Simple method for extracting plasmid DNA from lactic acid bacteria

Rapid screening and large-scale plasmid DNA isolation procedures are described for lactic acid bacteria, using glass beads to break cells. The rapid screening procedure allows one to obtain plasmid DNA pellets in less than 1 h. This method has been successfully tested on various bacteria from the genera Lactococcus, Leuconostoc, Lactobacillus, Pediococcus, Streptococcus, Enterococcus and Propionibacterium. This procedure yields plasmid DNA with minor chromosomal and plasmid DNA-degraded form contaminations.     

生长猪结肠乳酸菌菌群的昼夜节律性变化

【目的】本文旨在研究生长猪在24 h内结肠乳酸菌的多样性变化、丰富度变化与节律性变化。【方法】选取6头装有结肠瘘管的“杜×长×大”三元杂交生长猪,于清晨6:00开始,每隔3 h连续采集一天内生长猪的结肠食糜,提取DNA,用乳酸菌特异性引物进行高通量测序,在属和种水平上分析结肠乳酸菌菌群丰度与节律性变化。【结果】生长猪结肠乳酸菌在24 h内Chao1和Simpson指数发生显著性变化(P<0.05);属水平上Lactobacillus相对丰度最高,一天内在6:00时相对丰度最低,为94.15%,18:00时相对丰度最高,为97.46%;种水平上Lactobacillus johnsonii相对丰度最高,一天内在3:00时相对丰度最低,为47.66%,18:00时相对丰度最高,为71.59%,Lactobacillus reuteri丰度次之。生长猪结肠乳酸菌中46个核心OTU具有节律性,均为Lactobacillus;而在种水平上Lactobacillus gasseri、Lactobacillus johnsonii、Lactobacillus sp. KC45a和Lactobacillus reuteri显现出节律性(P<0.05)。【结论】生长猪结肠乳酸菌在24 h内多样性发生显著性变化,在种水平上显现出节律性,丰富了我们对猪肠道微生物昼夜节律的了解。     

Characterization of chloramphenicol resistance inLactobacillus plantarum caTC2

A strain ofLactobacillus plantarum caTC2R isolated from a meat source was resistant to chloramphenicol (30 g/ml). Resistance was mediated through an inducible chloramphenicol acetyltransferase. Plasmid analysis of this strain showed three plasmids, of which the 8.5-kb plasmid apparently encodes the gene for chloramphenicol resistance. This plasmid was lost at high frequency (25%) when theLactobacillus was subcultured at a higher than optimal temperature (40°C).     

A new runaway type episomal vector for mammalian cells based on a temperature-sensitive simian virus 40 and inducible erythropoietin production

A runaway vector for mammalian cells was constructed from the simian virus 40 (SV40) genome with a temperature-sensitive mutation of the large T antigen and bacterial neo ^r gene. Replication of this plasmid was repressed above 39°C and vigorous DNA propagation was observed below 33°C in simian CV-1 cells. The human erythropoietin gene was inserted downstream of the SV40 late promoter of the plasmid and the recombinant plasmid was introduced into CV-1 cells. By a temperature shift from 37 to 33°C, the plasmid copy number increased from 5 × 10² to 5 × 10³ copies per cell and the specific production rate of erythropoietin increased more than ten-fold. The bacterial-derived sequences such as the neo ^r gene and vector pUC sequences were prone to delete but the main body of the recombinant plasmid such as SV40 and the erythropoietin-coding sequences were stably maintained at either 33 or 37°C.     

A Simple Method for the Efficient Isolation of Genomic DNA from Lactobacilli Isolated from Traditional Indian Fermented Milk (<Emphasis Type="Italic">dahi</Emphasis>)

A simple, inexpensive and effective genomic DNA isolation procedure for Lactobacillus isolates from traditional Indian fermented milk (dahi) is described. A total of 269 Lactobacillus isolates from fermented milk collected from four places in North and west India were tested for lysis by an initial weakening of the Gram positive cell wall with Ampicillin followed by Lysozyme treatment. The average genomic DNA yield was ~50 μg/ml log phase culture. Quality and repeatability of the method was found to be adequate for subsequent molecular applications. The quality of the genomic DNA isolated by this method was verified by restriction digestion and polymerase chain reaction (PCR). No inhibition was observed in subsequent PCR amplification and restriction digestion. The presented method is rapid, cheap and useful for routine DNA isolation from gram positive bacteria such as Lactobacillus.     

Rapid Mini-Prep Isolation of High-Quality Plasmid DNA from Lactococcus and Lactobacillus spp.

