Measure of molecular diversity within the Trypanosoma brucei subspecies Trypanosoma brucei brucei and Trypanosoma brucei gambiense as revealed by genotypic characterization

We have evaluated whether sequence polymorphisms in the rRNA intergenic spacer region can be used to study the relatedness of two subspecies of Trypanosoma brucei. Thirteen T. brucei isolates made up of 6 T. b. brucei and 7 T. b. gambiense were analyzed using restriction fragment length polymorphism (RFLP). By PCR-based restriction mapping of the ITS1-5.8S-ITS2 ribosomal repeat unit, we found a fingerprint pattern that separately identifies each of the two subspecies analyzed, with unique restriction fragments observed in all but 1 of the T. b. gambiense "human" isolates. Interestingly, the restriction profile for a virulent group 2 T. b. gambiense human isolate revealed an unusual RFLP pattern different from the profile of other human isolates. Sequencing data from four representatives of each of the two subspecies indicated that the intergenic spacer region had a conserved ITS-1 and a variable 5.8S with unique transversions, insertions, or deletions. The ITS-2 regions contained a single repeated element at similar positions in all isolates examined, but not in 2 of the human isolates. A unique 4-bp [C(3)A] sequence was found within the 5.8S region of human T. b. gambiense isolates. Phylogenetic analysis of the data suggests that their common ancestor was a nonhuman animal pathogen and that human pathogenicity might have evolved secondarily. Our data show that cryptic species within the T. brucei group can be distinguished by differences in the PCR-RFLP profile of the rDNA repeat.     

Partial characterization of a bacteriocin produced by Lactobacillus helveticus

AIMS: To investigate the antimicrobial activity of a strain of Lactobacillus helveticus. METHODS AND RESULTS: The culture supernatant fluid Lact. helveticus G51 showed antimicrobial activity against thermophilic strains of Lactobacillus. Purification of the active compound was achieved after gel filtration and ion exchange chromatography. As revealed by SDS-PAGE, active fractions were relatively homogeneous, showing a protein with a molecular mass of 12.5 kDa. The antimicrobial compound was heat labile, inactivated by proteolytic enzymes and had a bactericidal mode of action. CONCLUSION: The antimicrobial activity expressed by Lact. helveticus G51 was correlated with the production of a bacteriocin with properties that were different to other helveticins. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has provided further data on Lact. helveticus bacteriocins. The strong activity of the bacteriocin towards various thermophilic lactobacilli warrants further investigation for its potential to obtain attenuated cultures for the enhancement of the cheese-ripening process.     

Biodiversity among Lactobacillus helveticus strains isolated from different natural whey starter cultures as revealed by classification trees

Lactobacillus helveticus is a homofermentative thermophilic lactic acid bacterium used extensively for manufacturing Swiss type and aged Italian cheese. In this study, the phenotypic and genotypic diversity of strains isolated from different natural dairy starter cultures used for Grana Padano, Parmigiano Reggiano, and Provolone cheeses was investigated by a classification tree technique. A data set was used that consists of 119 L. helveticus strains, each of which was studied for its physiological characters, as well as surface protein profiles and hybridization with a species-specific DNA probe. The methodology employed in this work allowed the strains to be grouped into terminal nodes without difficult and subjective interpretation. In particular, good discrimination was obtained between L. helveticus strains isolated, respectively, from Grana Padano and from Provolone natural whey starter cultures. The method used in this work allowed identification of the main characteristics that permit discrimination of biotypes. In order to understand what kind of genes could code for phenotypes of technological relevance, evidence that specific DNA sequences are present only in particular biotypes may be of great interest.     

Molecular diversity among marine picophytoplankton as revealed by psbA analyses

Photosynthetic microorganisms play a crucial role in the marine environment. In vast areas of the oceans, marine primary productivity is performed by cells smaller than 2-3 micro m (picoplankton). Here, we report on molecular analyses of the conserved photosynthetic psbA gene (coding for protein D1 of photosystem II reaction centre) as a diversity indicator of naturally occurring marine oxygenic picophytoplankton. The psbA genes proved to be good indicators of the presence of a wide variety of photosynthetic marine microbial groups, including new cyanobacterial groups and eukaryotic algae (prasinophytes). Furthermore, using environmental bacterial artificial chromosome (BAC) libraries, we were able to correlate psbA genes with small subunit rRNAs and, therefore, to confirm their phylogenetic affiliation.     

