Determination of ribonucleosides,deoxyribonucleosides, and purine and pyrimidine bases in adult rabbit cerebrospinal fluid and plasma

Purine and pyrimidine base and nucleoside levels were measured in adult rabbit cisternal CSF and plasma by reversed-phase high-performance liquid chromatography. The concentrations of bases, nucleosides, and nucleoside phosphates were similar in plasma and CSF except for the adenosine phosphates and uracil which were higher in the plasma. In plasma and CSF, adenosine levels were low (0.12 microM) and guanosine, deoxyadenosine, deoxyguanosine, and deoxyinosine were not detectable (less than 0.1 microM); inosine and xanthine concentrations were 1-2 microM and hypoxanthine concentrations were approximately 5 microM; uridine (approximately 8 microM), cytidine (2-3 microM), and thymidine, deoxyuridine, and deoxycytidine (0.5-1.4 microM) were easily detectable. In both plasma and CSF, guanine, and thymine were undetectable (less than 0.1 microM), adenine and cytosine were less than 0.2 microM, but uracil was present (greater than 1 microM). Adenosine, inosine, and guanosine phosphates were also detectable at low concentrations in CSF and plasma. These results are consistent with the hypothesis that purine deoxyribonucleosides are synthesized in situ in the adult rabbit brain. In contrast, pyrimidine deoxyribonucleosides and ribonucleosides, and purine and pyrimidine bases are available in the CSF for use by the brain.     

Inhibition of Growth of Lactobacillus bulgaricus by Purine Deoxyribonucleotides.

Morris, George K. (University of Georgia, Athens), and William L. Williams. Inhibition of growth of Lactobacillus bulgaricus by purine deoxyribonucleotides. J. Bacteriol. 90:715-719. 1965.-An inhibition of growth of Lactobacillus bulgaricus GS was observed with deoxyadenylic acid and deoxyguanylic acid. Deoxynucleotides of cytosine, thymine, and uracil, and the deoxynucleosides of adenine, guanine, cytosine, and thymine were inactive as inhibitors. The inhibition was reversed by liver extract (a crude source of two unidentified growth factors for this organism). With suboptimal concentrations of liver extract, the inhibition was reversed by nucleotides of adenine, guanine, uracil, cytosine, and thymine. When the medium contained partially purified sources of the two growth factors rather than crude liver extract, fewer compounds reversed the inhibition. Adenylic acid and guanylic acid reversed the action of either inhibitor. Inosinic acid reversed inhibition caused by deoxyguanylic acid, but not that caused by deoxyadenylic acid. Thymidylic acid reversed inhibition caused by deoxyadenylic acid better than that caused by deoxyguanylic acid. Uridylic acid and cytidylic acid were no longer effective in reversing the inhibitions. This organism preferentially responded to monophosphorylated compounds as inhibitors and as reversers of inhibitions. Studies on the acid-soluble nucleotide pool revealed an accumulation of adenosine triphosphate, guanosine triphosphate, and an unidentified compound which resembled a nucleotide in its physical properties. These data cannot be explained by known metabolic pathways of nucleic acid biosynthesis. This organism responds differently from other related organisms to nucleic acid derivatives; therefore, it may be a new useful tool for studying nucleic acid metabolism and biosynthesis.     

Use of folic acid casei medium reveals trimethoprim susceptibility of Lactobacillus species

AIM: Lactobacilli have been reported to have intrinsic resistance to trimethoprim. The susceptibility of lactobacilli to trimethoprim on different media was investigated in order to search for a phenotypic test method that could indicate the presence of acquired resistance genes. METHODS AND RESULTS: Strains of Lactobacillus acidophilus, Lact. paracasei, Lact. rhamnosus and Lact. plantarum were susceptibility tested with E-tests on folic acid casei medium (FACM), MRS and defined medium 1. The effects of addition or removal of nucleosides and thymidine phosphorylase were investigated. E-tests on FACM yielded reproducible minimal inhibitory concentrations (MICs) for trimethoprim but addition of nucleosides was necessary for growth of Lact. acidophilus. MICs for the tested strains were 0.125-0.19, 0.25-3 and 0.064-0.19 microg ml(-1) for Lact. paracasei, Lact. rhamnosus and Lact. plantarum, respectively. With the addition of deoxyuridine and deoxyadenosine to FACM the MICs of Lact. acidophilus were 0.064-1 microg ml(-1). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacilli do not have intrinsic resistance to trimethoprim. The results show that trimethoprim susceptibility testing of the tested Lactobacillus species is possible and indicate that transferable resistance genes are absent in all the tested strains.     