A simple, rapid plasmid mini-prep procedure for lactococci and lactobacilli which gives high yields and can be performed on overnight broth cultures is presented. Large plasmids were isolated from both lactococci and lactobacilli, including a 70-kb plasmid from Lactobacillus acidophilus C7. The purity of the resulting plasmid DNA makes it suitable for subsequent molecular manipulations. The convenience of the technique makes this rapid mini-prep procedure suitable for routine plasmid isolation from lactic acid bacteria.     

Analysis of functional properties of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis>

Metabolites from Lactobacillus acidophilus were analysed. The results showed that Lactobacillus acidophilus Ind-1 and Lactobacillus acidophilus Lakcid produced respectively 12.73 g and 13.33 g lactic acid l⁻¹ after incubating in skim milk at 37 °C for 36 h; and 2.229 unit and 1.808 unit β-galactosidase l⁻¹ in an MRS medium. The proteolytic activity of Lactobacillus acidophilus was high and the content of 17 free amino acids in the fermented milk of Lactobacillus acidophilus Ind-1 and Lactobacillus acidophilus Lakcid was 394.4 mg l⁻¹ and 563.2 mg l⁻¹, respectively. Meantime, Lactobacillus acidophilus reduced cholesterol level in an MRS medium supplemented with cholesterol. Furthermore, Lactobacillus acidophilus Ind-1 and Lactobacillus acidophilus Lakcid showed antimicrobial activity against Bacillus anthracis and Escherichia coli.     

Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L.

The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.     

Rapid species identification within two groups of closely related lactobacilli using PCR primers that target the 16S/23S rRNA spacer region

A rapid and reliable PCR-based method for distinguishing closely related species within two groups of lactobacilli is described. Primers complementary to species-specific sequences in the 16S/23S rDNA spacer regions were designed after sequencing and sequence comparison of the spacer regions of 32 strains. The strains belong to two groups of closely related Lactobacillus species; one composed of Lactobacillus curvatus, Lactobacillus graminis and Lactobacillus sake, the other of Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus plantarum. PCR assays with the designed primers and subsequent agarose gel analysis of the amplified fragments allowed the same species identification as the DNA/DNA hybridization procedure.     

Plasmid instability inLactobacillus plantarum strain caTC2

The stability of plasmids inLactobacillus plantarum was investigated by extended incubation of bacterial cells in the presence of different carbohydrates. Strain caTC2, carrying a plasmid-encoded chloramphenicol-resistant (Cm^r) phenotype, was grown overnight (16–18 h) in MRS, MRS-L, and MRS-M broths containing 2% glucose, lactose, and maltose respectively at 30°C. The cultures were subsequently held at 30°C and room temperature (21±1°C) for an extended period (7 days). The total viable cell counts were assayed on MRS agar plates and tested for sensitivity to 30 g chloramphenicol/ml by replica plating. The plasmid profiles of the chloramphenicol-sensitive strains showed that there was a loss of the 8.5-kb plasmid, but not the 10.6 or 6.5 kb plasmids. Concomitant loss of the chloramphenicol resistance phenotype and plasmid at high frequency, particularly by using MRS-L at 21°C method, suggests that this would be a simple and efficient method for curing selected plasmids in lactobacilli.Contribution No. 2039 from the Centre for Food and Animal Research.     

Properties of a bacteriocin produced by Carnobacterium piscicola CP5

Summary Carnobacterium piscicola CP5 produced a bacteriocin named carnocin CP5 that inhibited Carnobacterium, Enterococcus and Listeria spp. and among the Lactobacillus spp. only Lactobacillus delbrueckii ssp. Carnocin CP5 was stable 1h at 100°C at pH 7.0. It was inactivated by numerous proteolytic enzymes. Production of carnocin, CP5 occured in MRS broth regulated at pH 7.0. The apparent molecular weight of the bacteriocin in the crude extract was greater than 10 kDa, but around 5 kDa after action of SDS or urea. Novobiocin treatment led to non-producer variants.     

Rapid Mini-Prep Isolation of High-Quality Plasmid DNA from Lactococcus and Lactobacillus spp

A simple, rapid plasmid mini-prep procedure for lactococci and lactobacilli which gives high yields and can be performed on overnight broth cultures is presented. Large plasmids were isolated from both lactococci and lactobacilli, including a 70-kb plasmid from Lactobacillus acidophilus C7. The purity of the resulting plasmid DNA makes it suitable for subsequent molecular manipulations. The convenience of the technique makes this rapid mini-prep procedure suitable for routine plasmid isolation from lactic acid bacteria.     