Plasmids from Lactobacillus helveticus: distribution and diversity among natural isolates

AIMS: To investigate the distribution and the level of diversity of extrachromosomal molecules in Lactobacillus helveticus strains in relation to their different ecological niches. METHODS AND RESULTS: The plasmid profile of 22 Lact. helveticus strains, isolated from five different Italian cheeses, was determined. Among the tested strains, there was a variable presence of plasmids: eight plasmid-free strains and the remaining with several plasmids that could be differentiated on the basis of number and molecular weight. The profiles showed between one and five plasmid bands, which size ranged between 2.3 and 31 kb. Four of these plasmids were further analyzed by restriction digestion and compared with the plasmids from Lact. helveticus ATCC 15009(T). Analyses and comparison of their primary structures and hybridization experiments revealed the presence of different DNA homology groups. CONCLUSIONS: This study indicates that within Lact. helveticus species, there is a high degree of variability in relation to the presence of plasmid molecules. Moreover, the structural diversity found among some of these plasmids allows to hypothesize the presence of different evolutionary lineages. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies on plasmid distribution and diversity should be considered as an essential component in a continuing effort to explore microbial diversity as well as to understand the real role of plasmids in the flow of genetic information in natural bacterial communities.     

Exopolysaccharide biosynthesis by Lactobacillus helveticus ATCC 15807

Exopolysaccharide (EPS) production and the activities of the enzymes involved in sugar nucleotide biosynthesis in Lactobacillus helveticus ATCC 15807 under controlled pH conditions were investigated. Batch fermentations using lactose as energy source showed higher EPS synthesis by L. helveticus ATCC 15807 at pH 4.5 with respect to pH 6.2, the enzyme -phosphoglucomutase (-PGM) being correlated with both total and specific EPS production. When glucose was used as carbon source instead of lactose, the lower EPS synthesis obtained was linked to a decrease in -PGM and galactose 1-phosphate-uridyltransferase (GalT) activities, the reduction of the latter being more pronounced. Higher EPS production by L. helveticus ATCC 15807 at the acidic constant pH of 4.5 requires that both -PGM and GalT activities are high. These enzymes are needed to synthesize UDP-glucose and UDP-galactose for supplying the corresponding monomers for EPS biosynthesis. Although differences are observed in EPS production by this strain regarding the energy source (lactose or glucose), the monomeric composition of the polymers produced is independent of the carbohydrate used. The obtained results contribute to a better understanding of the physiological factors that affect EPS biosynthesis by lactobacilli, which could help in the correct handling of the fermentation parameters within the fermented dairy industry.     

Cholesterol assimilation and biotransformation by Lactobacillus helveticus

Lactobacillus helveticus, grown at 37°C in MRS medium supplemented with 3 mM cholesterol, assimilated all the cholesterol in 42 h having 68 U mg⁻¹ of intracellular cholesterol oxidase activity. The strain transformed 1 g cholesterol to 0.05 g of androsta-1, 4-diene-3, 17-dione and 0.04 g of androst-4-ene-3, 17 dione within 48 h at 37°C with extracellular cholesterol oxidase activity at 12 U mg⁻¹ and intracellular oxidase at 0.5 U mg⁻¹.     

Physicochemical characterization of phage adsorption to Lactobacillus helveticus ATCC 15807 cells

Characterization of the adsorption process by the phages hv and ATCC 15807-B1 to Lactobacillus helveticus ATCC 15807 was carried out. For this purpose, the influence of Ca²⁺ ions, temperature and physiological cell state were studied. The ability of several saccharides and related compounds to inactivate the phages hv and ATCC 15807-B1 was determined to investigate their potential role as phage receptors. Furthermore, several chemical treatments on the sensitive strain cells were carried out to study their influence on phage adsorption. Cell lysis and plaque formation were independent of Ca²⁺ ions for phage hv, but the cation was indispensable for completion of the lytic cycle of phage ATCC 15807-B1. However, for this phage, Ca²⁺ was not necessary for the adsorption process. The adsorption rates were almost normal for both phages within the temperature range examined (0 – 50 °C) and the adsorption kinetics were practically identical on viable and non-viable cells. The saccharides and related compounds used did not produce inactivation of the phages, suggesting that they were not essential components of phage receptor structures. Lactobacillus helveticus ATCC 15807 cells treated with SDS 1%, SDS 0·5% -EDTA 50 mmol l⁻¹ or NaOH 50 mmol l⁻¹ exhibited reduced adsorption of the phages, indicating possible damage or extraction of receptors from the cell wall. Phage adsorption presents an extremely attractive target for interfering in the lytic cycle of phages.     

