Complete genome sequence of Lactobacillus delbrueckii subsp. bulgaricus strain ND02

Lactobacillus delbrueckii subsp. bulgaricus strain ND02 is a Chinese commercial dairy starter used for the manufacture of yoghurt. It was isolated from naturally fermented yak milk in Qinghai, China. Here, we report the main genome features of ND02 and several differences with two other published genomes of Lactobacillus delbrueckii subsp. bulgaricus strains.     

Capillary zone electrophoresis for enumeration of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in yogurt

Enumeration of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus is a priority due to their importance in yogurt production. Capillary electrophoresis (CE) of both bacteria could be achieved in 7.2 min with a resolution of 3.2 in the background electrolyte (BGE) containing 4.5 mM Tris(hydroxymethyl) amminomethane (TRIS)–4.5 mM boric acid–0.1 mM ethylenediamine tetraacetate (EDTA) (TBE) buffer (pH 8.4) and 0.05% (v/v) polyethylene oxide (PEO), using a capillary of 47.5 cm (effective length) × 100 μm i.d., injection of 50 mbar × 3 s followed by ?5 kV × 120 s, a voltage and temperature of 20 kV and 25 °C, respectively. Appropriate amounts of PEO in the BGE, sample preparation (i.e. vortex) and introduction were key factors for their separation. A short hydrodynamic injection followed by applying reversed polarity voltage could compress the bacteria into narrow zones, which were detected as separated single peaks. Method linearity (r² > 0.99), precision (%RSDs < 9.3%), recovery (%R = 91.7–106.7%) and limit of quantitation (1.0 × 10⁶ colony forming unit per mL (CFU/mL)) were satisfactory. Results from the CE analysis of both bacteria in yogurt were not statistically different from those of the plate count method (P > 0.05). The CE method can be used as an alternative for quantitation of L. delbrueckii subsp. bulgaricus and S. thermophilus in yogurt since it was reliable, simple, cost and labor effective and rapid, allowing the analysis of 3 samples/h (comparing to 2d/sample by plate count method).     

Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis by SDS-PAGE of cell-wall proteins

AIMS: In the present study, a method based on SDS-PAGE fingerprinting of surface layer proteins was developed to identify Lactobacillus delbrueckii subsp. bulgaricus and subsp. lactis dairy isolates. METHODS AND RESULTS: The two subspecies, identified by species-specific PCR, were characterized by different SDS-PAGE cell-wall protein profiles; subspecies bulgaricus showed one band of about 31 kDa which, in some cases, was observed at a doublet, and subspecies lactis showed one band of about 21 kDa or 18 kDa. CONCLUSION: The sensitivity of this procedure for discriminating between the two subspecies was very high. The different types of SDS-PAGE profile for cell-wall proteins of the strains studied in this work did not seem to be correlated to the different dairies of origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The method appears to be an efficient taxonomic tool. It has the advantage of easy gel interpretation over fingerprinting of whole-cell protein extracts, and may be used as an alternative to established PCR-based techniques which, though rapid and safe, require expensive instruments and reagents.     

Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus survive gastrointestinal transit of healthy volunteers consuming yogurt

To date, there is significant controversy as to the survival of yogurt bacteria (namely, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) after passage through the human gastrointestinal tract. Survival of both bacterial species in human feces was investigated by culture on selective media. Out of 39 samples recovered from 13 healthy subjects over a 12-day period of fresh yogurt intake, 32 and 37 samples contained viable S. thermophilus (median value of 6.3 x 10(4) CFU g(-1) of feces) and L. delbrueckii (median value of 7.2 x 10(4)CFU g(-1) of feces), respectively. The results of the present study indicate that substantial numbers of yogurt bacteria can survive human gastrointestinal transit.     

德氏乳杆菌保加利亚亚种电转化平台的构建和优化

德氏乳杆菌保加利亚亚种(Lactobacillus delbrueckiisub sp.bulgaricus)是最具经济价值的乳酸菌之一,在世界上广泛应用于酸奶和其它发酵乳生产。当前对该菌的代谢机制研究甚少。外源基因的转化效率是制约其分子代谢机制研究的重要因素。本研究以pMG36c为材料,对L.delbrueckiisub sp.bulgaricus CH3进行电转化条件研究。结果表明,在电转化过程中,电场强度、质粒的浓度、细胞生长状态均对转化效率有明显影响,得到了该菌株的最适电转化条件为:对数初期的细胞,质粒浓度为100 ng/50μl菌液,在10 kV/cm电场强度下电转化,转化后细胞在复壮培养液中培养3 h后涂布选择性培养基,转化率可达2.6×103CFU/μg DNA。以甘氨酸、醋酸锂、二硫苏糖醇处理细胞壁,发现醋酸锂和二硫苏糖醇共同处理对转化效率有明显改善,可提高转化效率。     

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 10⁴ transformants per μg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii.     