Microbial products from probiotic bacteria inhibit Salmonella enteritidis 857-induced IL-8 synthesis in Caco-2 cells

Oral administration of Lactobacillus spp. as probiotics is gaining importance in the treatment of intestinal inflammations. However, their mechanism of action is unknown. We investigated whether nonspecific binding Lactobacillus casei Shirota (LcS) and mannose-specific Lactobacillus plantarum 299v (Lp) and their spent culture supernatant (SCS) affect Salmonella enteritidis 857 (Se) growth, IL-8 and Hsp70 syntheses. In one set of experiments human enterocyte-like Caco-2 cells were infected with LcS, Lp or Se at 1–500 bacteria per cell for 1 h. In another set, cells were exposed to Se (0–200 per cell, 1 h) after exposure to lactobacilli (LB) (500 per cell, 30 min) or by co-incubation of Se and LB (1 h). The third set of experiments involved exposure of cells for 1 h to SCS or Se (100 per cell) pretreated (1 h) in SCS. The effect of LB SCS on Se growth was evaluated by agar plate diffusion test. IL-8 and Hsp70 were assessed over 2–24 h using ELISA and Western blotting, respectively. Neither LcS nor Lp affected the Se growth and IL-8 production. In addition, they did not induce Hsp70 expression by Caco-2 cells. Instead, their SCS inhibited the Se growth and IL-8 production and induced the expression of Hsp70 by both crypt- and villus-like cells. The beneficial effect of Lactobacillus spp. to the intestinal inflammations might be associated with a decrease in IL-8 levels. This effect could be mediated, at least in part, via a secreted antimicrobial product(s) either directly against the pathogens or indirectly through the synthesis of Hsp70.     

Relationship between DNA damage,DNA repair,metabolic state and cell lethality

Summary Induction of unrepairable DNA damage, accumulation of misrepaired DNA damage, and generation of imbalances in competing biochemical and/or metabolic processes have been proposed to explain the relationship between radiation-induced DNA damage and cell lethality. Theoretically, the temperature dependence of the critical DNA repair process(es) should be 1) either independent of or identical to the temperature dependence of cell killing if the first two hypotheses are correct, and 2) different if the third hypothesis is correct. To test this, exponentially growing rat 9L brain tumor cells were left at 37°C or equilibrated for 3–14 h at 20°C before irradiation. Cells were irradiated and allowed to repair at either 20°C or 37°C. Alternatively, the cells were irradiated at one of these temperatures and immediately shifted to the other temperature for repair. DNA damage was assessed by the alkaline elution technique; cell kill was assessed by a clonogenic assay. 9L cells maintained at 20°C or 37°C sustained the same amount of DNA damage as measured by alkaline elution. DNA repair instantaneously assumed the rate characteristic of the postirradiation temperature. For 9L cells equilibrated, irradiated, and repaired at 20°C, the half-time of the fast phase of the DNA repair decreased by a factor of 2 and the half-time of the slow phase decreased by a factor of 5 over that measured in cells incubated, irradiated and repaired at 37°C. Although the rate of DNA repair decreased substantially at 20°C, the survival of 9L cells that were equilibrated and irradiated at 20°C was greater (p <10^–4) than those incubated and irradiated at 37°C, when assayed by an immediate plating protocol. In addition, the survival of 9L cells equilibrated and irradiated at 20°C and then shifted to 37°C immediately after irradiation was greater (p <10^–2) than that obtained with any other delayed plating protocol. Thus, the temperature dependence of the DNA repair processes measured by alkaline elution was different from the temperature dependence of cell killing measured either by an immediate or delayed plating protocol. These data support the hypothesis that many irradiated 9L tumor cells die because of imbalances in sets of competing biochemical and/or metabolic processes.Presented at the 81st Annual Meeting of the American Association for Cancer Research, May 23–26, 1990 in Washington, DC     

Structural and functional analysis of two cryptic plasmids from Lactobacillus pentosus MD353 and Lactobacillus plantarum ATCC 8014.

Summary The DNA sequences of a 2.4 kb plasmid (p353-2) from Lactobacillus pentosus MD353 and a 1.9 kb plasmid (p8014-2) from Lactobacillus plantarum ATCC 8014 show 81.5% overall similarity. Both plasmids carry elements (replication protein gene, plus-origin and minus-origin of replication), which are typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR). Direct evidence for an RCR mechanism was obtained by showing the accumulation of single-stranded plasmid intermediates in the presence of rifampicin. A minus-origin of replication was defined for plasmids p353-2 and p8014-2 based on DNA sequence analysis and on its ability to convert single-stranded into double-stranded plasmid DNA. Plasmids pLPE323, pLPE350 and pLPC37 that are derived from the p353-2 or p8014-2 replicon are structurally and segregationally stable in L. pentosus MD353, L. plantarum ATCC 8014 and in Lactobacillus casei ATCC 393. The presence of Escherichia coli or DNA fragments in vectors derived from p353-2 or p8014-2 does not affect the structural stability but results in segregational instability of the vectors. The instability increases with increasing size of the inserted DNA fragment. Since vectors based on these replicons can be efficiently propagated in a wide variety of Lactobacillus species, they are highly suitable for cloning and expression of foreign DNA in Lactobacillus, provided that selective pressure is applied.This paper is dedicated with great appreciation to Dr. Frits Berends on the occasion of his retirement as Head of the Biochemistry Department of the TNO Medical Biological Laboratory     

