Three new insertion sequence elements ISLdl2, ISLdl3, and ISLdl4 in Lactobacillus delbrueckii: isolation,molecular characterization,and potential use for strain identification

A group of new insertion sequence (IS) elements, ISLdl2, ISLdl3, and ISLdl4, from Lactobacillus delbrueckii subsp. lactis ATCC 15808 was isolated, characterized, and used for strain identification together with ISLdl1, recently characterized as an L. delbrueckii IS element belonging to the ISL3 family. ISLdl2 was 1367 bp in size and had a 24 bp IR and an 8 bp DR. The single ORF of ISLdl2 encoded a protein of 392 aa similar to transposases of the IS256 family. ISLdl3 had a single ORF encoding a protein of 343 aa similar to transposases of the IS30 family. Finally, ISLdl4 had a single ORF encoding a protein of 406 aa and displayed homology to the transposases of the IS110 family. ISLdl4 was only slight different from ISL4 (Accession No. AY040213). ISLdl1, ISLdl2, and ISLdl4 were present in all of the 10 L. delbrueckii subsp. lactis and subsp. delbrueckii strains tested, as well as in three of the 11 L. delbrueckii subsp. bulgaricus strains tested. ISLdl3 was present only in four closely related strains of L. delbrueckii subsp. lactis. These IS elements were not observed in Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus helveticus, or Lactobacillus plantarum. A cluster of IS elements, ISLdl1, ISLdl2, ISLdl3, ISLdl4, and ISL6, was observed in L. delbrueckii subsp. lactis strain ATCC 15808. Within this cluster, ISLdl4 was inserted into ISLdl1 between the left IR and the start codon of ORF455, encoding a putative transposase. Most of the integration sites of the IS elements were strain-specific. We have observed that IS elements can migrate from one strain to another as integral parts of bacterial DNA by using phage LL-H as a vehicle. We demonstrate for the first time that inverse PCR and vectorette PCR methods with primers based on sequences of the IS elements could be used for identification of L. delbrueckii strains.     

Effective plasmid pX3 transduction in Lactobacillus delbrueckii by bacteriophage LL-H

High-frequency plasmid transductions in Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus strains mediated by pac-type bacteriophages were observed and further investigated. The frequency of plasmid transduction by phages LL-H and LL-S attained levels of from 0.10 to about 1 with plasmid pX3, but only about 2 × 10⁻² with plasmid pJK650. Infection of L. delbrueckii subsp. lactis strain LKT(pX3) or ATCC 15808(pX3) with phage LL-H resulted in intensive concatemerization of plasmid pX3, and most progeny phage particles contained concatemers of plasmid DNA instead of phage LL-H DNA. The synthesis of phage LL-H DNA was depressed. No evident homology or recombination was observed between phage LL-H DNA and plasmid pX3. The unusually high frequency of plasmid pX3 transduction by phage LL-H could be considered to result from specific interaction(s) between a particular phage and plasmid. These interactions may include pX3-mediated blockage of phage LL-H DNA replication and effective use of a particular pac-like site located about 1 kb from BglII in the smaller NdeI-BglII fragment of plasmid pX3. Phage LL-H together with plasmid vector pX3 could be used as effective plasmid transduction tools for genetic engineering of L. delbrueckii subsp. lactis and subsp. bulgaricus strains.     

A new methodology for rapid detection of Lactobacillus delbrueckii subsp. bulgaricus based on multiplex PCR

In this study we present a novel multiplex PCR assay for rapid and efficient detection of Lactobacillus delbrueckii subsp. bulgaricus. The accuracy of our method was confirmed by the successful identification of L. delbrueckii subsp. bulgaricus in commercial yoghurts and food supplements and it may be readily applied to the food industry.     

Production of l (+) lactic acid by Lactobacillus delbrueckii immobilized in functionalized alginate matrices

The role of functionalized alginate gels as immobilized matrices in production of l (+) lactic acid by Lactobacillus delbrueckii was studied. L. delbrueckii cells immobilized in functionalized alginate beads showed enhanced bead stability and selectivity towards production of optically pure l (+) lactic acid in higher yields (1.74Yp/s) compared to natural alginate. Palmitoylated alginate beads revealed 99% enantiomeric selectivity (ee) in production of l (+) lactic acid. Metabolite analysis during fermentation indicated low by-product (acetic acid, propionic acid and ethanol) formation on repeated batch fermentation with functionalized immobilized microbial cells. The scanning electron microscopic studies showed dense entrapped microbial cell biomass in modified immobilized beads compared to native alginate. Thus the methodology has great importance in large-scale production of optically pure lactic acid.     