Pathways of Nucleotide Biosynthesis in Mycoplasma mycoides subsp. mycoides

By measuring the specific activity of nucleotides isolated from ribonucleic acid after the incorporation of (14)C-labeled precursors under various conditions of growth, we have defined the major pathways of ribonucleotide synthesis in Mycoplasma mycoides subsp. mycoides. M. mycoides did not possess pathways for the de novo synthesis of nucleotides but was capable of interconversion of nucleotides. Thus, uracil provided the requirement for both pyrimidine ribonucleotides. Thymine is also required, suggesting that the methylation step is unavailable. No use was made of cytosine. Uridine was rapidly degraded to uracil. Cytidine competed effectively with uracil to provide most of the cytidine nucleotide and also provided an appreciable proportion of uridine nucleotide. In keeping with these results, there was a slow deamination of cytidine to uridine with further degradation to uracil in cultures of M. mycoides. Guanine was capable of meeting the full requirement of the organism for purine nucleotide, presumably by conversion of guanosine 5'-monophosphate to adenosine 5'-monophosphate via the intermediate inosine 5'-monophosphate. When available with guanine, adenine effectively gave a complete provision of adenine nucleotide, whereas hypoxanthine gave a partial provision. Neither adenine nor hypoxanthine was able to act as a precursor for the synthesis of guanine nucleotide. Exogenous guanosine, inosine, and adenosine underwent rapid cleavage to the corresponding bases and so show a pattern of utilization similar to that of the latter.     

Comparative study of permanganate oxidation reactions of nucleotide bases by spectroscopy

The permanganate oxidation of free nucleotide bases was successfully studied in aqueous solution of tetraethylammonium chloride using spectroscopic techniques. The reaction was highly selective toward thymine and uracil, less with cytosine, very little reaction on guanine, and no reaction on adenine.     

Growth and exopolysaccharide production by Lactobacillus helveticus ATCC 15807 in an adenine-supplemented chemically defined medium

AIMS: To analyse the exopolysaccharide (EPS) production by Lactobacillus helveticus ATCC 15807 in a chemically defined medium (CDM) and the effect of nutrients and stress culture conditions on cell growth and EPS formation. METHODS AND RESULTS: Cultures were conducted in CDM: (i) containing essential and nonessential bases and vitamins; (ii) without nonessential bases and vitamins [Simplified CDM (SCDM)]; (iii) SCDM supplemented individually with vitamins and bases. The influence of carbohydrates, pH and osmotic culture conditions on growth and polymer formation was analysed. Adenine and lactose stimulated both growth and EPS production. Constant pH fermentations (4.5 and 6.2) did not improve EPS synthesis while NaCl and glycerol were detrimental for growth and polymer formation. In all media the EPS monomer composition was glucose and galactose (2.5 : 1). CONCLUSIONS: A SCDM containing adenine and lactose was optimal for cell growth and EPS formation by Lact. helveticus ATCC 15807. Controlled pH (6.2 and 4.5) and osmotic stress culture conditions did not improve polymer production. The EPS characteristics were identical in all media. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides a better knowledge on EPS synthesis by Lact. helveticus. A CDM to perform regulation studies on EPS production by Lact. helveticus species is now available.     