Sequence Analyses of a Broad Host-Range Plasmid Containing ermT from a Tylosin-Resistant Lactobacillus sp. Isolated from Swine Feces

Anaerobic bacteria resistant to the macrolide antibiotics tylosin and erythromycin were isolated from the feces of swine. One of the strains, 121B, was initially identified by 16S rDNA sequence analysis as an unknown Lactobacillus sp. The strain was found to contain at least two plasmids, one of which was capable of replicating and providing erythromycin and tylosin resistance to Bacillus subtilis, Streptococcus gordonii, and Escherichia coli. DNA sequence analyses of the 4,232-bp plasmid, p121BS, identified one open reading frame encoding a methylase gene highly similar (>98% amino acid identity, >99% DNA sequence identity) to the ermT gene from the Lactobacillus reuteri plasmid pGT633. This is only the second ermT gene to be reported. p121BS also contains two additional open reading frames with significant amino acid similarities to replication proteins from Lactobacillus and other Gram-positive bacteria. Received: 13 October 2000 / Accepted: 4 December 2000     

Rapid micropreps and minipreps of Ti plasmids and binary vectors fromAgrobacterium tumefaciens

A simple and versatile procedure has been developed for the isolation of both large helper/Ti plasmids and binary vectors fromAgrobacterium tumefaciens. Using a slightly modified alkaline lysis protocol, intact plasmid can be recovered from cultures grown in standard micro-centrifuge tubes or culture tubes in sufficient yield and purity to allow for restriction analysis on ethidium bromide stained gels of the >200 kb Ti plasmid DNA. Contamination by chromosomal DNA is minimal and there is thus no need for isopycnic gradient purification. This same procedure can be combined with a high temperature treatment (37°C) and antibiotic selection to generate preparations containing binary vector DNA that are virtually free of interfering Ti plasmid DNA. Restriction patterns produced from these binary vector DNA preparations are unambiguous and therefore preliminary screening by Southern hybridization can be eliminated.     

Characterization of a cryptic plasmid from Lactobacillus fermentum KC5b and its use for constructing a stable Lactobacillus cloning vector

Lactobacillus fermentum KC5b, a strain originally isolated from the human vagina, contains a cryptic plasmid pKC5b. The sequence and genetic organization of the 4392-bp plasmid were determined. It contains two convergently oriented replicons, which are homologous to each other and to the stable replicon of the Enterococcus faecium plasmid pMBB1. The two replicons of pKC5b were used either individually or together to construct Lactobacillus–Escherichia coli shuttle plasmids. Only the plasmid pSP1 that carried both replicons transformed lactobacilli, suggesting a complementary function between the two replicons. Since the replicons had a high homology to those of other plasmids that replicate via a theta-like mechanism and no detectable single-stranded intermediates were found for the plasmid, it is possible that pKC5b may replicate via a theta-like mechanism. The new shuttle plasmid pSP1 has been transformed and stably maintained in several Lactobacillus strains. As an initial application, pSP1 was used to clone the S-layer protein gene (slpA) of Lactobacillus acidophilus ATCC 4356 into a heterologous vaginal Lactobacillus strain and achieved surface-bound expression of the protein.     

Some properties of arctic rhizobia

Forty-eight strains of Rhizobium isolated from the root nodules of three species of legumes indigenous to the high tundra (Astragalus alpinus, Oxytropis maydelliana andOxytropis arctobia) are phenotypically heterogenous with respect to intrinsic antibiotic resistance, expression of nitrogenase activityex planta and plasmid content. All of the strains possess a 250–300 kb plasmid and are homologous to each other on the genomic DNA level but have little DNA homology with selected reference strains of well characterized species of rhizobia. The arctic rhizobia have an optimum growth temperature of 23°C and can grow slowly at 5°C. The DNA from four of the isolates, which were selected for detailed investigation, have sequences homologous tonif andnod genes fromRhizobium trifolii.