactobacilli culture with assessment of the ratio of peroxide to nonperoxide-producing organisms . Colposcopy revealed mild erythema in five of 24 subjects receiving active gel and in none of the 12 placebo recipients, but histology in all subjects revealed no evidence of inflammation . No impact on vaginal lactobacilli was found . We conclude that D2S gel is safe and well tolerated intravaginally at the dosing schedule used in this study. J Dent Res, 1997 Mar, 76(3), 768 - 72 Incorporation of antibacterial monomer MDPB into dentin primer; Imazato S et al.; The polymerizable monomer methacryloyloxydodecylpyridinium bromide (MDPB) shows antibacterial activity when immobilized in a resin-based material . In this study, the antibacterial effect of a dentin primer incorporating MDPB was investigated . The influence of incorporation of MDPB on bond strength to dentin and on the curing performance of the adhesive system was also evaluated . Experimental primers were prepared by addition of MDPB into a proprietary primer at 1, 2, or 5% . Antibacterial effects of experimental primers were compared with those of control primer and two other proprietary primers by an agar disc-diffusion method and bactericidal activity test . Experimental primers produced greater inhibition zones against Streptococcus mutans, Actinomyces viscosus, and Lactobacillus casei than any of three proprietary primers, and inhibition increased as the concentration of MDPB was increased . Bactericidal activity of MDPB-containing primers against Streptococcus mutans was greater than those of the other three primers, with incorporation of MDPB at 5% showing complete killing of bacteria after 30 s contact . No decrease in tensile bond strength was observed for materials containing MDPB . On the contrary, the primer incorporating 1 and 2% MDPB showed higher bond strength than all the others, including the control (p < 0.05) . When the degree of conversion of the complex of primer and adhesive resin was determined with Fourier Transform Infrared Spectroscopy, there were no significant differences between any of the experimental primers and the control (p > 0.05) . These results indicate that incorporation of the antibacterial monomer MDPB enhanced the antibacterial effect of a proprietary dentin primer before curing, and had no adverse influence on bond strength to dentin and curing of the adhesive system. Lett Appl Microbiol, 1997 Mar, 24(3), 180 - 4 A zinc-dependent proteinase from the cell wall of Lactobacillus delbrueckii subsp . bulgaricus; Stefanitsi D et al.; A zinc-dependent proteinase was extracted from the cell wall of Lactobacillus delbrueckii subsp . bulgaricus and partially purified despite a marked unstability . The caseinolytic activity was associated with a polypeptide chain of 65 kDa that belonged to the M1 family of zinc-dependent proteases . This zinc-dependent proteinase could degrade intact caseins, with a significant preference for beta-casein . The pH-profile of its activity indicated that its relative contribution to the caseinolytic activity increased at acidic pH, suggesting that this zinc proteinase could be involved in the late stages of milk fermentation. Lett Appl Microbiol, 1997 Mar, 24(3), 153 - 8 Incorporation of nisin in micro-particles of calcium alginate; Wan J et al.; Nisin was successfully incorporated into a matrix of calcium alginate and ground into micro-particles smaller than 150 microns . Formation of micro-particles and incorporation of nisin was verified by scanning electron microscopy and by the reduction in the inactivation of nisin activity with proteolytic enzymes . Incorporation efficiency was 87-93% and the nisin in the alginate-incorporated form was 100% active against an indicator culture of Lactobacillus curvatus both in MRS broth and reconstituted skim milk. Poult Sci, 1997 Mar, 76(3), 463 - 8 Counteracting Fusarium proliferatum toxicity in broiler chicks by supplementing drinking water with Poultry Aid Plus; Wu W; To test whether Poultry Aid Plus (PAP, a commercial product for drinking water application) could reduce the stress on broiler chicks caused by Fusarium proliferatum contamination of feed, water (with or without PAP application, according to the manufacturer's instructions), and feed (experimentally infected with F . proliferatum fermented and dried corn culture material, CM) were provided to broiler chicks for 3 wk . Eight treatments consisting of a 2 (with or without PAP in water) x 4 (0, 1, 2, and 4% CM in feed) factorial design were tested in four replicate cages of six chicks each . The diet with 2% CM reduced weight gain by 23%; this reduction was preventable by PAP water application . The diet with 4% CM caused a cumulative mortality of 87.5%, which was reduced by PAP water application to 50% . The population half-life of the chicks on the diet with 4% CM was 6.5 d; this half-life was prolonged to at least 21 d by PAP water application . The PAP application also reduced the relative weight of the small intestine and promoted Lactobacillus colonization of the large intestine regardless of the level of CM in feed . Therefore, water application of PAP can be a prophylactic measure for F . proliferatum toxicity in poultry production. FEMS Microbiol Lett, 1997 Mar 1, 148(1), 97 - 100 Isolation and partial characterization of bacteriocins produced by Lactobacillus gasseri JCM 2124; Tahara T et al.; Four antibacterially active peptides (B1 to B4) were purified from the culture broth of L . gasseri JCM 2124 . The B2 peptide (gassericin B2) was determined to be 4400 Da by mass spectrometry and partially sequenced . Gassericin B2 did not show any sequence similarities to other known bacteriocins . The B1 and B3 peptides shared identical sequences with two peptides of a two-component bacteriocin from Lactobacillus acidophilus . However, synergistic activity upon complementation of B1 and B3 was not observed . Based on amino acid sequencing and molecular mass, it is suggested that B1 and B4 peptides were derived from B3 (gassericin B3). FEMS Microbiol Lett, 1997 Mar 1, 148(1), 83 - 9 Establishing a model to study the regulation of the lactose operon in Lactobacillus casei; Gosalbes MJ et al.; The chromosomally encoded lactose-specific phosphoenol pyruvate-dependent phosphotransferase system (PTS) has been investigated in Lactobacillus casei ATCC 393 {pLZ15-} and it was considered an excellent system to study the regulation of the lactose operon . This chromosomal operon has been cloned and sequenced, being 99% homologous to that encoded on the plasmid pLZ64 . Expression of the lactose operon in different mutants of L . casei ATCC 393 {pLZ15-} and primer extension analysis revealed that it is subject to a dual regulation: (i) glucose repression possibly mediated by CcpA and PTS elements, and (ii) induction by lactose through transcriptional antitermination. Curr Microbiol, 1997 Mar, 34(3), 186 - 91 Purification of glucoamylase from Lactobacillus amylovorus ATCC 33621; James JA et al.; An intracellular glucoamylase (E.C . 3.2.1.3) was purified to homogeneity from Lactobacillus amylovorus on a Fast Protein liquid chromatography System (FPLC) with a Mono Q ion-exchanger and two Superose 12 gel filtration columns arranged in series . The enzyme activity was quantified with a specific, chromogenic substrate, p-nitrophenyl-beta-maltoside.Preparative gel electrophoresis was then used to further purify active enzyme fractions . Native polyacrylamide gel electrophoresis (Native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of molecular weight 47 kDa . Glucoamylase activity of the purified protein was confirmed by its ability to degrade starch on a 0.025% starch-polyacrylamide gel stained with I2/KI . Glucoamylase exhibited optimum catalytic activity at pH 6.0 and 45 degrees C, and the enzyme had an isoelectric point near 4.39 . The glucoamylase contained high levels of hydrophilic amino acids, comparable to fungal glucoamylases. Curr Microbiol, 1997 Mar, 34(3), 180 - 5 Lactobacillus helveticus: strain typing and genome size estimation by pulsed field gel electrophoresis; Lortal S et al.; Genomic DNAs of 22 strains of Lactobacillus helveticus of various geographical origins were analyzed by pulsed-field gel electrophoresis . Two endonucleases, SmaI and SgrAI, of the 19 tested produced DNA fragments useful for strain comparison . With the endonuclease SmaI, a characteristic restriction pattern was identified for 18 of the 22 strains . The percentage of similarity (Dice coefficient) between the profiles varied between 26% and 100%, and clustering was accomplished by using the unweighted pair group method with arithmetic averages (UPGMA) . For the strains showing identical profiles,the high genomic similarity was confirmed when the endonuclease SgrAI was used instead of SmaI . From summation of SmaI and SgrAI fragments from three L . helveticus strains(CNRZ 241, CNRZ 303, and CIP 57.15), the genomic length was estimated at ca.1 . 