Effects of culture conditions on the growth and auto-aggregation ability of vaginal Lactobacillus johnsonii CRL 1294

AIMS: To evaluate the effects of different physico-chemical factors on the growth and auto-aggregating ability of vaginal Lactobacillus johnsonii CRL 1294. METHODS AND RESULTS: L. johnsonii CRL 1294 was cultivated in different culture media, initial pH and temperature of incubation. The growth parameters were estimated by the Gompertz model, being optimal (higher final biomass and growth rate, and shorter lag phase) at an initial pH of 6.5 and at a temperature of 37 degrees C, both in LAPTg and MRS. The auto-aggregation ability, which was assessed by a model of exponential association, was evidenced in all the growth phases, being higher at pH 5 or 6.5. CONCLUSIONS: The growth of L. johnsonii CRL 1294 was affected in different way by all the physico-chemical factors tested. However, the auto-aggregation ability increased mainly at low initial pH of growth media. SIGNIFICANCE AND IMPACT OF THE STUDY: The auto-aggregation ability under different culture conditions of a vaginal Lactobacillus strain was systematically and statistically evaluated for the first time. The higher cellular aggregation evidenced at low pH could be a fundamental characteristic in the acidic vaginal environment to promote the protective role of lactobacilli.     

Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens

AIM: To develop in vitro assays for comparing the antagonistic properties and anti-oxidative activity of probiotic Lactobacillus and Bifidobacterium strains against various entero- and urinary pathogens. METHODS AND RESULTS: The antagonistic activity of five probiotic lactobacilli (Lactobacillus rhamnosus GG, Lactobacillus fermentum ME-3, Lactobacillus acidophilus La5, Lactobacillus plantarum 299v and Lactobacillus paracasei 8700:2) and two bifidobacteria (Bifidobacterium lactis Bb12, Bifidobacterium longum 46) against six target pathogens was estimated using different assays (solid and liquid media, anaerobic and microaerobic cultivation) and ranked (low, intermediate and high). Bacterial fermentation products were determined by gas chromatography, and the total anti-oxidative activity of probiotics was measured using linolenic acid test. Pyelonephritic Escherichia coli was highly suppressed by GG and both bifidobacteria strains. Lactobacilli strains 8700:2, 299v and ME-3 were the most effective against Salmonella enterica ssp. enterica in microaerobic while ME-3 and both bifidobacteria expressed high activity against Shigella sonnei in anaerobic milieu. Lact. paracasei, Lact. rhamnosus and Lact. plantarum strains showed intermediate antagonistic activity against Helicobacter pylori under microaerobic conditions on solid media. The highest anti-oxidative activity was characteristic for Lact. fermentum ME-3 (P < 0.05). No efficient antagonist against Clostridium difficile was found. The positive correlations between the pH, lactic acid production and anti-microbial activity for all tested probiotics were assessed. CONCLUSIONS: Developed experimental assays enable to compare the anti-microbial and -oxidative activity of Lactobacillus and/or Bifidobacterium probiotics, which have been claimed to possess the ability of suppressing the growth of various enteric and urinary pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Screening Lactobacillus and Bifidobacterium sp. strains according to their activity in various environmental conditions could precede the clinical efficacy studies for adjunct treatment with probiotics in cure of different gastrointestinal and urinary tract infections.     

A survey of lactic acid bacteria in Italian silage

G razia , L. & S uzzi , G. 1984. A survey of lactic acid bacteria in Italian silage. Journal of Applied Bacteriology 56 , 373–379.
Lactic acid bacteria, isolated from Italian ensiled products, were represented by strains of the genera Lactobacillus and Leuconostoc . The predominant strains were heterofermentative lactobacilli, with Lactobacillus buchneri being the most frequent. Among homofermentative lactic acid bacteria, strains of Lact. plantarum and Lact. casei were recovered. Almost all strains utilized malic acid and showed good acid-tolerance, but only some of them were able to metabolize malic acid at extremely low pH; these were five homofermentative lactobacilli (4 Lact. plantarum and 1 Lacr. casei var. casei ) and two heterofermentative lactobacilli ( Lact. cellobiosus and Lactobacillus sp.).     

Expanding the Genetic Code: Unnatural Base Pairs in Biological Systems

Molecular Biology - The genetic code is considered to use five nucleic bases (adenine, guanine, cytosine, thymine and uracil), which form two pairs for encoding information in DNA and two pairs for...     