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids.

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 10(4) transformants per microg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii.     

Two-Dimensional Electrophoresis Study of Lactobacillus delbrueckii subsp. bulgaricus Thermotolerance

The response of Lactobacillus delbrueckii subsp. bulgaricus cells to heat stress was studied by use of a chemically defined medium. Two-dimensional electrophoresis (2-DE) analysis was used to correlate the kinetics of heat shock protein (HSP) induction with cell recovery from heat injury. We demonstrated that enhanced viability, observed after 10 min at 65°C, resulted from the overexpression of HSP and from mechanisms not linked to protein synthesis. In order to analyze the thermoadaptation mechanisms involved, thermoresistant variants were selected. These variants showed enhanced constitutive tolerance toward heat shock. However, contrary to the wild-type strain, these variants were poorly protected after osmotic or heat pretreatments. This result suggests that above a certain threshold, cells reach a maximum level of protection that cannot be easily exceeded. A comparison of protein patterns showed that the variants were able to induce more rapidly their adaptive mechanisms than the original strain. In particular, the variants were able to express constitutively more HSP, leading to the higher level of thermoprotection observed. This is the first report of the study by 2-DE of the heat stress response in L. delbrueckii subsp. bulgaricus.     

Two-dimensional electrophoresis study of Lactobacillus delbrueckii subsp. bulgaricus thermotolerance

The response of Lactobacillus delbrueckii subsp. bulgaricus cells to heat stress was studied by use of a chemically defined medium. Two-dimensional electrophoresis (2-DE) analysis was used to correlate the kinetics of heat shock protein (HSP) induction with cell recovery from heat injury. We demonstrated that enhanced viability, observed after 10 min at 65 degrees C, resulted from the overexpression of HSP and from mechanisms not linked to protein synthesis. In order to analyze the thermoadaptation mechanisms involved, thermoresistant variants were selected. These variants showed enhanced constitutive tolerance toward heat shock. However, contrary to the wild-type strain, these variants were poorly protected after osmotic or heat pretreatments. This result suggests that above a certain threshold, cells reach a maximum level of protection that cannot be easily exceeded. A comparison of protein patterns showed that the variants were able to induce more rapidly their adaptive mechanisms than the original strain. In particular, the variants were able to express constitutively more HSP, leading to the higher level of thermoprotection observed. This is the first report of the study by 2-DE of the heat stress response in L. delbrueckii subsp. bulgaricus.     

Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412^T, which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.     

Polymer production by Lactobacillus delbrueckii ssp. bulgaricus

A polymer-forming strain of Lactobacillus delbrueckii ssp. bulgaricus was grown under differing conditions. It was found that at higher temperatures and slower growth the production of the polymer per cell was greater. Polymer-producing ability seems to be unstable with cells losing the phenotype faster at 48 than at 40°C. Specific production of polymer was increased in the presence of hydrolysed casein early in the growth phase when growing in milk, but production of polymer in MRS broth + lactose was reduced compared with milk. Furthermore, addition of hydrolysed casein to MRS did not increase specific production of polymer. Preliminary results suggest that the polymer is a glycoprotein, although the protein may be loosely associated with the carbohydrate.     

Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412(T), which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.     

Acidification improves cryotolerance of Lactobacillus delbrueckii subsp. bulgaricus CFL1

The effect of acidification of the fermented broth at the end of the culture was examined on the growth and the cryotolerance of Lactobacillus bulgaricus CFL1, as a new means to better preserve lactic acid bacteria. Cryotolerance was investigated by evaluating the loss of specific acidification activity during freezing and frozen storage. An experimental design made it possible to determine optimal acidification conditions that improved cryotolerance, such as pH 5.15 for 30min. These conditions were also conducive to high biomass productivity. By considering the type of acid used, H(2)SO(4) enabled us to obtain cells with better cryotolerance, as compared to HCl. It was also observed that increasing the pH after acidification slightly minimised the acid shock, thus improving cryotolerance. Moreover, it was concluded that this improvement was related to a physiological adaptation of L. bulgaricus CFL1 during the 30-min acidification at pH 5.15.     

Screening and selection of exopolysaccharide-producing strains of Lactobacillus delbrueckii subsp. bulgaricus

AIMS: The selection of exopolysaccharide (EPS)-producing strains of Lactobacillus delbrueckii subsp. bulgaricus. METHODS AND RESULTS: Improved EPS-overproducing strains of L. delbrueckii subsp. bulgaricus were derived by chemical mutagenesis and selection. Initial screening of the chemically induced mutant pool relied primarily on the selection of strains with raised levels of lactic acid and reduced biomass formation. Supporting selection criteria used were ropiness and colonial mucoidy. Final screening of candidate strains undertaken in a semi-defined medium in batch culture, resulted in the selection of a mutant with a 35% improvement in specific EPS yield relative to the parent strain. CONCLUSIONS: Initial selection of mutants of L. delbrueckii subsp. bulgaricus on the basis of enhanced formation of lactate and reduced biomass formation, coupled with a ropy or mucoid phenotype, proved to be a satisfactory means of isolating strains with the potential for a higher level of specific EPS production than the parent strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay protocol allowed for the selection of an EPS-overproducing strain of L. delbrueckii subsp. bulgaricus. Such strains are useful for the purposes of metabolic studies related to EPS-production.     