85-2.0 Mb. Curr Microbiol, 1997 Mar, 34(3), 173 - 9 Purification and partial amino acid sequence of brevicin 27, a bacteriocin produced by Lactobacillus brevis SB27; Benoit V et al.; Brevicin 27, a bacteriocin produced by Lacto bacillus brevis SB27, is inhibitory mainly against closely related Lactobacillus brevis and Lactobacillus buchneri strains . It was purified from the culture supernatant by a four-step purification procedure including ammonium sulfate precipitation, cation exchange, hydrophobic interaction, and reverse-phase, high performance liquid chromatographies . The purified bacteriocin was subjected to mass spectrometry, amino acid composition analysis, and sequencing by Edman degradation . It was shown to be an about 5200-Da basic protein containing a high proportion of lysine and of hydrophobic amino acids . The partial N-terminal amino acid sequence (25 residues) was unique when compared with the Protein Data Bank (PDB), Swiss Prot, and Protein Information Resource(PIR) data banks and to the translated Gen Bank. Gene, 1997 Feb 28, 186(2), 255 - 62 High level heterologous protein production in Lactococcus and Lactobacillus using a new secretion system based on the Lactobacillus brevis S-layer signals; Savijoki K et al.; A secretion cassette, based on the expression and secretion signals of a S-layer protein (SlpA) from Lactobacillus brevis, was constructed . E . coli beta-lactamase (Bla) was used as the reporter protein to determine the functionality of the S-layer signals for heterologous expression and secretion in Lactococcus lactis, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus gasseri and Lactobacillus casei using a low-copy-number plasmid derived from pGK12 . In all hosts tested, the bla gene was expressed under the slpA signals and all Bla activity was secreted to the culture medium . The Lb . brevis S-layer promoters were very efficiently recognized in L . lactis, Lb . brevis and Lb . plantarum, whereas in Lb . gasseri the slpA promoter region appeared to be recognized at a lower level and in Lb . casei the level of transcripts was below the detection limit . The production of Bla was mainly restricted to the exponential phase of growth . The highest yield of Bla was obtained with L . lactis and Lb . brevis . Without pH control, substantial degradation of Bla occurred during prolonged cultivations with all lactic acid bacteria (LAB) tested . When growing L . lactis and Lb . brevis under pH control, the Bla activity could be stabilized also at the stationary phase . L . lactis produced up to 80 mg/l of Bla which to our knowledge represents the highest amount of a heterologous protein secreted by LAB so far . The short production phase implied a very high rate of secretion with a calculated value of 5 x 10(5) Bla molecules/cell per h . Such a high rate was also observed with Lb . plantarum, whereas in Lb . brevis the competition between the wild type slpA gene and the secretion construct probably lowered the rate of Bla production . The results obtained indicate wide applicability of the Lb . brevis slpA signals for efficient protein production and secretion in LAB. Mol Gen Genet, 1997 Feb 27, 253(6), 674 - 86 Organization and expression of a gene cluster involved in the biosynthesis of the lantibiotic lactocin S; Skaugen M et al.; Some 8.8 kb of the Lactobacillus sake plasmid pCIM1 was sequenced, revealing eight tightly clustered open reading frames (ORFs) downstream from lasA, which encodes pre-lactocin S . Transcription analyses demonstrated that the genes are expressed as an operon, with transcription initiating upstream of lasA and terminating immediately 3' to the ninth ORF x lasA is also represented by two small RNAs (RNAI and RNAII) which differ in size by approximately 90 nucleotides, and primer extension experiments demonstrated a corresponding difference in the 5' termini . A palindromie sequence constitutes the 3' terminus of both RNAI and RNAII, and we propose that this sequence has a dual regulatory function in controlling the expression of las operon, acting both as a barrier to 3'-5' exonuclease degradation of the lasA-specific transcript(s), and as a "leaky" transcriptional terminator which limits the expression of down-stream genes . Three of the genes in the las operon have identifiable counterparts in other lantibiotic systems: lasM is likely to be involved in prepeptide modification, lasT, which encodes an ATP-dependent transport protein, is probably involved in the secretion of lactocin S, while lasP specifies a subtilisin-type serine protease which may be the lactocin S leader peptidase . Insertional mutation of either lasT or lasM by the resident transposable element IS1163 abolishes lactocin S production . The remaining five ORFs in the las operon are apparently unique, and their significance with respect to the lactocin S phenotype is presently not known. Eur J Biochem, 1997 Feb 15, 244(1), 213 - 9 D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp . bulgaricus . II . Mutagenic analysis of catalytically important residues; Bernard N et al.; Five residues involved in catalysis and coenzyme binding have been identified in D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp . bulgaricus by using biochemical and genetical methods . Enzyme inactivation with diethylpyrocarbonate indicated that a single histidine residue was involved in catalysis . Since H296 is the only conserved histidine in the whole family of NAD-dependent D-2-hydroxyacid dehydrogenases, we constructed the H296Q and H296S mutants and showed that their catalytic efficiencies were reduced 10(5)-fold compared with the wild-type enzyme . This low residual activity was shown to be insensitive to diethylpyrocarbonate . Taken together these data demonstrate that H296 is responsible for proton exchange in the redox reaction . Two acidic residues (D259 and E264) were candidates for maintaining H296 in the protonated state and their roles were examined by mutagenesis . The D259N and E264Q mutant enzymes both showed similar and large reductions in their Kcat/K(m) ratios (200-800-fold, depending on pH), indicating that either D259 or E264 (or both) could partner H296 . The conserved R235 residue was a candidate for binding the alpha-carboxyl group of the substrate and it was changed to lysine . The R235K mutant showed a 104-fold reduced Kcat/K(m) due to both an increased K(m) and a reduced Kcat for 2-oxo-4-methylvalerate . Thus R235 plays a role in binding the substrate carboxylate similar to R171 in the L-lactate dehydrogenases . Finally, we constructed the H205Q mutant to test the role of this partially conserved histidine residue (in 10/13 enzymes of the family) . This mutant enzyme displayed a 7.7-fold increased Kcat and a doubled catalytic efficiency at pH 5, was as sensitive to diethylpyrocarbonate as the wild-type but showed a sevenfold increased K(m) for NADH and a 100-fold increase in Kd for NADH together with 10-30-fold lower substrate inhibition . The transient kinetic behaviour of the H205Q mutant is as predicted from our previous study on the enzymatic mechanism of D-2-hydroxy-4-methylvalerate dehydrogenase which showed that coenzyme binding is highly pH dependent and indicated that release of the oxidised coenzyme is a significant component of the rate-limiting processes in catalysis at pH 6.5. Eur J Biochem, 1997 Feb 15, 244(1), 203 - 12 D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp . bulgaricus . I . Kinetic mechanism and pH dependence of kinetic parameters, coenzyme binding and substrate inhibition; Alvarez JA et al.; The steady-state kinetics of