Metabolism associated with raised metabolic flux to sugar nucleotide precursors of exopolysaccharides in Lactobacillus delbrueckii subsp. bulgaricus

Exopolysaccharide (EPS) metabolism was studied in a galactose-negative strain of Lactobacillus delbrueckii subsp. bulgaricus, using two different approaches. Firstly, using both the parent strain and a chemically induced mutant with higher yield and specific productivity of EPS than the parent, comparative information was obtained relating to enzyme activities and metabolite levels associated with EPS formation when grown on lactose. Under continuous culture conditions (D=0.10 h⁻¹), the higher metabolic flux towards EPS formation in the mutant strain relative to the parent appeared to be mediated by raised levels of UDP-glucose pyrophosphorylase (UGP). Marginally raised UDP-galactose 4-epimerase (UGE) activity in the mutant strain suggested that this enzyme could also play a role in EPS overproduction. The second approach involved investigating the effect of growth rate on sugar nucleotide metabolism in the parent, as it is known that EPS production is growth-associated in this strain. UGE activity in the parent strain appeared to increase when the growth rate was elevated from 0.05 to 0.10 h⁻¹, and further to 0.35 h⁻¹, conditions that can be associated with higher levels of metabolic flux to EPS formation. Concurrent with these increments, intracellular ATP levels in the cell were raised. In both investigations glucose-6-phosphate accumulated pointing to a constriction at this branch-point, and a limitation in the flow of carbon towards fructose-6-phosphate or glucose-1-phosphate. The changes in metabolism associated with enhanced flux to EPS provide guidance as to how the yield of Lactobacillus delbrueckii subsp. bulgaricus EPS can be improved.     

Exopolysaccharide and extracellular metabolite production by Lactobacillus delbrueckii subsp. bulgaricus, grown on lactose in continuous culture

Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483, when grown on lactose in continuous culture, showed increasing specific yields and volumetric productivities of exopolysaccharide (EPS) with increasing dilution rate. Specific and volumetric productivities of lactate and galactose, as extracellular metabolites, increased in response to the incremental changes in the dilution rate up to 0.4 h^–1. Elevated Y_p/s values determined for EPS (0.025 g EPSg lactose^–1) at the dilution rates of 0.3 h^–1–0.4 h^–1, relative to those determined at lower dilution rates, suggest a diversion of carbon flux towards EPS being associated with the higher rates of growth.     

Strain improvement of Lactobacillus delbrueckii using nitrous acid mutation for l-lactic acid production

Insertional mutagenesis is impractical in the mechanisms for protection against low pH, high solute concentration etc. due to the involvement of large number of loci and multiple genes. An attempt was made to improve Lactobacillus delbrueckii NCIM 2025 strain by classical mutation using nitrous acid. In the present investigation, classical mutation was proved to be successful and the selected mutants had improved qualities like increased lactic acid productivity, acid tolerance and sugar tolerance. Mutants showed better growth rate and lesser generation time than the wild type.     

Galacto-oligosaccharides as protective molecules in the preservation of Lactobacillus delbrueckii subsp. bulgaricus

In this work, the protective capacity of galacto-oligosaccharides in the preservation of Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333 was evaluated.Lactobacillus bulgaricus was freeze-dried or dried over silica gel in the presence of three commercial products containing galacto-oligosaccharides. The freeze-dried samples were stored at 5 and 25 °C for different periods of time. After desiccation, freeze-drying or storage, samples were rehydrated and bacterial plate counts were determined.According to the results obtained, all galacto-oligosaccharides assays demonstrated to be highly efficient in the preservation of L. bulgaricus. The higher content of galacto-oligosaccharides in the commercial products was correlated with their higher protective capacity.Galacto-oligosaccharides are widely known by their prebiotic properties. However, their role as protective molecules have not been reported nor properly explored up to now. In this work the protective capacity of galacto-oligosaccharides in the preservation of L. bulgaricus, a strain particularly sensitive to any preservation process, was demonstrated.The novel role of galacto-oligosaccharides as protective molecules opens up several perspectives in regard to their applications. The supplementation of probiotics with galacto-oligosaccharides allows the production of self-protected synbiotic products, galacto-oligosaccharides exerting both a prebiotic and protecting effect.     

