Effects of Ethidium Bromide and Several Acridine Dyes on the Kinetoplast DNA of Leishmania tropica*†

Whole cell DNA from Leishmania tropica has 2 peaks when banded by CsCl equilibrium density centrifugation. The main band has a buoyant density of 1.721 and the satellite band a buoyant density of 1.705, with Clostridium perfringens DNA (ρ= 1.6915) used as a reference. The satellite band has been identified as the kinetoplast DNA by purifying DNA from isolated kinetoplasts. L. tropica has the highest G + C content of both nuclear and kinetoplastic DNA thus far reported for trypanosomatids. The effects of ethidium bromide, acriflavin, proflavin, and 5-aminoacridine on the kinetoplast of L. tropica have been compared. Ethidium bromide and acriflavin, but not proflavin or 5-aminoacridine, induce dyskinetoplasty. L. tropica is one of the most sensitive trypanosomatids to ethidium bromide and acriflavin. Examination of the DNA from drug-treated cells in CsCl gradients revealed a loss of the satellite band after ethidium bromide or acriflavin treatment, but not after proflavin or 5-aminoacridine treatment. Cell division was required to produce these effects on the kinetoplast.     

Plasmid-encoded ropiness production inLactobacillus casei SSP.casei

Summary Genetic determinants of the Muc⁺ character were investigated in two ropy strains,Lactobacillus delbrueckii ssp.bulgaricus 201 andL. casei ssp.casei NCIB 4114, which secrete a large amount of slime in culture media. Plasmid DNA analysis revealed the presence of two plasmids (4.5 and 2.3 Mdal) inL. casei ssp.casei, whileL. delbrueckii ssp.bulgaricus was plasmid free, suggesting a chromosomal location of Muc⁺ character in this strain. Curing experiments carried out onL. casei ssp.casei NCIB 4114 indicated a correlation between the Muc⁺ phenotype and the 4.5 Mdal plasmid.     

The role of the HCR system in the repair of lethal lesions ofBacillus subtilis phages and their transfecting DNA damaged by radiation and alkylating agents

The role of the HCR system in the repair of prelethal lesions induced by UV-light, γ-rays and alkylating agents was studied in theBacillus subtilis SPP1 phage, its thermosensitive mutants (N3, N73 endts ₁) and corresponding infectious DNA. The survival of phages and their transfecting DNA after treatment with UV light is substantially higher inhcr ⁺ cells than inhcr cells, the differences being more striking in intact phages than in their transfecting DNA’s. Repair inhibitors reduce the survival inhcr ⁺ cells: caffeine lowers the survival of UV-irradiated phage SPP1 in exponentially growinghcr ⁺ cells but has no effect on its survival in competenthcr ⁺ cells; acriflavin and ethidium bromide decrease the survival of UV-irradiated SPP1 phage in both exponentially growing and competenthcr ⁺ cells to the level of survival observed inhcr cells; moreover, ethidium bromide lowers the number of infective centres inhcr ⁺ cells of UV-irradiated DNA of the SPP1 phage. Repair inhibitors do not lower the survival of UV-irradiated phages or their DNA inhcr cells. The repair mechanism under study repairs effectively also lesions induced by polyfunctional alkylating agents in transfecting DNA’s ofB. subtilis phages but is not functional with lesions induced by these agents in free phages and lesions caused in phages and their DNA by ethyl methanesulphonate or γ-rays.     

Carbon Source Requirements for Exopolysaccharide Production by Lactobacillus casei CG11 and Partial Structure Analysis of the Polymer

Exopolysaccharide production by Lactobacillus casei CG11 was studied in basal minimum medium containing various carbon sources (galactose, glucose, lactose, sucrose, maltose, melibiose) at concentrations of 2, 5, 10, and 20 g/liter. L. casei CG11 produced exopolysaccharides in basal minimum medium containing each of the sugars tested; lactose and galactose were the poorest carbon sources, and glucose was by far the most efficient carbon source. Sugar concentrations had a marked effect on polymer yield. Plasmid-cured Muc^- derivatives grew better in the presence of glucose and attained slightly higher populations than the wild-type strain. The values obtained with lactose were considerably lower for both growth and exopolysaccharide yield. The level of specific polymer production per cell obtained with glucose was distinctively lower for Muc^- derivatives than for the Muc⁺ strain. The polymer produced by L. casei CG11 in the presence of glucose was different from that formed in the presence of lactose. The polysaccharide produced by L. casei CG11 in basal minimum medium containing 20 g of glucose per liter had an intrinsic viscosity of 1.13 dl/g. It was rich in glucose (76%), which was present mostly as 2- or 3-linked residues along with some 2,3 doubly substituted glucose units, and in rhamnose (21%), which was present as 2-linked or terminal rhamnose; traces of mannose and galactose were also present.     