D-2-hydroxy-4-methylvalerate dehydrogenase have been studied at pH 8.0 by initial velocity, product inhibition, and dead-end inhibition techniques . The mechanism is rapid-equilibrium ordered in the NAD+ plus D-2-hydroxy-4-methylvalerate direction, and steady-state ordered in the other direction . In both cases coenzyme is the first substrate added and both the E-NADH-D-2-hydroxy-4-methylvalerate and E-NAD+-2-oxo-4-methylvalerate give rise to abortive complexes which cause excess substrate inhibition . Steady-state measurements show that the rate-limiting step in both directions at pH 8.0 is between formation of the enzyme-coenzyme-substrate ternary complex and the release of the first product of the reaction . Transient kinetics combined with primary kinetic deuterium isotope effects show that in the NADH-->NAD+ direction there is a slow, rate-limiting rearrangement of the E-NADH-oxoacid complex while hydride transfer is very fast . The release of NAD+ at pH 8.0 is 200-times faster than Kcat (NADH-->NAD+) whereas the release of NADH is only 5-times faster than Kcat (NAD+-->NADH) . The pH dependence of NADH binding depends upon the presence of two ionizable residues with a pKa of about 5.9 . The pH dependence of kinetic parameters is explained by a third ionizable residue with pKa values 7.2 (in the E-NADH complex) and < or = 6.4 (in the E-NAD+ complex) which may be the proton donor and acceptor for the chemical reaction . At pH 6.5 the mechanism changes in the NADH-->NAD+ direction to be partly limited by the chemical step with a measured primary kinetic isotope effect of 5.7 and partly by an only slightly faster dissociation of NAD+ . In addition the inhibition by excess oxo-4-methylvalerate is more pronounced . The mechanism implies that removing the positive charges created by the two groups which control coenzyme affinity could both enhance the catalytic rate at pH 6.5 and diminish excess substrate inhibition to provide an enzyme better suited to the bulk synthesis of D-2-hydroxyacids. FEBS Lett, 1997 Feb 3, 402(2-3), 157 - 61 The influence of aspartate 26 on the tautomeric forms of folate bound to Lactobacillus casei dihydrofolate reductase; Birdsall B et al.; The ternary complex of Lactobacillus casei dihydrofolate reductase (DHFR) with folate and NADP+ exists as a mixture of three interconverting forms (I, IIa and IIb) whose relative populations are pH dependent, with an effective pK of approx . 6 . To investigate the role of Asp26 in this pH dependence we have measured the 13C chemical shifts of {2,4a,7,9-(13)C4}folate in its complex with the mutant DHFR Asp26 --> Asn and NADP+ . Only a single form of the complex is detected and this has the characteristics of form I, an enol form with its N1 unprotonated . A study of the pH dependence of the 13C chemical shifts of DHFR selectively labelled with {4-(13)C}aspartic acid in its complex with folate and NADP+ indicates that no Asp residue has a pK value greater than 5.4 . Two of the Asp CO2 signals appear as non-integral signals with chemical shifts typical of non-ionised COOH groups and with a pH dependence characteristic of the slow exchange equilibria previously characterised for signals in forms I and IIb (or IIa) . It is proposed that the protonation/deprotonation controlling the equilibria involves the O4 position of the folate and that Asp26 influences this indirectly by binding in its CO2 form to the protonated N1 group of folate in forms I and IIa thus reducing the pK involving protonation at the O4 position to approx . 6 . These findings indicate that, in forms I and IIa of the ternary complex, folate binds to DHFR in a very similar way to methotrexate. Arch Oral Biol, 1997 Feb, 42(2), 181 - 4 Interindividual differences in degradation of sodium monofluorophosphate by saliva in relation to oral health status; Klimek J et al.; The enzymatic degradation of sodium monofluorophosphate by whole saliva in patients with differing oral health status was compared Ten patients each with good or poor oral health were selected . Assessment of oral health status included indices of the amount of dental plaque, caries experience and counts of Streptococcus mutans and lactobacilli . Whole-saliva samples were collected under standardized conditions and monofluorophosphatase (MFPase) activity was measured in 2-ml saliva samples during 2 h of incubation at 37 C with 1 ml of monofluorophosphate solution (equivalent 31.5 mmol/1 F) . MFPase activity was found in all the saliva samples . The values ranged from 0.6 to 17.8 nmol F/ml saliva per min . High MFPase activities correlated well with large amounts of plaque with the D component of the DMFT index and with high counts of salivary S . mutans and lactobacilli . The results show a wide range of MFPase activity in individuals and a statistically significant correlation (p < 0.05) between high MFPase activity and poor oral health status. Nahrung, 1997 Feb, 41(1), 18 - 21 Effect of bacterial galactosidase treatment on the nutritional status of soybean seeds and its milk derivative; Sanni AI et al.; Four field strains of Lactobacillus plantarum (LS 4, 19, 21, 133) obtained from fufu (a semi-solid product obtained by boiling fermented cassava--Manihot esculenta Crantz) and a type strain DSM 2017 were grown on different carbon sources to induce galactosidase production . LS 21 produced the highest concentration of alpha- and beta-galactosidase with 0.28 mumol/l and 0.28 mumol/l respectively on lactose and galactose . Milk obtained from soybean seeds treated with the enzyme mixture for 24 h showed a 99, 98 and 96% reduction respectively in the raffinose, stachyose and sucrose content when compared with the dry soybean seed . Glucose and galactose which were not detected in the dry seeds became readily available after soaking in both enzyme mixture and distilled water . Although there was reduction in the nutritional composition of both milk samples, reduction of phytic acid and trypsin inhibitor is beneficial to the consumers . The result of the sensory evaluation showed that the milk prepared from enzyme-treated soybean seeds was rated better in terms of flavour, texture, appearance and palatability. J Pediatr Gastroenterol Nutr, 1997 Feb, 24(2), 153 - 61 Enteral nutrition modifies gut-associated lymphoid tissue in rat regardless of the molecular form of nitrogen supply; Guihot G et al.; BACKGROUND: It has been suggested that beneficial effect of elemental enteral diets in the treatment of inflammatory bowel diseases could be mediated by the suppression of protein dietary antigens . The objective of the present work was to study the effect of enteral diet on gut associated lymphoid tissue and on gastric Lactobacillus flora, in rat . METHODS: The effects of three molecular forms of nitrogen supply: amino acids, oligopeptides or whole casein, were compared in rats on continuous enteral diet . Frozen sections of small bowel were studied with monoclonal antibodies anti-CD5, -CD4, -CD8, -CD25, -macrophages, -MHC II . The Lactobacillus flora was also enumerated in the stomach, in order to assess the effect of ED on rat flora . RESULTS: Growth and mucosa morphology were identical in control and enteral groups . Rats on enteral diet showed, whatever was the molecular form of nitrogen supply, a decrease in CD5+, CD4+ and CD8+ intraepithelial cell numbers, but not in lamina propria cell number, and a decreased MHC II epithelial expression, when compared to control rats . The enterally fed rats also showed a decrease in Lactobacillus gastric contents . CONCLUSIONS: The current study demonstrates that continuous enteral nutrition modifies MHC II epithelial expression and gut associated lymphoid tissue cell number in rat, whatever is the molecular form of nitrogen supply . Intestinal flora could be responsible, at least for part, for these results. Microbiol Res, 1997 Feb, 152(1), 87 - 92 Quantitation by enzyme immunoassay of spirosin from Lactobacillus reuteri and Escherichia coli; Yamato M et al.; Three EIA methods (Direct, Indirect and Sandwich EIA) were studied to quantify spirosin in Lactobacillus reuteri and Escherichia coli cultured under various conditions in an attempt to get some insight into the function of spirosome . Both Direct and Indirect EIA were suited well for the quantitation of L . reuteri