16S ribosomal DNA analysis of the faecal lactobacilli composition of human subjects consuming a probiotic strain Lactobacillus acidophilus NCFM

AIMS: The aims of this study were to evaluate the ability of exogenous Lactobacillus acidophilus strain NCFM to survive through the human gastro-intestinal (GI) tract, and to evaluate the selectivity of Rogosa SL medium for faecal lactobacilli. METHODS AND RESULTS: The composition of the faecal lactobacilli of 10 healthy subjects was monitored for two weeks prior to, two weeks during and two weeks after the administration of the Lact. acidophilus strain NCFM consumed with skim milk (daily dose 10(10) viable cells). Fresh faecal samples were collected, processed and cultured on Rogosa SL selective medium for lactobacilli enumeration. Colonies demonstrating various morphologies were identified and purified for 16S ribosomal DNA sequence analysis for speciation of colonial genotype. The species composition of cultivable faecal lactobacilli changed considerably during consumption of the strain NCFM. CONCLUSIONS: The probiotic Lact. acidophilus strain NCFM can survive through the human GI tract, but cannot colonize itself during the two-week consumption. Rogosa SL medium is selective for faecal lactobacilli. However, genetic analysis is required for colony speciation. SIGNIFICANCE AND IMPACT OF THE STUDY: It is demonstrated that continuous consumption is necessary to maintain a high population of the probiotic strain, and that the Rogosa SL medium is reliable.     

Pyrimidine scavenging by Mycobacterium leprae

Mycobacterium leprae incorporated exogenously supplied pyrimidines as bases or nucleosides, but not as a nucleotide, into its nucleic acids. Notably, thymine was incorporated approximately 5 times more rapidly than thymidine by both suspensions of, or intracellular M. leprae. Thymine incorporation was significantly inhibited by clofazamine and dapsone at near-pharmacological levels. Therefore, incorporation of thymine is preferable as an activity for assessing viability of M. leprae. Nucleosides were converted to nucleotides through kinases, bases through phosphoribosyltransferases. Alternatively, thymine and uracil could first be converted to nucleosides. Cytosine and uracil bases were interconvertible, and uracil alone could supply all the pyrimidine requirements of M. leprae, though conversion to the thymine base was extremely slow. Overall, pyrimidine scavenging occurs at a slower rate than, and appears not to be so important as purine scavenging in M. leprae.     

Presence in the composition of bacterial DNA of covalently bound RNA fragments

The nucleotide composition of chromosome and plasmid DNA free of hybrid RNA isolated from resting Escherichia coli cells preliminary cultivated with the help of [14C] uracil has been studied. It has been established that DNA contains [14C]uracil side by side with adenine, thymine, guanine and cytosine. It confirms the presence of RNA fragments in the composition of bacterial DNA which are connected with it covalently.     

Selenium and Lactobacillus species

Selenium (Se) is an essential element for man and animal. Supplementation of Se has been done with Se-enriched yeast and inorganic Se compounds. In the present study a quantitative and qualitative evaluation was made of whether lactobacilli are able to concentrate Se. A high correlation was found between the bacterial Se concentration and the concentration of Se in the medium. The Se concentration in biomass was respectively 253 ± 50, 375 ± 33 and 407 ± 108 μg g⁻¹ dry weight for Lactobacillus delbrueckii subsp. bulgaricus, Lact. plantarum and Lact. casei subsp. casei when 1 mg 1⁻¹ Se⁴⁺ was present in the medium. Manganese (Mn) was concentrated in Lact. plantarum and Lact. casei subsp. casei but not in Lact. delbrueckii subsp. bulgaricus. The Mn concentration in biomass was higher compared to the Se concentration but this difference decreased when the concentration of Mn/Se increased in the culture medium. Copper, zinc and iron were also concentrated in biomass of Lact. delbrueckii subsp. bulgaricus. Characterization of the bacterial selenocompound revealed that ⁷⁵Se was generally incorporated, as selenocysteine, into protein of Lact. delbrueckii subsp. bulgaricus. Addition of L-cysteine to the medium decreased the bacterial Se content. It was concluded that Lact. delbrueckii subsp. bulgaricus incorporated Se non-specifically into bacterial protein. The application of Se-enriched lactobacilli (Se-Lb) in Se supplementation would be an interesting approach since selenomethionine, which is the major selenocompound in commercialized Se-yeast, was not detected in Se-Lb and because lactobacilli are already widely used in human nutrition.     