Polyphasic investigation of the diversity within Lactobacillus plantarum related strains revealed two L. plantarum subgroups

The diversity of 140 strains related to Lactobacillus plantarum was investigated using a polyphasic approach combining two molecular techniques: randomly amplified polymorphic DNA fingerprinting (RAPD) and Southern hybridisation with a pyr probe on BglI digests of chromosomal DNA, as well as phenotypic characterization. The RAPD technique allowed us to classify a subset of 60 representative strains into four groups. One group belonged to Lactobacillus paraplantarum, the second to Lactobacillus pentosus and the two remaining groups to L. plantarum (G(L)p1 and G(L)p2). The Southern hybridisation technique (F. Bringel, M.-C. Curk and J.-C. Hubert, Int. J. Syst. Bacteriol. 46: 588-594, 1996) revealed nine groups of profiles (I to IX). Results indicated an excellent convergence between RAPD and hybridisation classifications for more than 93% (56/60) of the strains studied. When we compared the fermentation patterns of the L. plantarum strains, three differences were found. Melezitose fermentation was not fermented by the G(L)p2 RAPD group, unlike the G(L)p1 RAPD group which included L. plantarum type strain NCIMB11974T. Second, alpha-methyl-D-mannoside was fermented by a majority of the strains of the G(L)p1 RAPD group but by none of the strains in the G(L)p2 RAPD group. Third, dulcitol was catabolized by nearly half of the strains of the G(L)p2 RAPD group but by none of the strains in the G(L)p1 RAPD group. Molecular diversity within L. plantarum was confirmed using Southern profiles, PCR amplification and subsequent sequencing of these PCR products. A 773 bp sequence overlapping the pyrDF genes showed high homology: at least 97% identical in L. plantarum strains (V to IX) and 99.9% identical in hybridisation groups VII and VIII. The same G-T transversion which destroyed the pyrF BglI site was found in 11 strains (hybridisation groups VI, VII and VIII). DNA rearrangements were identified downstream from the pyr genes, by PCR amplification and Southern hybridisation profile analysis in three strains of hybridisation groups VIII and IX, two of which also harboured the G-T transversion.     

Molecular cloning,purification, and characterization of a novel thermostable cinnamoyl esterase from Lactobacillus helveticus KCCM 11223

A gene encoding cinnamoyl esterase (CE), which breaks down chlorogenic acid (ChA) into caffeic and quinic acids, was cloned from Lactobacillus helveticus KCCM 11223. The gene with an open reading frame of 759 nucleotides was expressed in Escherichia coli, which resulted in a 51.6-fold increase in specific activity compared to L. helveticus KCCM 11223. The recombinant CE exists as a monomeric enzyme having a molecular weight of 27.4?kDa. Although the highest activity was observed at pH 7, the enzyme showed stable activity at pH 4.0–10.0. Its optimum temperature was 65°C, and it also possessed a thermophilic activity: the half-life of CE was 24.4?min at 65°C. The half-life of CE was 145.5, 80.5, and 24.4?min at 60, 62, and 65°C, respectively. The K_m and V_max values for ChA were 0.153?mM and 559.6?µM/min, respectively. Moreover, the CE showed the highest substrate specificity with methyl caffeate among other methyl esters of hydroxycinnamic acids such as methyl ferulate, methyl sinapinate, methyl p-coumarate, and methyl caffeate. Ca²⁺, Cu²⁺, and Fe²⁺ significantly reduced the relative activity on ChA up to 70%. This is the first report on a thermostable CE from lactic acid bacteria that can be useful to hydrolyze ChA from plant cell walls.     

Lactobacillus helveticus heterogeneity in natural cheese starters: the diversity in phenotypic characteristics

The study of wild strains from natural habitats is a useful means of understanding better the heterogeneity within a species of biotechnological importance, and of obtaining atypical isolates with unknown capabilities. In the present research carried out on different Lactobacillus helveticus strains isolated from natural cheese starters, it was observed that several biotechnologically important characteristics can differ greatly between strains. Biotypes were found which differ in terms of fructose, maltose and trehalose fermentation, acidifying activity, proteolytic and peptidase activity, and antibiotic and lysozyme resistance. The possibility of choosing Lact. heleveticus strains with specific biotechnological profiles will influence the quality and the variety of dairy products.     

Lactobacillus helveticus heterogeneity in natural cheese starters: the diversity in phenotypic characteristics

The study of wild strains from natural habitats is a useful means of understanding better the heterogeneity within a species of biotechnological importance, and of obtaining atypical isolates with unknown capabilities. In the present research carried out on different Lactobacillus helveticus strains isolated from natural cheese starters, it was observed that several biotechnologically important characteristics can differ greatly between strains. Biotypes were found which differ in terms of fructose, maltose and trehalose fermentation, acidifying activity, proteolytic and peptidase activity, and antibiotic and lysozyme resistance. The possibility of choosing Lact. heleveticus strains with specific biotechnological profiles will influence the quality and the variety of dairy products.     