spirosin while Direct EIA was appropriate for spirosin of E . coli . Sandwich EIA could not be applied successfully in either case . By use of these methods, the amounts of spirosin produced by E . coli were determined to be 1.4, 36.2 and 46.5 micrograms per mg protein of the cell lysate under aerobic, standing and anaerobic culture conditions, respectively . Since the production profile of spirosin coincided entirely with that of alcohol dehydrogenase, these findings supported the identity of spirosin to alcohol dehydrogenase in E . coli . In the same way, L . reuteri spirosin was quantified to be 73.5 and 65.4 micrograms/ mg protein of the lysate in standing and anaerobic culture, respectively . The production pattern of spirosin did not parallel that of alcohol dehydrogenase among three strains of L . reuteri, suggesting that spirosin might not be identical to alcohol dehydrogenase. Can J Microbiol, 1997 Feb, 43(2), 157 - 63 A specific oligonucleotide primer for the rapid detection of Lactobacillus lindneri by polymerase chain reaction; Yasui T et al.; A polymerase chain reaction (PCR) method was developed for the rapid detection of the beer-spoilage heterofermentative lactic acid bacterium Lactobacillus lindneri . Three strains, the Chinese brewery isolate DA1, the Japanese commercial beer isolate BG2, and the Japanese brewery isolate SE3, which were serologically classified as belonging to L . lindneri, were used in this study . After sequencing the 16S rDNA of the isolates DA1 and BG2 and the typical beer-spoilage heterofermentative Lactobacillus brevis L63, these sequences were compared with published data . A L . lindneri specific PCR primer, DA-40, was then constructed based on the V1 variable region of 16S rDNA . The specificity of PCR using the L . lindneri specific primer DA-40 and the universal primer 907r was examined using five L . lidneri strains: the three isolates described above and two strains from culture collection, DSM 20690 and DSM 20692 . A variety of beer-spoilage lactic acid bacteria, including 71 Lactobacillus strains and 13 Pediococcus strains, were also included in this examination . No PCR product was obtained from any DNA with the exception of the five L . lindneri strains, indicating that the L . lindneri specific primer DA-40 was highly specific . The detection limit for L . lindneri in beer was 63 CFU/100 mL of beer. Can J Microbiol, 1997 Feb, 43(2), 143 - 8 Rapid procedure for acid adaptation of oral lactic-acid bacteria and further characterization of the response; Ma Y et al.; Acid-adaptive responses could be induced readily in oral lactic-acid bacteria by growing them in batch cultures with excess sugar or more conveniently and rapidly by transferring cells to acidified growth media for the time required for biomass doubling . The response of Streptococcus mutans GS-5 was induced in a progressive rather than all-or-nothing way, and the extent of acid tolerance was inversely related to the pH of the inducing medium over a range from 8.5 to 5 . The weak acids fluoride, acetate, or lactate did not measurably enhance acid adaptation, and so the response did not appear to depend primarily on changes in delta pH or the proton motive force across the cell membrane . Transcription and translation to form new proteins did appear to be necessary, as indicated by inhibition of adaptation by rifampin or chloramphenicol and by lack of adaptation by cells suspended in phosphate buffer at pH 5 . Streptococcus salivarius and Lactobacillus casei were acid adapted by the rapid method, and the method appeared to be generally useful for oral lactic-acid bacteria . The rapid induction of the response in multiple oral lactic-acid bacteria suggests that it is of general importance for maintaining a diversity of organisms in the oral microbiota, which is regularly subjected to acid stresses. Eur J Oral Sci, 1997 Feb, 105(1), 27 - 35 Effects of chlorhexidine on the bacterial colonization and degradation of dentin and completely demineralized dentin in situ; van Strijp AJ et al.; The effects of 0.2% chlorhexidine on selected plaque microorganisms were studied in an intraoral dentin caries model . In 8 individuals wearing partial dentures, sound and completely demineralized dentin specimens were placed consecutively in 2 periods of 4 weeks, respectively . Throughout the experimental period, the specimens were treated 2 x daily with 0.2% chlorhexidine; control specimens were treated with water . Plaque accumulation on the specimens was left undisturbed . No protection against demineralization of the dentin or degradation of the dentin collagen by the chlorhexidine treatment was observed . The chlorhexidine treatment did not result in a reduction of the total cultivable flora when compared with the control specimens . A significant reduction of mutans streptococci and total streptococci recovered from completely demineralized dentin treated with chlorhexidine was observed, but the proportions of Actinomyces and lactobacilli were not affected significantly . It is speculated that areas of exposed roots, which are difficult to reach by oral hygiene measurements, such as approximal surfaces, will not be protected by a 0.2% chlorhexidine mouthrinse against the caries process. J Hepatol, 1997 Feb, 26(2), 417 - 24 Effect of oral supplementation of lactobacilli on bacterial translocation in acute liver injury induced by D-galactosamine; Kasravi FB et al.; BACKGROUND/AIM: Bacterial infections and sepsis are frequent complications of acute liver injury, with a high share in the mortality and morbidity of this condition . Bacterial translocation from the gut may play an important role in the high rate of infections observed . In this experiment the effect of different oral supplementation on bacterial translocation was evaluated in acute liver injury induced by D-galactosamine in the rat . METHODS: Rats were given oral supplements of lactulose, neomycin, Lactobacillus reuteri R2LC, and Lactobacillus plantarum DSM 9843 for 1 week . Liver injury was induced by intraperitoneal administration of 1.1 g/kg D-galactosamine . Twenty-four hours later, rats were sacrificed and liver enzymes and histology, intestinal bacterial count and microflora, intestinal mucosal histology, DNA and RNA content, bacterial translocation to blood, mesenteric lymph nodes, and liver, and serum endotoxin were studied or measured . RESULTS: Lactulose was highly effective in prevention of liver injury and bacterial translocation . Neomycin and Lactobacillus plantarum DSM 9843 showed a moderate effect in prevention of liver injury and bacterial translocation . Intestinal bacterial count and microflora were affected by different treatment modalities . No endotoxin concentration was found in any of the experimental groups . Both lactobacilli could significantly improve the mucosal proliferative state . CONCLUSIONS: Oral supplementation of lactulose with anti-endotoxin effect could successfully prevent the liver injury and the subsequent bacterial translocation in acute liver injury induced by administration of D-galactosamine in the rat . This effect was irrespective of the intestinal bacterial alteration or mucosal proliferative state. J Reprod Med, 1997 Feb, 42(2), 91 - 8 Cesarean delivery . Microbial colonization in amniotic fluid; Keski-Nisula L et al.; OBJECTIVE: To determine the frequency, clinical significance and causative factors behind intraamniotic microbial colonization in uninfected parturients at the time of cesarean delivery . STUDY DESIGN: Amniotic fluid specimens for bacterial and mycoplasmal cultures were obtained by direct aspiration at cesarean section from 251 pregnant women (24-43 completed weeks) who had no clinical infection at the time of the operation . The symptoms of maternal infection were followed postoperatively for the first week of the puerperium . RESULTS: The prevalence of amniotic fluid microbial invasion was 29% (72/251) . In patients not in labor and with intact membranes, it was 13% (20/158); in patients in labor and with intact membranes, 23% (5/22); and in those with ruptured membranes, 66% (47/71) . The most common species isolated were Ureaplasma urealyticum, Lactobacillus species and coagulase-negative staphylococci . In the total 251 patients, clinically