Rapid identification of Lactobacillus nantensis, Lactobacillus spicheri and Lactobacillus hammesii species using species-specific primers.

Based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR), an identification tool for rapid differentiation of Lactobacillus nantensis, Lactobacillus spicheri and Lactobacillus hammesii, species isolated recently from French sourdough was developed. The DNA fragments containing ISRs were amplified with primers pairs 16S/p2 and 23S/p7. Clone libraries of the PCR-amplified rDNA with these primers were constructed using a pCR2.1 TA cloning kit and sequenced. The DNA sequences obtained were analyzed and species-specific primers were designed from these sequences. Two PCR amplicons, which were designated small ISR (S-ISR) and large ISR (L-ISR), were obtained for all Lactobacillus species studied. The L-ISR sequence reveale2d the presence of two tRNA genes, tRNAAla and tRNAIle. Species-specific primers designed allowed rapid identification of these species. The specificity of these primers was positively demonstrated as no response was obtained for more than 200 other species tested.

A one-step reaction for the rapid identification of Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16S-23S rRNA intergenic sequences.

AIMS: Species-specific primers targeting the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR) were designed to rapidly discriminate between Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti species recently isolated from French sourdough. METHODS AND RESULTS: The 16S-23S ISRs were amplified using primers 16S/p2 and 23S/p7, which anneal to positions 1388-1406 of the 16S rRNA gene and to positions 207-189 of the 23S rRNA gene respectively, Escherichia coli numbering (GenBank accession number V00331). Clone libraries of the resulting amplicons were constructed using a pCR2.1 TA cloning kit and sequenced. Species-specific primers were designed based on the sequences obtained and were used to amplify the 16S-23S ISR in the Lactobacillus species considered. For all of them, two PCR amplicons, designated as small ISR (S-ISR) and large ISR (L-ISR), were obtained. The L-ISR is composed of the corresponding S-ISR, interrupted by a sequence containing tRNA(Ile) and tRNA(Ala) genes. Based on these sequences, species-specific primers were designed and proved to identify accurately the species considered among 30 reference Lactobacillus species tested. CONCLUSIONS: Designed species-specific primers enable a rapid and accurate identification of L. mindensis, L. paralimentarius, L. panis, L. pontis and L. frumenti species among other lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed method provides a powerful and convenient means of rapidly identifying some sourdough lactobacilli, which could be of help in large starter culture surveys.

[Arabian variant of Kenny syndrome: a familial case in Tunisia]. / Variant arabe du syndrome de Kenny: à propos d'une famille maghrébine.

Kenny syndrome is rare. Clinical feature include severe dwarfism, growth retardation macrocephaly, episodic hypocalcemia, internal cortical thickening and medullary stenosis of tubular bones. Genetic and phenotypic polymorphisms are characteristic. We report the observation of a Tunisian girl with the arabic variant of Kenny syndrome. She had chronic hypoparathyroidism, classic dwarfism, short stature with hormone deficiency, mental retardation and low helper/suppressor ratio. Our patient had two sisters and one brother with the same dysmorphic face and a marked intra-uterine growth retardation. They died from severe infections. Hypoparathyroidism was established in one sister.

Rehabilitation of El Yahoudia dumping site, Tunisia.

As in all developing countries, cities in Tunisia face serious problems of environmental pollution caused mainly by the inadequate and inefficient final disposal of their generated solid wastes. The Tunisian government launched a development program including the construction of landfills in the main cities and the closure of the contaminated sites issued from solid wastes landrising practice. The project of the Henchir El Yahoudia landfill restoration is the first experience in this programme. It has been suggested to convert the site to a green park and to implement an ornamental plant nursery. The whole surface of the landfill is approximately 100 ha from which 30 ha have been already transformed to an urban recreational area and the remaining 70 ha have to be characterized for the project extension. A field investigation by boring was conducted in order to define the geological and the hydrogeological conditions, the vertical and horizontal wastes layer extension, content and degree of decomposition and the composition and quantities of leachate and landfill gas. Representative samples of waste, soil, groundwater and leachate were collected for laboratory analyses. Several of these borings were converted to piezometers to define the flow regime in the site. The results showed that the biogas (CH4, H2S, and CO2), leachate and waste, distribution in the site is mainly affected by the temporal variation of the site operating method. The underlying fissured clay layer facilitated leachate infiltration into the groundwater where high BOD, COD and nitrogen concentrations were registered.