Exopolysaccharides production in Lactobacillus bulgaricus and Lactobacillus casei exploiting microfiltration

The physiology of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus casei, extensively used in the dairy industry, was studied in order to evaluate key parameters in the synthesis of exopolysaccharides and to improve their production through novel fermentation processes. Selected strains were studied in shake flasks and in fermentor experiments using glucose and lactose as main carbon sources and bacto casitone as the only complex component, in a temperature range between 35 and 42°C. The production of exopolysaccharides was monitored and correlated to the growth conditions using both a colorimetric assay and chromatographic methods. Fermentor experiments in batch mode yielded 100 mg l⁻¹ of EPS from L. bulgaricus and 350 mg l⁻¹ from L. casei. Moreover, the use of a microfiltration (MF) bioreactor resulted in exopolysaccharides (EPS) concentrations threefold and sixfold those of batch experiments, respectively. The monosaccharidic composition of the two analyzed polymers differed from those previously reported. The optimization of the production of EPSs using the MF fermentation strategy could permit the use of these molecules produced by generally recognised as safe (GRAS) microorganisms in the place of other polysaccharides in the food industry.     

Integrative Food-Grade Expression System Based on the Lactose Regulon of Lactobacillus casei

The lactose operon from Lactobacillus casei is regulated by very tight glucose repression and substrate induction mechanisms, which made it a tempting candidate system for the expression of foreign genes or metabolic engineering. An integrative vector was constructed, allowing stable gene insertion in the chromosomal lactose operon of L. casei. This vector was based on the nonreplicative plasmid pRV300 and contained two DNA fragments corresponding to the 3′ end of lacG and the complete lacF gene. Four unique restriction sites were created, as well as a ribosome binding site that would allow the cloning and expression of new genes between these two fragments. Then, integration of the cloned genes into the lactose operon of L. casei could be achieved via homologous recombination in a process that involved two selection steps, which yielded highly stable food-grade mutants. This procedure has been successfully used for the expression of the E. coli gusA gene and the L. lactis ilvBN genes in L. casei. Following the same expression pattern as that for the lactose genes, β-glucuronidase activity and diacetyl production were repressed by glucose and induced by lactose. This integrative vector represents a useful tool for strain improvement in L. casei that could be applied to engineering fermentation processes or used for expression of genes for clinical and veterinary uses.     

Lactic acid fermentation by Lactobacillus casei in free cell form and immobilised on gluten pellets

A comparative study of the fermentation of a range of carbohydrate substrates, at various temperatures, was carried out using a commercial Lactobacillus casei strain in a free cell form and immobilised on gluten pellets. This strain required yeast extract, l-cysteine HCl and Mn²⁺ at 5, 0.5 and 0.1 g l^–1, respectively, for maximum growth and lactic acid production. Sugar fermentation using free cells showed preference in the order glucose, sucrose, fructose while lactose was poorly utilised. Optimum temperature for growth and lactic acid production over (18–30 h) was 43 °C. L. casei was successfully immobilised on gluten pellets and fermented glucose and sucrose in a shorter time (18 h) with increased lactic acid production (42 and 41 g l^–1 on glucose and sucrose, respectively).     

Exopolysaccharide production by Lactobacillus casei under controlled pH

Summary The exopolysaccharide (EPS) production and growth characteristics of Lactobacillus casei CRL 87 under pH control were studied. Maximum polymer synthesis (488 mg/l) and cell viability (2.4×10¹⁰ cfu/ml) occurred when L. casei was cultured at a constant pH of 6.0 and 30°C for 24 h. However, the optimum specific EPS production (3.9×10^-5 g EPS/g cell dry weigt) and EPS yield (4.3%) were found at a pH of 4.0.     