evident postoperative endometritis was observed in 6 (2%) and wound infection in 10 (4%) . In patients operated on and with intact membranes, no risk factors were found as regards amniotic fluid microbial colonization . In patients operated on after rupture of the membranes, the only significant risk factor as regards amniotic fluid microbial invasion was use of an internal monitor before the operation (P < .0003) (relative risk 10.7, 95% confidence limit 2.9-39.4) . The relative risk of postoperative endometritis was 2.3 (95% confidence limit 1.3-4.3) in patients with microbial invasion of the amniotic cavity as compared to patients without invasion . The corresponding risk value for post-operative wound infection was 1.4 (95% confidence limit 0.6-3.1) . CONCLUSION: Though the incidence of microbial invasion of the amniotic fluid before surgery was unexpectedly high, its clinical significance as regards maternal puerperal morbidity appeared to be low . The use of internal monitoring during labor was the only significant risk factor as regards amniotic fluid microbial colonization in patients operated on after membrane rupture. Poult Sci, 1997 Feb, 76(2), 381 - 5 Effect of feeding diets containing an antibiotic, a probiotic, or yucca extract on growth and intestinal urease activity in broiler chicks; Yeo J et al.; A 6-wk study was conducted to determine the effect of feeding diets containing an antibiotic, a probiotic, or yucca extract on daily gain, feed conversion ratio, and urease activity and ammonia production in intestinal contents of broiler chicks . Four replicates of 10 broiler chicks (average body weight, 48 g) each were assigned to a control or diets containing 0.1% chloroxytetracycline (antibiotic), 0.1% Lactobacillus casei (probiotic), or 0.2% yucca extract . Feeding a diet containing the probiotic significantly (P < 0.05) increased average daily gain during the first 3-wk period compared to the control (30.7 vs 28.7 g) . This increase was partly accounted for by increased feed intake . During the first 3 wk, feeding the diet containing probiotic significantly (P < 0.05) decreased urease activity (per gram of collected contents) in small intestinal contents but not in large intestinal contents, compared with the control . Urease activity determined at 6 wk of age was not significantly affected by diet . Our studies indicate that dietary probiotic decreases urease activity in the small intestinal contents of young chicks and thus may be beneficial for improving animal health and growth, especially during early life. Mol Microbiol, 1997 Feb, 23(3), 505 - 14 Molecular analysis of the cos region of the Lactobacillus casei bacteriophage A2 . Gene product 3, gp3, specifically binds to its downstream cos region; Garcia P et al.; The terminal nucleotide sequence of the Lactobacillus casei bacteriophage A2 DNA revealed a single-stranded extension 13 bases in length (5'-AACGGTCGGCCTC-3') at its 3' termini that defines the packaging initiation nicking site (cosN) . The cosN sequence is bisected by an axis of hyphenated twofold rotational symmetry . Directly and inverted repeated sequences located to the left (cosL) and the right (cosR) of the cosN site were observed . Analysis of the 3.4 kb EcoRI DNA sequence surrounding the cos region revealed four complete and one incomplete open reading frames (orfs) . Northern blots indicated that all were cotranscribed in a single mRNA molecule in excess of 10 kb that appeared late during infection . Minicell studies indicated that the four orfs were translated into protein . From the ORF3 amino acid sequence DNA-binding and NTP-binding domains can be predicted . The purified ORF3 (predicted molecular mass 16.8 kDa) shows specific binding to the A2 cos region, so it was renamed gp3 . Gp3 forms a specific complex with a 369 bp cos DNA segment in the presence of ATP . Gp3 interaction with the intrinsically bent cos DNA segment induces intramolecular ligation in the presence of T4 DNA ligase . The data presented here suggest that gp3 is the small subunit of the terminase enzyme. Microbiology, 1997 Feb, 143 ( Pt 2), 527 - 37 Lactobacillus delbrueckii subsp . lactis DSM7290 pepG gene encodes a novel cysteine aminopeptidase; Klein JR et al.; A number of Escherichia coli clones were isolated from a Lactobacillus delbrueckii subsp . lactis gene library capable of hydrolysing the chromogenic substrate Gly-Ala-beta-naphthylamide (Gly-Ala-beta NA) . Some of the recombinant plasmids carried by these clones have been shown to encode the cysteine aminopeptidase gene pepC . Nucleotide sequence analyses of the plasmid inserts of the remaining clones resulted in the identification of two adjacent ORFs encoding proteins exhibiting a high degree of similarity between themselves (72.6%) and with PepC . One gene, designated pepG, was overexpressed in E . coli and the crude extracts obtained were shown to be peptidolytically active both against chromogenic substrates and peptides, and in a Salmonella typhimurium growth test . PepC and PepG activities were compared using chromogenic beta NA and p-nitroanilide substrates and leucine or proline-containing peptides were applied in growth experiments of recombinant Sal . typhimurium . The results indicate that the enzymes, although structurally related, have different substrate preferences . No enzyme activity could be ascribed to the second ORF (orfW), despite the production of a visible protein using a T7 RNA polymerase system . Primer extension analysis, using mRNA isolated from Lb . delbrueckii subsp . lactis DSM7290 did establish that orfW was transcribed. Int J Food Microbiol, 1997 Feb, 34(2), 157 - 70 Insufficient antilisterial capacity of low inoculum Lactobacillus cultures on long-term stored meats at 4 degrees C; Buncic S et al.; Two of the 210 lactobacilli strains isolated from chilled meats produced antilisterial bacteriocins: Lactobacillus sake 265 (Lb 265) and Lactobacillus casei 52 (Lb 52) . Factors affecting antilisterial effectiveness of these and two other bacteriocin-producing (Bac+) strains (Lactobacillus sake 706, Lb 706; and Lactobacillus sake 148, Lb 148) at refrigeration temperature (4 degrees C) were studied in laboratory media and meat systems . At both 4 degrees C and 25 degrees C, these Bac+ strains grown in buffered MRS broths (pH 5.4 or 6.5) showed longer lag phases and shorter generation times than Listeria monocytogenes (mixture of strains NCTC 7973 and two food derived strains, L70 and L72) when grown in buffered BHI broths at the same pH values . These differences were more significant at 4 degrees C than at 25 degrees C . The highest concentrations of bacteriocin in MRS broth were produced at 25 degrees C and 4 degrees C by strain Lb 265 and Lb 706, respectively . Generally, production of bacteriocins was more efficient at lower pH (in buffered MRS broths of pH 5.4 and unbuffered MRS broths), than at higher pH (in buffered broths of pH 6.5) . On vacuum packaged, raw beef (pH 5.3-5.4) initial numbers of L . monocytogenes (10(3)/g) did not change significantly during 23-days storage at 4 degrees C, when inoculated either alone or in the presence of Bac+ strains inoculated at initial levels of 10(3)/g . On vacuum packaged emulsion-type of sausages (pH 6.4) inoculated with L . monocytogenes and stored at 4 degrees C for 23 days growth was not significantly affected by addition of Bac+ strains at initial levels of 10(3)/g . These results indicated that amounts of bacteriocins produced in situ by low initial numbers (10(3)/g) of the protective strains tested were not sufficient to inhibit and/or reduce L . monocytogenes on these chilled meats, where high initial numbers of lactic acid bacteria are not desirable for product quality resons . To achieve these effects, higher concentrations of active (free) bacteriocins in meats must be provided. Int J Food Microbiol, 1997 Feb, 34(2), 145 - 56 Production, purification and characterization of reutericin 6, a bacteriocin with lytic activity produced by Lactobacillus reuteri LA6; Kabuki T et al.; A bacteriocin (Reutericin 6) produced by Lactobacillus reuteri LA6, was purified by hydrophobic chromatography from the modified MRS broth (D'-MRS) with 6180-fold increase in specific activity with 14% recovery . The molecular weight of reutericin 6 was determined to be 2.7 kDa by SDS-PAGE and ESI-MS . By amino acid analysis, reutericin 6 comprised of 67% hydrophobic and polar neutral amino acids . Lanthionine was not detected . The lytic activity against Lactobacillus delbrueckii subsp . bulgaricus JCM 1002T and N1A1 B6 was detected by the decrease of both turbidity and the number of viable cells, and by leaking of beta-galactosidase. Int J Food Microbiol, 1997 Feb, 34(2), 131 - 43 Anti-aflatoxigenic activity of Lactobacillus casei pseudoplantarum; Gourama H et al.; Lactobacillus casei pseudoplantarum 371 isolated from a silage inoculant was found to inhibit aflatoxins B1 and G1 biosynthesis by Aspergillus flavus subsp . parasiticus NRRI . 