Enzymatic properties of lipase and characteristics production byLactobacillus delbrueckii subsp.bulgaricus

Some properties of an extracellular lipase produced byLactobacillus delbrueckii subsp.bulgaricus were studied. Maximum enzyme activity was found against olive and butter oil as enzyme substrates. Addition of 9% acacia gum, 0.1% Na-deoxycholate and 0.01 M CaCl₂ to the enzyme reaction mixture increased-lipase activity from 5.3 to 14.5 (FFA/mg protein/minute) at pH 6.0 and at 40° C. Maximum lipase production was reached in the presence of glucose as a sole source of carbon, wheat bran as nitrogen source, olive oil as a sole lipid source and butyric acid as fatty acid supporting the growth medium. An initial pH value of the culture medium of 6.0 and a temperature of 35° C gave the highest lipolytic activity.     

Structure of a neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B26

The neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B26 in skimmed milk was found to be composed of d-glucose and d-galactose in a molar ratio of 2:3. Linkage analysis and 1D/2D NMR ((1)H and (13)C) studies performed on the native polysaccharide, and on an oligosaccharide obtained from a partial acid hydrolysate of the native polysaccharide, showed the polysaccharide to consist of branched pentasaccharide repeating units with the following structure. [structure: see text]     

Structural determination of a neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332

The neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332 in skimmed milk was found to be composed of d-glucose, d-galactose, and l-rhamnose in a molar ratio of 1:2:2. Linkage analysis and 1D/2D NMR (1H and 13C) studies carried out on the native polysaccharide as well as on an oligosaccharide generated by a periodate oxidation protocol, showed the polysaccharide to consist of linear pentasaccharide repeating units with the following structure: -->3-alpha-D-Glcp-(1-->3)-alpha-D-Galp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->.     

Optimization of lactic acid production from beet molasses by Lactobacillus delbrueckii NCIMB 8130

Production of lactic acid from beet molasses by Lactobacillus delbrueckii NCIMB 8130 in static and shake flask fermentation was investigated. Shake flasks proved to be a better fermentation system for this purpose. Substitution of yeast extract with other low cost protein sources did not improve lactic acid production. The maximum lactic acid concentration was achieved without treatment of molasses. A Central Composite Design was employed to determine the maximum lactic acid concentration at optimum values for the process variables (sucrose, yeast extract, CaCO₃). A satisfactory fit of the model was realized. Lactic acid production was significantly affected both by sucrose–yeast extract and sucrose–CaCO₃ interactions, as well as by the negative quadratic effects of these variables. Sucrose and yeast extract had a linear effect on lactic acid production while the CaCO₃ had no significant linear effect. The maximum lactic acid concentration (88.0 g/l) was obtained at concentrations for sucrose, yeast extract and CaCO₃ of 89.93, 45.71 and 59.95 g/l, respectively.     

Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus: a chronicle of evolution in action

Background

Lactobacillus delbrueckii ssp. lactis and ssp. bulgaricus are lactic acid producing bacteria that are largely used in dairy industries, notably in cheese-making and yogurt production. An earlier in-depth study of the first completely sequenced ssp. bulgaricus genome revealed the characteristics of a genome in an active phase of rapid evolution, in what appears to be an adaptation to the milk environment. Here we examine for the first time if the same conclusions apply to the ssp. lactis, and discuss intra- and inter-subspecies genomic diversity in the context of evolutionary adaptation.

Results

Both L. delbrueckii ssp. show the signs of reductive evolution through the elimination of superfluous genes, thereby limiting their carbohydrate metabolic capacities and amino acid biosynthesis potential. In the ssp. lactis this reductive evolution has gone less far than in the ssp. bulgaricus. Consequently, the ssp. lactis retained more extended carbohydrate metabolizing capabilities than the ssp. bulgaricus but, due to high intra-subspecies diversity, very few carbohydrate substrates, if any, allow a reliable distinction of the two ssp. We further show that one of the most important traits, lactose fermentation, of one of the economically most important dairy bacteria, L. delbruecki ssp. bulgaricus, relies on horizontally acquired rather than deep ancestral genes. In this sense this bacterium may thus be regarded as a natural GMO avant la lettre.