Nutritional Requirements of <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> in a Chemically Defined Medium

This study was undertaken to determine the nutritional requirements of Lactobacillus delbrueckii subsp. lactis and to develop a minimal chemically defined medium that supports sustained growth of these microorganisms. The single-omission technique was applied to each component of complete chemically defined medium in order to determine the nutritional requirements. L. delbrueckii subsp. lactis was prototrophic for alanine, glycine, aspartic acid, asparagine, glutamine, threonine, and proline. The lysine requirement was strain-dependent. Magnesium was the only essential oligoelement. These microorganisms also required uracil and guanine and adenine as pyrimidine and purine sources, respectively. In view of the nutritional requirements we designed a new minimal defined medium which supports sustained growth of L. delbrueckii subsp. lactis. This medium is simple and well defined, and should be preferable to complex media for conducting future biochemical, physiological, and genetic studies on L. delbrueckii subsp. lactis.     

Radioprotective effect of nucleosides and nucleotides and the mechanism of action of adenosine

Adenosine and a majority of adenine mononucleotides have a radioprotective action; adenine and 2'-deoxyadenosine have no radioprotective effect, and that of 3',5'-cAMP only approaches the detectable level. Ribo- and deoxyribonucleosides and nucleotides of guanine, uracil, thymine, and cytosine have no protective action. Dipyridamole increases and alkylxanthines block the radioprotective effect of adenosine. So it follows that the radioprotective effect is realized via A-receptors of the plasmatic membrane external surface.     

A new, completely defined medium for meat lactobacilli

A new defined medium (DML) for meat lactobacilli is presented. This succinate buffered medium with as few as 34 components supported good growth for 61 strains of typical meat lactobacilli, both isolated from meat and used as meat starters. For many of the strains tested, cell densities in DML were similar to those achieved in MRS medium. The high buffer capacity of DML and the balanced amino acid content allowed growth to continue to higher densities than eight other defined media. DML is compared with a previously published defined medium for Lactobacillus sake and shown to support higher cell densities of all strains tested. DML will allow a better determination of the nutritional requirements of different strains of meat lactobacilli as all strains can be tested under the same conditions, and will also be suitable for the study of growth kinetics and production of different metabolites.     

A survey of the microbial and chemical composition of seven semi-ripened Provola dei Nebrodi Sicilian cheeses

AIMS: The microbial and chemical composition of seven different semi-ripened (45 days) Provola dei Nebrodi Sicilian cheese samples were assessed in order to investigate the diversity of the microbial population in cheese made from different geographical areas throughout Sicily. METHODS AND RESULTS: The samples, which were obtained from seven different Provola dei Nebrodi manufacturers, were assessed using selective media. Interestingly, concentrations of presumptive lactobacilli represented over 90% of the total microbial population. In total, 105 presumptive Lactobacillus isolates were characterized to determine the relatedness of the isolates between the seven different cheeses. Randomly amplified polymorphic DNA polymerase chain reaction (RAPD PCR) analysis of the 105 presumptive lactobacilli indicated the presence of 22 distinct isolates. Further investigation of the isolates using pulsed field gel electrophoresis (PFGE) following restriction with the enzyme ApaI revealed the presence of 19 distinct macrorestriction patterns and the presence of between one and four distinct isolates per cheese sample (out of a total of 15 isolates per cheese randomly taken from Lactobacillus selective media plates). Analysis of the 16S rDNA sequence of each genetically distinct isolate demonstrated the dominance of the Lactobacillus casei species in all cheese samples assessed. Lactobacillus delbrueckii and Pediococcus pentosaceus species were also detected. The concentration of free amino acids, used to estimate the extent of proteolysis in each cheese, ranged from 59 to 433 mg 100 g(-1) cheese. CONCLUSIONS: Microbiological assessment of the cheeses demonstrated the dominance of Lactobacillus species after 45 days of ripening with levels ranging from 8.3 to 9.4 log CFU g(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information on the diversity of lactobacilli within an artisanal Sicilian cheese, enabling the identification of 17 strains of Lact. casei, one strain of Lact. delbrueckii and Ped. pentosaceus through the combined use of RAPD PCR, PFGE and 16S rDNA sequencing.     

Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates

AIMS: The overall growth kinetics of four potentially probiotic strains (Lactobacillus fermentum, Lact. reuteri, Lact. acidophilus and Lact. plantarum) cultured in malt, barley and wheat media were investigated. The objectives were to identify the main factors influencing the growth and metabolic activity of each strain in association with the cereal substrate. METHODS AND RESULTS: All fermentations were performed without pH control. A logistic-type equation, which included a growth inhibition term, was used to describe the experimental data. In the malt medium, all strains attained high maximum cell populations (8.10-10.11 log10 cfu ml(-1), depending on the strain), probably due to the availability of maltose, sucrose, glucose, fructose (approx. 15 g l(-1) total fermentable sugars) and free amino nitrogen (approx. 80 mg l(-1)). The consumption of sugars during the exponential phase (10-12 h) resulted in the accumulation of lactic acid (1.06-1.99 g l(-1)) and acetic acid (0.29-0.59 g l(-1)), which progressively decreased the pH of the medium. Each strain demonstrated a specific preference for one or more sugars. Since small amounts of sugars were consumed by the end of the exponential phase (17-43%), the decisive growth-limiting factor was probably the pH, which at that time ranged between 3.40 and 3.77 for all of the strains. Analysis of the metabolic products confirmed the heterofermentative or homofermentative nature of the strains used, except in the case of Lact. acidophilus which demonstrated a shift towards the heterofermentative pathway. All strains produced acetic acid during the exponential phase, which could be attributed to the presence of oxygen. Lactobacillus plantarum, Lact. reuteri and Lact. fermentum continued to consume the remaining sugars and accumulate metabolic products in the medium, probably due to energy requirements for cell viability, while Lact. acidophilus entered directly into the decline phase. In the barley and wheat media all strains, especially Lact. acidophilus and Lact. reuteri, attained lower maximum cell populations (7.20-9.43 log10 cfu ml(-1)) than in the malt medium. This could be attributed to the low sugar content (3-4 g l(-1) total fermentable sugar for each medium) and the low free amino nitrogen concentration (15.3-26.6 mg l(-1)). In all fermentations, the microbial growth ceased at pH values (3.73-4.88, depending on the strain) lower than those observed for malt fermentations, which suggests that substrate deficiency in sugars and free amino nitrogen contributed to growth limitation. CONCLUSIONS: The malt medium supported the growth of all strains more than barley and wheat media due to its chemical composition, while Lact. plantarum and Lact. fermentum appeared to be less fastidious and more resistant to acidic conditions than Lact. acidophilus and Lact. reuteri. SIGNIFICANCE AND IMPACT OF THE STUDY: Cereals are suitable substrates for the growth of potentially probiotic lactic acid bacteria.     

Exclusion of vanA, vanB and vanC type glycopeptide resistance in strains of Lactobacillus reuteri and Lactobacillus rhamnosus used as probiotics by polymerase chain reaction and hybridization methods

Strains of Lactobacillus reuteri and Lact. rhamnosus are used as probiotics in man and animal. The aim of this study was to determine whether the glycopeptide resistance in these lactobacilli has a similar genetic basis as in enterococci. Five Lact. reuteri strains and one Lact. rhamnosus, as well as four Enterococcus control strains, were probed for the vanA gene cluster, the vanB gene and the vanC gene by PCR and Southern hybridization, and DNA/DNA hybridization. Their resistance and plasmid patterns were also investigated. All Lactobacillus strains were resistant to vancomycin but susceptible to a broad range of antibiotics. Four of the Lactobacillus strains (including the Lact. rhamnosus strain) did not harbour any plasmid and two of them contained five and 6 plasmid bands respectively. None of the Lactobacillus strains possessed the vanA, vanB or vanC gene. These findings indicate that the glycopeptide resistance of the Lactobacillus strains analysed is different from the enterococcal type. The study provides reassurance on the safety of the Lactobacillus strains used as probiotics with regard to their vancomycin resistance.