Glutamate uptake in Lactobacillus delbrueckii subsp. bulgaricus CNRZ 208 and its enhancement by a combination of Mn2+ and Mg2+.

AIMS: To demonstrate the mechanism of glutamate uptake in the dairy strain Lactobacillus delbrueckii subsp. bulgaricus CNRZ 208, and to characterize key aspects of the system. METHODS AND RESULTS: Glutamate uptake proceeded via an active transport system requiring an exogenous source of energy. The system also transported aspartate and glutamine. It was unique, with a Kt of 2.8 micro mol l-1 and a Vmax of 900 micro mol s-1 (g dry weight)-1. The activity was optimal at pH 7.3 and 50 degrees C, was independent of the glutamate charge, and was enhanced by Mn2+ + Mg2+ in combination. Inhibition of the activity by uncouplers and ionophores showed that transport was driven by an ATP-dependent mechanism involving the proton-motive force. This inhibition was partially abolished in the presence of both Mn2+ and Mg2+. CONCLUSIONS: We demonstrated for the first time that an active transport system governs the uptake of the essential amino acid glutamate in Lact. delbrueckii subsp. bulgaricus CNRZ 208, the activity of which is enhanced by a combination of Mn2+ and Mg2+. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the growth of Lact. delbrueckii subsp. bulgaricus in mixed-strain cultures for the dairy industry.

Characterization of amino acid transport in the dairy strain Leuconostoc mesenteroides subsp. mesenteroides CNRZ 1273.

AIMS: To identify and characterize amino acid transport in Leuconostoc mesenteroides. METHODS AND RESULTS: The transport of labelled amino acids was measured in whole cells of Leuc. mesenteroides CNRZ 1273. Systems were operative under physiological conditions of growth, energy dependent and differed from peptide transport. Some of the systems were shared by several amino acids. Kinetic analysis indicated the presence of three transport systems with very high (VH), high (H) and low affinity (H) for the 11 amino acids studied. The K(t) values (micromol l(-1)) ranged from 0.088 to 0.815 (VH), 6-390 (H) and 320-4500 (L) and the V(max) values [nmol s(-1) (g dry weight)(-1)] from 0.015 to 0.8 (VH), 15-95 (H) and 90-470 (L). CONCLUSIONS: The study showed the presence of three transport systems in Leuc. mesenteroides for all amino acids tested, some of them being shared by several amino acids. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings are discussed with reference to the growth of Leuc. mesenteroides in milk as pure or in mixed-strain culture with Lactococcus lactis.

Lactococcin MMFII, a novel class IIa bacteriocin produced by Lactococcus lactis MMFII, isolated from a Tunisian dairy product.

A novel bacteriocin, lactococcin MMFII, produced by Lactococcus lactis MMFII isolated from a Tunisian dairy product had been identified. The bacteriocin was purified to homogeneity from fresh overnight M17 broth culture by sulfate ammonium precipitation, cation-exchange chromatography, sep-pack chromatography and two steps of reverse-phase chromatography. The purified bacteriocin was heat stable, pH resistant and protease sensitive. Its amino acid sequence, obtained by Edman degradation, revealed a 37-amino acid peptide with two cysteine residues in positions 9 and 14 and a calculated mass of 4144.6 Da. Laser desorption mass spectrometry analysis gave a molecular mass of 4142.6, suggesting the presence of a disulfide bond within the purified bacteriocin. Lactococcin MMFII contains the N-terminal YGNGV consensus motif and is active against Listeria. Thus, it belongs to the class IIa bacteriocins figuring the first example of such a bacteriocin produced by a lactococcal strain.