Batch fermentation with entrapped growing cells ofLactobacillus casei

Summary Growing cells ofLactobacillus casei were entrapped in-carrageenan/locust bean gum (LBG) (2:1 or 2.75%:0.25% w/w respectively) mixed gel beads (two ranges of diameter: 0.5–1.0 and 1.0–2.0 mm) to fermentLactobacillus Selection (LBS) medium and produce biomass. The results showed significant influence of initial cell loading of the beads and bead size on the fermentation rate. The highest cell release rates were obtained with 2.75%:0.25%-carrageenan/LBG small diameter gel beads. However, 17 h fermentation of LBS medium with immobilized cells resulted in substantial softening of the gel matrix, prohibiting reuse of immobilized biocatalysts as inoculum in subsequent batch fermentation. A dynamic shear rheological study showed that the gel weakness was related to chemical interactions with the medium. Results indicated that part of the matrix-stabilizing K⁺ ions diffused back to the medium. Stabilization of the gel was obtained by adding potassium ions to the LBS medium;L. casei growth was not altered by this supplementation. Fermentation of LBS medium supplemented with KCl byL. casei showed higher cell counts in the broth medium with immobilized cells than with free cells, reaching 10¹⁰ cells/ml after about 10 h with entrapped cells in 0.5–1.0 mm diameter beads and 17 h with free cells. Counts in the gel beads after fermentation were higher than 10¹¹ cells/ml and bead integrity was maintained throughout fermentation.     

Human papillomavirus type 16 E6-specific antitumor immunity is induced by oral administration of HPV16 E6-expressing Lactobacillus casei in C57BL/6 mice

Given that local cell-mediated immunity (CMI) against the human papillomavirus type 16 E6 (HPV16 E6) protein is important for eradication of HPV16 E6-expressing cancer cells in the cervical mucosa, the HPV16 E6 protein may be a target for the mucosal immunotherapy of cervical cancer. Here, we expressed the HPV16 E6 antigen on Lactobacillus casei (L. casei) and investigated E6-specific CMI following oral administration of the L. casei-PgsA-E6 to mice. Surface expression of HPV16 E6 antigens was confirmed and mice were orally inoculated with the L. casei-PgsA or the L. casei-PgsA-E6. Compared to the L. casei-PgsA-treated mice, significantly higher levels of serum IgG and mucosal IgA were observed in L. casei-PgsA-E6-immunized mice; these differences were significantly enhanced after boost. Consistent with this, systemic and local CMI were significantly increased after the boost, as shown by increased counts of IFN-γ-secreting cells in splenocytes, mesenteric lymph nodes (MLN), and vaginal samples. Furthermore, in the TC-1 tumor model, animals receiving the orally administered L. casei-PgsA-E6 showed reduced tumor size and increased survival rate versus mice receiving control (L. casei-PgsA) immunization. We also found that L. casei-PgsA-E6-induced antitumor effect was decreased by in vivo depletion of CD4⁺ or CD8⁺ T cells. Collectively, these results indicate that the oral administration of lactobacilli bearing the surface-displayed E6 protein induces T cell-mediated cellular immunity and antitumor effects in mice.     

Effect of lactic acid on diacetyl and acetoin production byLactobacillus casei subsp.rhamnosus ATCC 7469

Lactic acid or its acidity apparently play an important role in the regulation of the biosynthesis of flavor compounds inLactobacillus casei subsp.rhamnosus ATCC 7469. In pyruvate-containing media,L. casei produces lactic acid, acetoin, and diacetyl. A specific pH-dependent system is necessary for both the use of pyruvate and the induction of acetoin and diacetyl production. In cell extracts ofL. casei, lactic acid inhibits the enzymatic activity of acetolactate decarboxylase (ALD) and acetolactate synthetase (ALS); this effect does not occur in whole cells under standard physiological conditions. Lactic acid prevents the use of pyruvate, and the induction of acetoin and diacetyl production. When pyruvate-containing media are used, the pH must be kept close to 6.0 in order to obtain the best production of acetoin and diacetyl.     

Optimization of the fermentation of whey by Lactobacillus casei

Optimization studies have been carried out for the production of L-lactic acid from the fermentation of whey by Lactobacillus casei. Two Statistical Designs, a full 2⁴ Factorial Design and a full 3² Factorial Design, have been used to determine the most suitable values of the operational variables for obtaining the maximum lactic acid production rate. The variables selected for study were temperature, pH, lactose concentration, and inoculum concentration. Among them only temperature and pH were found to affect the response significantly. A second-order polynomial empirical model relating both the response (lactic acid production rate) and the variables was formulated. The maximum acid production rate was found when the values of pH and temperature were 5.4 and 38°C, respectively. All conclusions are restricted to the experimental range studied.     