Identification and characterization of helveticin V-1829, a bacteriocin produced by Lactobacillus helveticus 1829

Lactobacillus helveticus 1829 produced an antimicrobial agent, designated helveticin V-1829, that demonstrated antagonistic activity against closely-related species. The agent was excreted into MRS agar, and was present in the supernatant fluids from both overnight broth and clotted milk cultures. It was heat labile (inactivated by 50°C for 30 min) and was stable over the pH range 2.5 to 6.5. Production of the substance was pH-dependent and maximum yields were obtained in MRS broth cultures maintained at pH 5.5. Helveticin V-1829 was partially purified following growth of the producing strain in a semi-defined MRS medium and precipitating the cell-free filtrate with ammonium sulphate to 30% saturation. The cleared supernatant fluid was then brought to 60% saturation and the resulting precipitate pelleted and dialysed in 0.3 mol/l phosphate buffer. The partially purified inhibitor was sensitive to several proteolytic enzymes, and it was bactericidal in its mode of action against indicator cells of Lact. helveticus 1844 and Lact. delbrueckii subsp. bulgaricus 1489, indicating that it was a bacteriocin. A DNA probe specific for the helveticin J structural gene failed to hybridize to total genomic DNA of Lact. helveticus 1829, indicating that helveticin V-1829 is not significantly related to helveticin J.     

Molecular cloning and expression of the nucleoside deoxyribosyltransferase-II gene from Lactobacillus helveticus

Nucleoside deoxyribosyltransferase-II, which catalyzes transfer of glycosyl residues from a donor deoxynucleoside to an acceptor base, was purified from Lactobacillus helveticus and its gene was cloned. Analysis of the nucleotide sequence showed the presence of a 474-nucleotide open reading frame encoding a protein of 158 amino acids with a molecular weight of 18,317. The active enzyme can be produced in large quantities in E. coli cells using the cloned gene.     

Identification and characterization of helveticin V-1829, a bacteriocin produced by Lactobacillus helveticus 1829.

Lactobacillus helveticus 1829 produced an antimicrobial agent, designated helveticin V-1829, that demonstrated antagonistic activity against closely-related species. The agent was excreted into MRS agar, and was present in the supernatant fluids from both overnight broth and clotted milk cultures. It was heat labile (inactivated by 50 degrees C for 30 min) and was stable over the pH range 2.5 to 6.5. Production of the substance was pH-dependent and maximum yields were obtained in MRS broth cultures maintained at pH 5.5. Helveticin V-1829 was partially purified following growth of the producing strain in a semi-defined MRS medium and precipitating the cell-free filtrate with ammonium sulphate to 30% saturation. The cleared supernatant fluid was then brought to 60% saturation and the resulting precipitate pelleted and dialysed in 0.3 mol/l phosphate buffer. The partially purified inhibitor was sensitive to several proteolytic enzymes, and it was bactericidal in its mode of action against indicator cells of Lact. helveticus 1844 and Lact. delbrueckii subsp. bulgaricus 1489, indicating that it was a bacteriocin. A DNA probe specific for the helveticin J structural gene failed to hybridize to total genomic DNA of Lact. helveticus 1829, indicating that helveticin V-1829 is not significantly related to helveticin J.     

Molecular phylogenetic convergence within Elasmobranchii revealed by cytochrome oxidase subunits

The phylogenetic relationships within the subclass of Elasmobranchii are under question within the academic community and their systematic classification based on morphological, or physiological characteristics has not yet been fully justified. Modern cladistic studies suggested that batoids are derived sharks, a taxonomic status known as the Hypnosqualean hypothesis. The main purpose of this study was to address this issue using a data set of aligned, directly sequenced, mitochondrial cytochrome oxidase subunits I and II. Maximum Likelihood, Maximum Parsimony, Minimum Evolution and Bayesian inference were implemented for tree reconstructions. The results provided evidence that supported the rejection of the above hypothesis, in accordance with other recent molecular phylogenetic studies. More specifically Rajiformes species were presented as separate lineages from sharks. Prionace species on the other hand was grouped within Carcharhinoformes, which was clustered as sister group to Lamniformes. COI and COII regions supported, monophylies of Squaliformes and paraphylies of Carchariniformes.