2999, in liquid medium . The inhibitory activity in the Lactobacillus cell-free supernatant was found to be sensitive to proteolytic enzymes such as trypsin and alpha-chymotrypsin, but resistant to pepsin . Lab-Lemco tryptone broth (LTB), supplemented with 20% of dialyzed protein concentrate of the supernatant, totally inhibited the production of aflatoxins B1 and G1 . When the protein concentrate was digested with trypsin, the production of aflatoxins B1 and G1 was restored . The inhibitory activity of the supernatant was inactivated within 10 min at 100 degrees C . A . flavus grown in the Lactobacillus cell-free supernatant did not produce a mutagenic response in the Salmonella mutagenicity test . However, Lactobacillus casei pseudo plantarum 371 did not have an effect on aflatoxin production and mold growth as measured by ergosterol and plate count, when the organisms were inoculated together on sterile steamed rice. Appl Environ Microbiol, 1997 Feb, 63(2), 513 - 8 Antagonistic activity exerted in vitro and in vivo by Lactobacillus casei (strain GG) against Salmonella typhimurium C5 infection; Hudault S et al.; The aim of this study was to compare the antagonistic properties of Lactobacillus casei GG exerted in vitro against Salmonella typhimurium C5 in a cellular model, cultured enterocyte-like Caco-2 cells, to those exerted in vivo in an animal model, C3H/He/Oujco mice . Our results show that a 1-h contact between the invading strain C5 and either the culture or the supernatant of L . casei GG impeded the invasion by the Salmonella strain in Caco-2 cells, without modifying the viability of the strain . After neutralization at pH 7, no inhibition of the invasion by C5 was observed . The antagonistic activity of L . casei GG was examined in C3H/He/Oujco mice orally infected with C5 as follows: (i) L . casei GG was given daily to conventional animals as a probiotic, and (ii) it was given once to germ-free animals in order to study the effect of the population of L . casei GG established in the different segments of the gut . In vivo experiments show that after a single challenge with C5, this strain survives and persists at a higher level in the feces of the untreated conventional mice than in those of the treated group . In L . casei GG germ-free mice, establishment of L . casei GG in the gut significantly delayed the occurrence of 100% mortality of the animals (15 days after C5 challenge versus 9 days in germ-free mice {P < 0.01}) . Cecal colonization level and translocation rate of C5 to the mesenteric lymph nodes, spleen, and liver were significantly reduced during the first 2 days post-C5 challenge, although the L . casei GG population level in the gut dramatically decreased in these animals. Gene, 1997 Jan 31, 185(1), 119 - 25 Characterization of the genes encoding integrative and excisive functions of Lactobacillus phage øg1e: cloning, sequence analysis, and expression in Escherichia coli; Kakikawa M et al.; og1e is a temperate phage of the Lactobacillus strain G1e . The phage-host junctions attR and attL cloned from the lysogen have a 24-bp common (core) sequence implicated in recombination . DNA sequencing analysis of a 5.2-kbp SacI fragment of the og1e phage genome (42.5 kbp) revealed two possible open reading frames (ORF), xis and int, and the phage attachment (recombination) site (attP), whose 24-bp sequence is identical to the core sequence detected in attR and attL . The deduced int product (Int) is a basic protein of 391 amino acids with an estimated pI of 9.70, and significantly resembles other presumed integrases encoded by the Lactobacillus and Lactococcus phages including oadh and oLC3, as well as the Escherichia coli phages such as lambda . The predicted og1e xis protein (Xis) is small and very acidic (66 amino acids; pI 4.55), and shows a resemblance (32% overall identity) with a putative excisionase encoded by the Staphylococcus phage o11 . The og1e Int with a deduced molecular mass of 45.5 kDa was overproduced in E . coli cells, and electrophoretically analyzed. Proc Natl Acad Sci U S A, 1997 Jan 7, 94(1), 53 - 8 The B12-dependent ribonucleotide reductase from the archaebacterium Thermoplasma acidophila: an evolutionary solution to the ribonucleotide reductase conundrum; Tauer A et al.; A coenzyme B12-dependent ribonucleotide reductase was purified from the archaebacterium Thermoplasma acidophila and partially sequenced . Using probes derived from the sequence, the corresponding gene was cloned, completely sequenced, and expressed in Escherichia coli . The deduced amino acid sequence shows that the catalytic domain of the B12-dependent enzyme from T . acidophila, some 400 amino acids, is related by common ancestry to the diferric tyrosine radical iron(III)-dependent ribonucleotide reductase from E . coli, yeast, mammalian viruses, and man . The critical cysteine residues in the catalytic domain that participate in the thiyl radical-dependent reaction have been conserved even though the cofactor that generates the radical is not . Evolutionary bridges created by the T . acidophila sequence and that of a B12-dependent reductase from Mycobacterium tuberculosis establish homology between the Fe-dependent enzymes and the catalytic domain of the Lactobacillus leichmannii B12-dependent enzyme as well . These bridges are confirmed by a predicted secondary structure for the Lactobacillus enzyme . Sequence similarities show that the N-terminal domain of the T . acidophila ribonucleotide reductase is also homologous to the anaerobic ribonucleotide reductase from E . coli, which uses neither B12 nor Fe cofactors . A predicted secondary structure of the N-terminal domain suggests that it is predominantly helical, as is the domain in the aerobic E . coli enzyme depending on Fe, extending the homologous family of proteins to include anaerobic ribonucleotide reductases, B12 ribonucleotide reductases, and Fe-dependent aerobic ribonucleotide reductases . A model for the evolution of the ribonucleotide reductase family is presented; in this model, the thiyl radical-based reaction mechanism is conserved, but the cofactor is chosen to best adapt the host organism to its environment . This analysis illustrates how secondary structure predictions can assist evolutionary analyses, each important in "post-genomic" biochemistry. Caries Res, 1997, 31(5), 384 - 9 In vivo cariostatic effect of resin modified glass ionomer cement and amalgam on dentine; Kreulen CM et al.; Fluoride-releasing materials have been reported to be bactericidal in vitro . This may be of benefit to modern dentistry, which is directed to the preservation of tooth tissue during restorative treatment . Little is known about in vivo effects . The aim is to investigate the influence of a resin-modified glass ionomer cement (RM-GIC) on carious dentine that remains under restorations, compared to amalgam . Using a split month design, 40 molar pairs in 40 patients (mean age 14.9 years) were selected, based on clinically and radiographically diagnosed occlusal dentine caries . Under aseptic conditions, the enamel was removed and the carious dentine was sampled just beneath the dentino-enamel junction using a round bur . Without further removal of carious dentine, the molars of a pair were alternately restored with RM-GIC or amalgam . The colour and the consistency of the carious dentine were assessed . The samples were processed for microbiological determination of total viable counts (TVC), mutans streptococci (MS), and lactobacilli (LB) . After 6 months the molars were reopened, similarly sampled and evaluated, and