Chemical synthesis, molecular modeling, and antimicrobial activity of a novel bacteriocin, MMFII.

A new antimicrobial peptide, referred to as MMFII, was purified to homogeneity from lactic acid bacteria Lactococcus lactis, which were isolated from Tunisian dairy product. The complete amino acid sequence of the peptide has been established by amino acid analysis, Edman sequencing, and mass spectrometry and verified by solid-phase chemical synthesis. MMFII is a single-chain 37-residue polypeptide containing a single intramolecular disulfide bond, i.e., TSYGNGVHCNKSKCWIDVSELETYKAGTVSNPKDILW. It shares ca. 35% sequence identity with Leucocin A, a class IIa bacteriocin. Modeling based on the 3-D of Leucocin A shows three beta strands located in the N-terminal region (Thr1-Tyr3, Val7-Asn10, Lys13-Ile16) and an alpha helical domain from Asp17 to Asn31. When plotted as an alpha-helical wheel, the central alpha-helix of MMFII does not exhibit an amphipathic helical structure. The synthetic MMFII (sMMFII), obtained by the solid-phase method, was shown to be indistinguishable from the natural peptide. sMMFII is active against Lactococcus cremoris and Listeria ivanovii bacteria, whereas no activity was detected for any of the synthetic N-terminal truncated MMFII analogs Cys9-Trp37, Trp15-Trp37, and Val18-Trp37.

[Epidemiologic data in renal lithiasis in adults]. / Données épidémiologiques de la lithiase rénale chez l'adulte.

The morpho-costitutional analysis of 574 urinary lithiasis emitted by tunisean adults permitted to define an épidemiology's profile. This resemble to the épidemiology's profile of under-developed conry: Amore raised frequency of the renal lithiasis at the man than at the woman with a sec ratio of 2.4. An average age of +14 years with a peak to 4th decade in 2 sexes. The upper localitation of the calculi is founded in 94% cases. The fréquency of the relapses, the mode of expulsion and the size of calculi are différent of those published in the litérature. Probably because the time of study which last 4 years is too short, so it don't enable us to find a result like the literature. The surgery is the mode of most fréquent élimination (51%). This s dû to the présence great size calculi in our popûlation and to the récent introduction of the lithotritie in our country.

Valine transport and biodiversity of Leuconostoc wild strains from French raw milk cheeses.

The rate of L-valine transport in whole cells of Leuconostoc was at the maximum at 30 degrees C, pH 6.0 in the presence of an energy source. Transport was inhibited by 40-55%, in the presence of the ionophores (valinomycin, nigericin or monensin), and uncouplers (carbonyl cyanide-m-chloro-phenylhydrazone or 2,4-dinitrophenol) confirming the previously described delta p-driven branched-chain amino acid transport system described in cytoplasmic membranes (Winters et al., 1991, Appl. Environ. Microbiol., 57, 3350-3354). Sulfhydryl group reagents (p-chloro-mercuribenzoate, iodoacetate and N-ethyl maleimide) all inhibited valine transport by 60-70%, indicating that valine is actively transported at high valine concentration. Three kinetically distinguishable transport systems were identified for each strain using whole cells, confirming results obtained with membranes. L-valine transport Kt and Vmax could be an additional tool to estimate the biodiversity of 18 Leuconostoc strains belonging to the dominant flora of French raw milk cheeses. Kt values varied from 20 to 510 nmol/l for the very high affinity system, from 26 to 427 pmol/l for the high affinity system and from 0.65 to 4.40 mmol/l for the low affinity system. No correlation existed between valine transport rates and a particular strain's ability to acidify milk or complex media, suggesting that valine transport is not a growth-limiting function in species of the genus Leuconostoc.

Na-Stimulated Transport of l-Methionine in Brevibacterium linens CNRZ 918.