Comparative genome analysis of Lactobacillus casei strains isolated from Actimel and Yakult products reveals marked similarities and points to a common origin

The members of the Lactobacillus genus are widely used in the food and feed industry and show a remarkable ecological adaptability. Several Lactobacillus strains have been marketed as probiotics as they possess health‐promoting properties for the host. In the present study, we used two complementary next‐generation sequencing technologies to deduce the genome sequences of two Lactobacillus casei strains LcA and LcY, which were isolated from the products Actimel and Yakult, commercialized as probiotics. The LcA and LcY draft genomes have, respectively, an estimated size of 3067 and 3082 Mb and a G+C content of 46.3%. Both strains are close to identical to each other and differ by no more than minor chromosomal re‐arrangements, substitutions, insertions and deletions, as evident from the verified presence of one insertion‐deletion (InDel) and only 29 single‐nucleotide polymorphisms (SNPs). In terms of coding capacity, LcA and LcY are predicted to encode a comparable exoproteome, indicating that LcA and LcY are likely to establish similar interactions with human intestinal cells. Moreover, both L. casei LcA and LcY harboured a 59.6 kb plasmid that shared high similarities with plasmids found in other L. casei strains, such as W56 and BD‐II. Further analysis revealed that the L. casei plasmids constitute a good evolution marker within the L. casei species. The plasmids of the LcA and LcY strains are almost identical, as testified by the presence of only three verified SNPs, and share a 3.5 kb region encoding a remnant of a lactose PTS system that is absent from the plasmids of W56 and BD‐II but conserved in another smaller L. casei plasmid (pLC2W). Our observations imply that the results obtained in animal and human experiments performed with the Actimel and Yakult strains can be compared with each other as these strains share a very recent common ancestor.     

Effect of temperature and various nitrogen sources on L(+)-lactic acid production by Lactobacillus casei

 Two homofermentative strains, Lactobacillus casei NRRL B-441 and Lactobacillus casei subsp. rhamnosus NRRL B-445 were selected for further study from 17 lactic acid bacterial strains screened for lactic acid production. The effect of temperature on lactic acid production with the selected strains was investigated by adapting both strains to four different temperatures. The production of L(+)-lactic acid by both strains was most efficient at 37°C, although with L. casei the highest lactic acid concentration was obtained at 41°C. The maximal volumetric productivity with L. casei was 4.1 g l^-1 h^-1 and with L. casei subsp. rhamnosus 3.5 g l^-1 h^-1. The composition of the medium was studied in order to replace the costly yeast extract with less expensive sources of nitrogen and amino acids. From 11 different nitrogen sources investigated at 37°C, barley malt sprouts (88 g l^-1 lactic acid in 66 h) and grass extract (74 g l^-1 lactic acid in 73 h) were the best economic alternatives. The effect of different combinations of yeast extract, peptone and malt sprouts was further studied by using statistical experimental design, and an empirical second-order polynomial model was constructed on the basis of the results. With the right combination most of the yeast extract could be substituted by barley malt sprouts for efficient lactic acid production. A method for extraction of nutrients and growth factors from malt sprouts is also described. Received: 25 September 1995/Accepted: 24 October 1995     

Yeast mutants resistant to ethidium bromide

Summary Yeast mutants resistant to ethidium bromide have been isolated among sensitive grande cells (⁺) for their ability to grow on glycerol in the presence of the dye. Mutant cells are also resistant to acriflavin and do not yield petites (^-) when grown on galactose with the mutagen. Genetic analysis reveals that resistance to ethidium bromide is controlled by a cytoplasmic factor, carried by, or linked to, the determinant (mitochondrial DNA). The expression of resistance to ethidium bromide seems to be related to the presence in the cell of a product of mitochondrial protein synthesis. It is concluded that some mitochondrial DNA sequence is involved in the resistance to ethidium bromide of yeast mitochondria.     

Protoplast fusion between Lactobacillus casei and Lactobacillus acidophilus

Summary From the fusion between Lactobacillus casei and Lactobacillus acidophilus, 8 fusants were selected: Four were able to ferment maltose, lactose, galactose and mannose, but two had greater abilities of acid production than parents. Increased values of up to 7.6–8 % in -galactosidase activity were obtained from two when compared to that of L. acidophilus, whereas another 2 had activities of 800 and 548 nmol/mg protein/min comparable to that of L casei giving a value of 400 nmol/mg protein/min in phospho--galactosidase activity.     