then permanently restored after complete caries removal . For both materials a substantial decrease in the numbers of TVC, MS and LB was found after the 6-month period . Also a positive effect was observed on the colour and the consistency of the remaining carious dentine, which was comparable for the two materials . RM-GIC showed a significantly larger decrease in counts of MS and LB than amalgam, but not for TVC . Since in only few cavities the number of bacteria decreased under the level of detection, it is still considered essential to remove all carious dentine during restorative treatment. Caries Res, 1997, 31(5), 379 - 83 Fluoride and mutans streptococci levels in plaque on aged restorations of resin-modified glass ionomer cement, compomer and resin composite; van Dijken JW et al.; The use of fluoride-releasing restoratives such as glass ionomer cements (GICs) has increased during the last decade . The antibacterial effect of released fluoride is thought to be a possible caries-preventive effect of these restorations . In this study fluoride concentrations in plaque on 1-year old resin-modified GIC, compomer and resin composite restorations were compared intraindividually and related to the occurrence of caries-associated bacteria . Plaque from class III restorations of the three restorative materials and from a proximal enamel surface in 18 individuals was analysed . Low fluoride levels were detected in all the samples, while the resin-modified GIC samples showed significantly higher amounts . The distribution of oral streptococci, mutans streptococci and lactobacilli did not differ significantly among the surfaces and did not correlate to the fluoride levels in the samples . A good correlation was found between the counts of mutans streptococci in saliva and their proportions in the plaque . The results indicate that the fluoride concentrations released in vivo from 1-year-old restoratives are not high enough to affect the plaque levels of the caries-associated bacteria mutans streptococci and lactobacilli. Caries Res, 1997, 31(5), 349 - 55 Bacterial colonization of mineralized and completely demineralized dentine in situ; van Strijp AJ et al.; The changing environment in a developing root lesion may result in a succession of the microbial flora in the dentine . As demineralization proceeds, the collagenous matrix is exposed, which could be conducive to the growth of specific microorganisms . In this study both sound and completely demineralized dentine were placed together in the partial prothesis of 8 individuals to test whether the type of substrate influenced the composition of the bacterial flora . After 6 weeks the degradation of the collagenous matrix, the demineralization of the dentine and the microbial composition were assessed . The collagen loss varied between 0 and 69 wt% . Mineral loss from the originally sound dentine specimens ranged from virtually none to complete demineralization . Percentages of total streptococci, mutans streptococci, Actinomyces and lactobacilli isolated from both dentinal substrates did not differ significantly . The percentage of lactobacilli in the dentine specimens was positively correlated to the lesion depth . The percentage of Actinomyces species was significantly higher in both the dentine specimens that had been demineralized in vitro and those that were found to be completely demineralized in situ compared to the partially demineralized dentine specimens . In vitro, no collagenolytic activity of the predominant flora isolated from both dentinal substrates could be shown. Gynecol Obstet Invest, 1997, 44(1), 16 - 20 Microorganisms in vaginal fluid from women in prolonged pregnancy; Goffeng AR et al.; In order to compare the vaginal microflora of women in prolonged pregnancy with that of women who delivered at term, samples for quantitative aerobic and anaerobic microbiological culture were collected from 100 women at 42 weeks of gestation and from 60 women at term . The occurrence of lactobacilli-dominated flora was similar in women at term and women with prolonged pregnancy . However, non-hydrogen-peroxide-producing lactobacilli (p < 0.01) were significantly more common and Peptostreptococci species (p < 0.05) significantly less common in postterm women as compared with term controls . In postterm women, Candida albicans was more common (p < 0.001) in microfloras dominated by non-hydrogen-peroxide-producing lactobacilli than in floras dominated by hydrogen-peroxide-producing lactobacilli . The ecosystem of the vagina in asymptomatic postterm women was disrupted concerning the composition of lactobacilli as compared with term controls. Zh Mikrobiol Epidemiol Immunobiol, 1997 Jan-Feb, (1), 67 - 9 {The possibility of colonizing the intestines of white mice with Lactobacillus acidophilus during bacterial therapy}; Kruglikov VD et al.; The study revealed the possibility, on principle, for L . acidophilus strain VKM V-2020 D to colonize the intestine of white mice with the preservation of the viability of lactobacilli subjected to the action of antibiotics . The culture of this strain, isolated from the animals, showed the stability of its biological properties: resistance to polymyxin M, kanamycin, cyprofloxacin, nalidixic acid (including acquired resistance to rifampicin), as well as pronounced antagonism with respect to Vibrio cholerae . Good prospects for the use of L . acidophilus strain VKM V-2020 D for further studies regarding its use for prophylaxis and therapy were noted. Gastroenterol Clin Biol, 1997, 21(4), 293 - 8 Effects of intrajejunal perfusion and chronic ingestion of Lactobacillus johnsonii strain La1 on serum concentrations and jejunal secretions of immunoglobulins and serum proteins in healthy humans; Marteau P et al.; OBJECTIVES: Link-Amster reported an increase in serum IgA when healthy subjects ingested a fermented dairy product containing Lactobacillus johnsonii La1 . We aimed to assess the effects of La1 on the jejunal secretions and serum concentrations of total and specific immunoglobulins and proteins . METHODS: Twelve healthy volunteers ingested a fermented milk containing La1 or a control from day 1 till day 28, following a randomised double blind protocol . At days 0 and 28, the jejunum was successively perfused with a control solution and with a La1 suspension . The serum concentrations and jejunal secretions of albumin, orosomucoid, transferrin, alpha 2-macroglobulin, m-IgA, p-IgA, IgG, IgM, secretory component, and specific antibodies against La1 were assessed . RESULTS: Serum concentrations of IgA slightly increased between d0 and d28 in the group receiving La1 (1.85 +/- 0.64 g/L vs 1.76 +/- 0.76; P = 0.02) . The other parameters were not altered . CONCLUSION: This study shows that the immunomodulating effects of La1 ingestion in man are not due to modification of jejunal protein permeability. Arch Tierernahr, 1997, 50(1), 25 - 9 Effect of maduramicin and monensin on survival of Lactobacillus salivarius 51R administered in the crop and caeca of young chickens; Rada V et al.; A rifampicin-resistant Lactobacillus salivarius 51R was administered orally to newly hatched broiler chickens . The resistance to rifampicin enabled us to differentiate the organism administered from indigenous strains . One day after inoculation, Lactobacillus salivarius 51R dominated among lactobacilli in the crop and caeca of all inoculated chickens, even in those ones receiving maduramicin and monensin at 5 and 100 mg per kg of feed mixture, respectively . Coliform counts in both crop and caeca of inoculated chickens were significantly lowered on the first day after treatment . Also, counts of the crop enterococci were decreased in inoculated chickens . Rifampicin-resistant lactobacilli were still present in high numbers in the crop and caecal contents of inoculated chickens sampled 5 days after inoculation . Differences in counts of total lactobacilli, coliform bacteria, and enterococci were mostly nonsignificant in these samples . Our results demonstrate that (i) bacterial counts in the chicken gut were influenced by probiotic Lactobacillus administration, and (ii) chicken lactobacilli are resistant to ionophore coccidiostats under in vivo conditions. Plasmid, 1997, 37(3), 199 - 203 Isolation and characterization