The transport of l-methionine by the gram-positive species Brevibacterium linens CNRZ 918 is described. The one transport system (K(m) = 55 muM) found is constitutive for l-methionine, stereospecific, and pH and temperature dependent. Entry of l-methionine into cells is controlled by the internal methionine pool. Competition studies indicate that l-methionine and alpha-aminobutyric acid share a common carrier for their transport. Neither methionine derivatives substituted on the amino or carboxyl groups nor d-methionine was an inhibitor, whereas powerful inhibition was shown by l-cysteine, s-methyl-l-cysteine, dl-selenomethionine and dl-homocysteine. Sodium plays important and varied roles in l-methionine transport by B. linens CNRZ 918: (i) it stimulates transport without affecting the K(m), (ii) it increases the specific activity (on a biomass basis) of the l-methionine transport system when present with methionine in the medium, suggesting a coinduction mechanism. l-Methionine transport requires an exogenous energy source, which may be succinic, lactic, acetic, or pyruvic acid but not glucose or sucrose. The fact that l-methionine transport was stimulated by potassium arsenate and to a lesser extent by potassium fluoride suggests that high-energy phosphorylated intermediates are not involved in the process. Monensin eliminates stimulation by sodium. Gramicidin and carbonyl cyanide-m-chlorophenylhydrazone act in the presence or absence of Na. N-Ethylmaleimide, p-chloromercurobenzoate, valinomycin, sodium azide, and potassium cyanide have no or only a partial inhibitory effect. These results tend to indicate that the proton motive force reinforced by the Na gradient is involved in the mechanism of energy coupling of l-methionine transport by B. linens CNRZ 918. Thus, this transport is partially similar to the well-described systems in gram-negative bacteria, except for the role of sodium, which is very effective in B. linens, a species adapted to the high sodium levels of its niche.

Influence of Oxygen and pH on Methanethiol Production from l-Methionine by Brevibacterium linens CNRZ 918.

The effects of dissolved oxygen concentration and pH on the growth of Brevibacterium linens CNRZ 918 and its production of methanethiol from l-methionine were investigated. Optimal specific methanethiol production was obtained at 25% saturation of dissolved oxygen and at a pH between 8 and 9, whereas optimal cell growth occurred at 50% oxygen saturation and when the pH was maintained constantly at 7. Methanethiol production by nonproliferating bacteria required the presence of l-methionine (7 mM) in the culture medium. This was probably due to the induction of enzyme systems involved in the process. The intracellular concentration of l-methionine seemed to play a key role in this process. B. linens CNRZ 918 tolerated alkaline pHs with a maximal growth pH of approximately 9. Its orange pigmentation seemed to depend on the presence of l-methionine in the culture medium and on the concentration of dissolved oxygen.

Production of methanethiol from methionine by Brevibacterium linens CNRZ 918.

The conditions under which Brevibacterium linens CNRZ 918, a strain isolated from the surface smear flora of Gruyère de Comté cheese, produced methanethiol from methionine were studied. Demethiolation was estimated from the methanethiol production capacity of resting cells. Methionine was demethiolated mainly during the exponential growth phase of the organism during which time the cells were rod-shaped and had a generation time of 5 h, and the medium became alkaline. At the end of growth (pH 9) the cells were coccoid, and produced only very little methanethiol. The production of methanethiol required the presence of methionine in the culture medium, this reflecting the probable induction of the enzyme systems involved. Glucose favoured growth and inhibited production of methanethiol. Lactate favoured both growth and methanethiol production. Resting rod cells also produced methanethiol from structural analogues of methionine and from methionine-containing peptides. The apparent kinetic constants of the production of methanethiol for rod and coccoid cells were respectively Km = 14 mM and 46 mM, Vmax = 208 nkat g-1 and 25 nkat g-1. The optimum temperature and pH for production were 30 degrees C and pH 8. Azide or malonate favoured the production of methanethiol by resting cells, whereas chloramphenicol had no effect.