Attenuation of Colitis by Lactobacillus casei BL23 Is Dependent on the Dairy Delivery Matrix

The role of the food delivery matrix in probiotic performance in the intestine is not well understood. Because probiotics are often provided to consumers in dairy products, we investigated the contributions of milk to the health-benefiting performance of Lactobacillus casei BL23 in a dextran sulfate sodium (DSS)-induced murine model of ulcerative colitis. L. casei BL23 protected against the development of colitis when ingested in milk but not in a nutrient-free buffer simulating consumption as a nutritional supplement. Consumption of (acidified) milk alone also provided some protection against weight loss and intestinal inflammation but was not as effective as L. casei and milk in combination. In contrast, L. casei mutants deficient in DltD (lipoteichoic acid d-alanine transfer protein) or RecA (recombinase A) were unable to protect against DSS-induced colitis, even when consumed in the presence of milk. Mice fed either L. casei or milk contained reduced quantities of colonic proinflammatory cytokines, indicating that the L. casei DltD⁻ and RecA⁻ mutants as well as L. casei BL23 in nutrient-free buffer were effective at modulating immune responses. However, there was not a direct correlation between colitis and quantities of these cytokines at the time of sacrifice. Identification of the cecal microbiota by 16S rRNA gene sequencing showed that L. casei in milk enriched for Comamonadaceae and Bifidobacteriaceae; however, the consumption of neither L. casei nor milk resulted in the restoration of the microbiota to resemble that of healthy animals. These findings strongly indicate that probiotic strain efficacy can be influenced by the food/supplement delivery matrix.     

Enhancement of Host Resistance against Listeria Infection by Lactobacillus casei: Activation of Liver Macrophages and Peritoneal Macrophages by Lactobacillus casei

The functions of liver macrophages and peritoneal macrophages obtained after injection of Lactobacillus casei were examined. Listericidal activity in vivo was enhanced in liver macrophages 13 days after L. casei injection but was somewhat suppressed in the macrophages 2 days after the injection. The listericidal activity in vitro was enhanced in peritoneal macrophages obtained 13 days after L. casei injection but was suppressed in cells obtained 2 days later. The PMA-triggered respiratory burst in the liver macrophages elicited by L. casei was higher than that of resident macrophages. Alkaline phosphodiesterase activity in the liver macrophages was decreased by L. casei injection, as was also the case with peritoneal macrophages. These observations indicate that L. casei augmented cellular functions of both liver and peritoneal macrophages.     

Lactobacillus casei expressing Internalins A and B reduces Listeria monocytogenes interaction with Caco-2 cells in vitro

Listeria monocytogenes has been implicated in a number of outbreaks including the recent largest outbreak in South Africa. Current methods for prevention of foodborne L. monocytogenes infection are inadequate, thus raising a need for an alternative strategy. Probiotic bioengineering is considered a prevailing approach to enhance the efficacy of probiotics for targeted control of pathogens. Here, the ability of Lactobacillus casei expressing the L. monocytogenes invasion proteins Internalins A and B (inlAB) to prevent infection was investigated. The inlAB operon was cloned and surface-expressed on L. casei resulting in a recombinant strain, Lbc^InlAB, and subsequently, its ability to inhibit adhesion, invasion and translocation of L. monocytogenes through enterocyte-like Caco-2 cells was examined. Cell surface expression of InlAB on the Lbc^InlAB was confirmed by Western blotting and immunofluorescence staining. The Lbc^InlAB strain showed significantly higher (P < 0.0001) adherence, invasion and translocation of Caco-2 cells than the wild-type L. casei strain (Lbc^WT), as well as reduced L. monocytogenes adhesion, invasion and transcellular passage through the cell monolayer than Lbc^WT. Furthermore, pre-exposure of Caco-2 cells to Lbc^InlAB significantly reduced L. monocytogenes-induced cell cytotoxicity and epithelial barrier dysfunction. These results suggest that InlAB-expressing L. casei could be a potential practical approach for prevention of listeriosis.     

Production of Human Papillomavirus Type 16 L1 Virus-Like Particles by Recombinant Lactobacillus casei Cells

Infections with human papillomavirus type 16 (HPV-16) are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, the most promising vaccine against HPV-16 infection is based on the L1 major capsid protein, which self-assembles in virus-like particles (VLPs). In this work, we used a lactose-inducible system based on the Lactobacillus casei lactose operon promoter (plac) for expression of the HPV-16 L1 protein in L. casei. Expression was confirmed by Western blotting, and an electron microscopy analysis of L. casei expressing L1 showed that the protein was able to self-assemble into VLPs intracellularly. The presence of conformational epitopes on the L. casei-produced VLPs was confirmed by immunofluorescence using the anti-HPV-16 VLP conformational antibody H16.V5. Moreover, sera from mice that were subcutaneously immunized with L. casei expressing L1 reacted with Spodoptera frugiperda-produced HPV-16 L1 VLPs, as determined by an enzyme-linked immunosorbent assay. The production of L1 VLPs by Lactobacillus opens the possibility for development of new live mucosal prophylactic vaccines.