Permeabilization of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus with Ethanol

Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus cultures were treated with ethanol and tested for viability and β-galactosidase activity. Exposure of the biomass of test cultures to 30%–55% ethanol (vol/vol) caused a 100% loss of viability and up to 15-fold increase in measurable β-galactosidase activity in both streptococci and lactobacilli. Ethanol-treated cell suspensions could be stored for up to 6 months without loss of enzyme activity. The nonviable permeabilized biomass of the more active S. thermophilus was used to achieve up to 80% hydrolysis of lactose in aqueous solutions and non-fat milk. Received: 28 July 1997 / Accepted: 30 September 1997     

A new mobile genetic element inLactobacillus delbrueckii subsp.bulgaricus

A new IS element (ISL3) was discovered inLactobacillus delbrueckii subsp.bulgaricus during the characterization of the linkage relationships between the two genes important for milk fermentation,β-galactosidase (lacZ) and the cell-wall associated protease (prtP). ISL3 is a 1494 by element, flanked by 38 by imperfect inverted repeats, and generates an 8 by target duplication upon insertion. It contains one open reading frame, encoding a potential polypeptide of 434 amino acids, which shows significant homology (34% identity) to the transposase of theLeuconostoc mesenteroides element IS1165. Molecular analysis of spontaneouslacZ mutants revealed some strains that had sustained deletions of 7 to 30 kb in size, centered on and eliminating the copy of ISL3 next tolacZ. Other deletion endpoints were identified as located immediately adjacent to ISL3. Furthermore, genetic translocations that had occurred via transposition of ISL3 were observed fortuitously in cultures screened for deletion mutants. ISL3 can be found in one to several copies in various strains ofL. delbrueckii. However, it was not present in other dairy lactic acid bacteria tested.     

Strain dependency of the immunopotentiating activity of Lactobacillus delbrueckii subsp. bulgaricus

To obtain strains of Lactobacillus delbrueckii subsp. bulgaricus with high immunopotentiating activity, we screened 90 strains of this bacterial species for the proliferative response of murine spleen and beta-lactoglobulin-primed lymph node cells. In this screening, certain strains showed strong immunopotentiating activity. Among them, strain 1023 had the strongest mitogenic activity for murine Peyer's patch (PP) cells. Furthermore, strain 1023 induced IgA antibody production by PP cells as strongly as Bifidobacterium longum 6001, which had adjuvant activity when orally administered. Also in the assays using immune cells from human-flora-associated mice a few strains including 1023 showed strong immunopotentiating activity comparable to B. longum 6001. These results suggest that L. delbrueckii subsp. bulgaricus strains such as 1023 may be useful for the production of fermented milk with a more beneficial effect on the systemic and mucosal immune system.     

Differentiation of Lactobacillus helveticus,Lactobacillus delbrueckii subsp bulgaricus,subsp lactis and subsp delbrueckii using physiological and genetic tools and reclassification of some strains from the ATCC collection

Several physiological tests of glucose metabolism and genetic tools including species specific probes and 16S rDNA sequences were used to identify strains of L. helveticus and the group of L. delbrueckii with its three subspecies lactis, bulgaricus, and delbrueckii. These species are important for the milk industry as fermenting lactic acid bacteria. The identification procedure was applied to the different strains of these species available from the ATCC collection and allowed to reclassify part of them.     

Sequence analysis of pLBB1, a cryptic plasmid from Lactobacillus delbrueckii subsp. bulgaricus

The first report of the complete nucleotide sequence of a cryptic plasmid from Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus) is presented. The plasmid pLBB1 consists of 6127 bp with a GC content of 44.8%. No ssDNA was detected by hybridization experiments, which is consistent with the notion that pLBB1 does not replicate by a rolling circle mechanism. A putative replication region of pLBB1 was cloned and found to be functional in Lactobacillus johnsonii and Lactococcus lactis. Plasmid pLBB1 showed significant DNA sequence identity with plasmid pLL1212 from Lactobacillus delbrueckii subsp. lactis (Lactobacillus lactis) CRL1212 (GenBank accession No. AF109691). Four open reading frames (ORFs) larger than 100 amino acids were identified. ORFA shared similarity with a putative primase-helicase system, and ORFB and ORFC exhibited limited identity with a mobilization protein and a transposase, respectively. Curing experiments did not allowed us to assign a function to the ORFs.