of a plasmid from Lactobacillus fermentum conferring erythromycin resistance; Fons M et al.; Lactobacillus fermentum is a lactic acid bacterial species commonly found in the digestive tracts of pigs and rodents and also present in man . We characterized a 5.7-kb plasmid, pLEM3, conferring erythromycin resistance, which was isolated from a porcine strain of L . fermentum . Plasmid pLEM3 established efficiently in L . fermentum, conferred high-level erythromycin resistance (MIC > 1 mg/ml), and was segregationally stable . A deletion derivative of pLEM3, called pLEM5, was constructed and found to be as genetically stable as the parent . A multiple cloning site was inserted into pLEM5, generating plasmid pLEM7 . Nucleotide sequence determination of pLEM5 revealed similarities with known genes . The replicon itself is a member of the pC194 family of rolling circle plasmids . The region responsible for erythromycin resistance was 98.2% identical to the erm gene of conjugative transposon Tn1545. Folia Microbiol (Praha), 1997, 42(1), 31 - 4 Assessment of a bacteriocin-producing Lactobacillus strain in the control of spoilage of a cereal-based African fermented food; Olasupo NA et al.; As a part of a program to develop starter cultures aiding in the spoilage control and sanitation of African fermented foods, a cereal-based food ('ogi' and its solid form 'agidi' or 'eko') was prepared using a bacteriocin-producing Lactobacillus strain as the starter culture . The survival of an enterotoxigenic Escherichia coli strain was investigated in the naturally fermented food and in food fermented with the starter bacteriocin-producing Lactobacillus strain . An inhibition of E . coli was observed within 2 h of incubation in 'ogi' fermented with the bacteriocin producing strain . After 6 h, the viable count of E . coli in locally fermented 'ogi' was log 6.41 (2.54 X 10(6) CFU/mL), whereas in 'ogi' fermented with the bacteriocin producer it was reduced to log 1.70 (0.5 x 10(2) CFU/mL) . Comparison of the shelf life of 'agidi' prepared from the naturally fermented food with that bacteriocin-producing starter culture showed that the latter had a better shelf life (kept for 11 d before spoilage occurred as compared with 7 d for the natural one) . The results are discussed in terms of the potential of bacteriocin-producing cultures in the control and retardation of spoilage and food-forne infections in some African fermented foods. Microbiol Immunol, 1997, 41(4), 361 - 5 Experimental Helicobacter pylori infection in association with other bacteria; Isogai H et al.; We performed surgical treatment on normal ddY mice before Helicobacter pylori inoculation . The treatment was expected to obstruct bacterial flow out of the stomach and increase the chance of bacterial attachment to the gastric epithelium in mice . The bacterial challenge induced inflammation in the stomach . H . pylori was recovered from the stomach throughout the observation period . Lactobacilli and streptococci tended to relate to the increase in number of H . pylori recovered . Pretreatment with atropine was considered to confuse the gastric flora and affect the number of H . pylori recovered . These results suggested that a certain amount of time is necessary for H . pylori to contact with the gastric epithelium and that the composition of flora is important for the establishment of H . pylori infection. J Appl Microbiol, 1997 Jan, 82(1), 115 - 20 Viability of Cryptosporidium parvum during ensilage of perennial ryegrass; Merry RJ et al.; The survival of Cryptosporidium parvum during ensilage of perennial ryegrass was examined in laboratory silos with herbage prepared in one of three different ways; either untreated, inoculated with a strain of Lactobacillus plantarum or by direct acidification with formic acid . The pH values of all silages initially fell below 4.5, but only formic acid-treated silage remained stable at less than pH 4 after 106 d, with the pH of the untreated and inoculant-treated silages rising to above 6 . The formic acid-treated silage had a high lactic acid concentration (109 g kg-1 dry matter (DM)) and low concentrations of propionic and butyric acids after 106 d . However, the untreated and inoculant-treated silages showed an inverse relationship, with low lactic acid concentrations and high concentrations of acetic, propionic and butyric acids . These silages also contained ammonia-N concentrations in excess of 9 g kg-1 DM . In terms of the viability of Cryptosporidium parvum oocysts very few differences were seen after 14 d of ensilage with ca 50% remaining viable, irrespective of treatment and total numbers had declined from the initial level of 5.9 x 10(4) to 1 x 10(4) g(-1) fresh matter . Total oocyst numbers remained approximately the same until the end of the ensiling period, with the percentage of viable oocysts declining to 46, 41 and 32% respectively for formic acid, inoculant and untreated silages . The results are discussed in terms of changes occurring during the silage fermentation, in particular the products which may influence the survival of Cryptosporidium and implications for agricultural practice and the health of silage fed livestock. Vet Med (Praha), 1997 Jan, 42(1), 19 - 27 {Criteria for selection of lactobacilli for probiotic use}; Nemcova R; The present knowledge of research makes it possible to use a whole range of stabilized micro-organisms and their metabolites for the production of preparations known as probiotics . The mechanism of the effect of probiotics, although it is not fully elucidated, is closely connected with the properties of the production strains . When selecting them, it is necessary to respect the origin of the strain used, its ability to adhere to the epithelial cells of the gut and to produce the inhibitory substances, the ability to survive and grow in the respective ecological units . The strain should be genetically stable, it should have good growth properties in vitro and in vivo, to maintain its high viability at processing, lyophilization and storage . Lactobacilli belong to the micro-organisms most frequently used to prepare the probiotics . The statement that lactobacilli inhibit the colonization of pathogenic bacteria upon the intestinal epithelium has been confirmed by many experiments on experimental germ-free and/or gnotobiotic animals . This inhibitory process known as "competitive exclusion" can be explained by the competition for the adherence sites on the intestinal mucosa between pathogens and lactobacilli and by the production of inhibitory substances . The host specificity as well as different degree of the expression of adherent phenotype, which is conditioned, in addition to the effect of the external environment also by the presence of plasmids of adherence, present the important property of the adherence of lactobacilli . The host specificity of the lactobacilli is closely connected with the presence of the specific molecules of receptors on the host cells which can be distinguished by means of specific molecules of the bacterial cells . The competition for the nutrients to be found on the intestinal epithelium, which present the growth substrates for both the probiotic strains and the pathogens, may present an important factor influencing the colonization of lactobacilli . The inhibitory components of the lactobacilli comprise the production of bacteriocins, toxic metabolites of the oxygen and organic acids . The inhibitory spectrum of the bacteriocins is different; the overwhelming majority of them inhibits G+ bacteria within the genus or taxonomically close to the genus of Lactobacillus, others such as bulgaricin, acidolin, laktocidin, acidophilin and non-protein substance reuterin are active against a wide spectrum of G+, G- bacteria, yeasts and fungi . Of toxic oxygen metabolites, the production of hydrogen peroxide is of importance . The hydrogen peroxide along with lactoperoxidase-thiocyanate milk system exerts bacteriocid effects on most pathogens . The production of organic acids (lactic, acetic) by lactobacilli is considered to be the primary regulator of the gut microbial activity in animals . The lactic acid as a product of the intestinal fermentation is further metabolized by lactate utilizing bacteria to VFA, thus the env