Conclusions

The dairy lactic acid producing bacteria L. delbrueckii ssp. lactis and ssp. bulgaricus appear to represent different points on the same evolutionary track of adaptation to the milk environment through the loss of superfluous functions and the acquisition of functions that allow an optimized utilization of milk resources, where the ssp. bulgaricus has progressed further away from the common ancestor.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-407) contains supplementary material, which is available to authorized users.     

Growth and Proteolytic Activity of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus in Reduced Sodium Kashkaval Cheese

Summary Reduced sodium Kashkaval cheese was produced from cow’s milk. Mixtures of NaCl, NaCl:KCl (1:1, 2:1) and NaCl:KH₂PO₄ (1:1, 2:1) were used for hot brining and salting of the cheddarized cheese curd. There were no significant differences (P < 0.05) in the count of Lb. delbrueckii ssp. bulgaricus after aging of Kashkaval samples. At the end of the ripening process the counts of Lb. delbrueckii ssp. bulgaricus reached 10⁶ c.f.u./g and the counts of Streptococcus thermophilus varied from 10⁴ to 10⁵ c.f.u./g. Proteolysis during ripening of reduced sodium Kashkaval cheese, initiated by the starter microorganisms Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, was studied through the changes in the levels of non-casein and non-protein nitrogen. It was observed that non-casein and non-protein nitrogen increased significantly (P < 0.05) during ripening. The amounts of non-casein and non-protein nitrogen accumulated in the studied Kashkaval samples were similar. That indicates that the partial replacement of NaCl with KCl or KH₂PO₄ does not cause significant changes in the course of proteolysis of Kashkaval cheese by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus.     

Optimization of a protective medium for enhancing the viability of freeze-dried Lactobacillus delbrueckii subsp. bulgaricus based on response surface methodology

Response surface methodology (RSM) was used to optimize a protective medium for enhancing the cell viability of Lactobacillus delbrueckii subsp. bulgaricus LB14 during freeze-drying. Using a previous Plackett–Burman design, it was found that sucrose, glycerol, sorbitol and skim milk were the most effective freeze-drying protective agents for L. bulgaricus LB14. A full factorial central composite design was applied to determine the optimum levels of these four protective agents. The experimental data allowed the development of an empirical model (P<0.0001) describing the inter-relationships between the independent and dependent variables. By solving the regression equation, and analyzing the response surface contour and surface plots, the optimal concentrations of the agents were determined as: sucrose 66.40 g/L, glycerol 101.20 g/L, sorbitol 113.00 g/L, and skim milk 130.00 g/L. L. bulgaricus LB14 freeze-dried in this medium obtained a cell viability of up to 86.53%.     

Use of SDS-PAGE of cell-wall proteins for rapid differentiation of Lactobacillus delbrueckii subsp. lactis and Lactobacillus helveticus

Cell-wall protein profiles of different strains of Lactobacillus helveticus and L. delbrueckii subsp. lactis isolated from regional cheeses were studied by SDS-PAGE. The patterns were highly reproducible and the presence of numerous bands with molecular weight ranging from 14 to 160 kDa allowed L. delbrueckii subsp. lactis to be differentiated from L. helveticus. The method is a reliable and rapid way to identify thermophilic lactobacilli.     

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,-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.     

保加利亚乳杆菌ATCC11842 L-乳酸脱氢酶同工酶基因的克隆与表达分析

通过对保加利亚乳杆菌(Lactobacillus delbrueckii subsp.bulgaricus)L-乳酸脱氢酶(L-lactate dehydrogenase, L-LDH)同工酶基因的异源表达、酶活测定和摇瓶发酵研究L-LDH在乳酸合成中的作用。将保加利亚乳杆菌ATCC11842中L-乳酸脱氢酶基因ldb0120和ldb0094分别克隆至载体pET28a(+)中,构建重组表达载体pET28aldb0120和pET28aldb0094,并转化到大肠埃希菌(Escherichia coli) BL21(DE3)中进行表达。进一步对重组蛋白进行Ni-NTA柱亲和层析和酶学活性测定,结果显示,LDB0120和LDB0094的比活力分别为0和25 U/mg,表明LDB0094是具有低活性的L-乳酸脱氢酶,而LDB0120不具有活性。对两株重组菌分别进行好氧和微好氧发酵,重组菌E.coli BL21/pET28aldb0094在好氧和微好氧条件可以合成L-乳酸,浓度分别为41.9和227.9 mg/L,而菌株E.coli BL21/pET28aldb0120在两种培养条件下均基本不合...