Relationships between autonomous and integrated forms of tetracycline resistance plasmid in Staphylococcus aureus.

Recombinant plasmids carrying apparently the complete genome of a small staphylococcal plasmid, pT181, or of its temperature-sensitive replication mutant, pSA0301, were isolated and characterized; in these recombinants, pT181 or pSA0301 were considered as “integrated” into the other plasmid, inasmuch as they seem to have a subsidiary role in the replication of the respective recombinant plasmids. Using these recombinants, the incompatibility relationships between integrated and autonomous forms of the same plasmid were studied. The results obtained showed that, although integrated plasmids express their incompatibility toward autonomous ones, they are not susceptible to the incompatibility manifested by an autonomous or another integrated plasmid. No differences were observed between pT181 and pSA0301 in their response to the incompatibility manifested by recombinant plasmids. The expression of the incompatibility of an integrated plasmid did not require the function of the repC gene, involved in plasmid autonomous replication. Moreover, the pT181 repC⁺ gene seems not to be expressed when pT181 is integrated into another plasmid in that the integrated form does not complement autonomous pSA0301 for replication at nonpermissive temperature.     

Use of Lactobacillus plantarum LPCO10, a Bacteriocin Producer, as a Starter Culture in Spanish-Style Green Olive Fermentations

Bacteriocin-producing Lactobacillus plantarum LPCO10 and its non-bacteriocin-producing, bacteriocinimmune derivative, L. plantarum 55-1, were evaluated separately for growth and persistence in natural Spanish-style green olive fermentations. Both strains were genetically marked and selectively enumerated using antibiotic-containing media. Plasmid profile and bacteriocin production (bac⁺) were used as additional markers. When olive brines were inoculated at 10⁵ CFU/ml, the parent strain, LPCO10, proliferated to dominate the epiphytic microflora, sharing high population levels with other spontaneously occurring lactobacilli and persisting throughout the fermentation (12 weeks). In contrast, the derivative strain could not be isolated after 7 weeks. Stability of both plasmid profile and bac⁺ (LPCO10 strain) or bac^- (55-1 strain) phenotype was shown by L. plantarum LPCO10 and L. plantarum 55-1 isolated throughout the fermentation. Bacteriocin activity could be found in the L. plantarum LPCO10-inoculated brines only after ammonium sulfate precipitation and concentration (20 times) of the final brine. Spontaneously occurring lactobacilli and lactic coccus populations, which were isolated from each of the fermenting brines studied during this investigation, were shown to be sensitive to the bacteriocins produced by L. plantarum LPCO10 when tested by the drop diffusion test. The declines in both pH and glucose levels throughout the fermentative process were similar in L. plantarum LPCO10- and in L. plantarum 55-1-inoculated brines and were comparable to the declines in the uninoculated brines. However, the final concentration of lactic acid in L. plantarum LPCO10-inoculated brines was higher than in the L. plantarum 55-1-inoculated brines and uninoculated brines. These results indicated that L. plantarum LPCO10 may be useful as a starter culture to control the lactic acid fermentation of Spanish-style green olives.     

New incompatibility groups for Staphylococcus aureus plasmids

Incompatibility relationships between naturally occurring staphylococcal plasmids conferring erythromycin or kanamycin resistance have been studied making use of recombinants between these plasmids and pSA0301, a temperature-sensitive mutant plasmid determining tetracycline resistance. The four plasmids encoding kanamycin resistance fall in two incompatibility groups; similarly, the three plasmids responsible for erythromycin resistance belong to two other incompatibility groups. This brings the number of distinct incompatibility groups reported for Staphylococcus aureus plasmids to 13.     

Assessment of Pediococcus acidilactici as a Potential Silage Inoculant

Eighteen Pediococcus strains were screened for their potential as silage inoculants. Pediococcus acidilactici G24 was found to be the most suitable, exhibiting a short lag phase on both glucose and fructose, a rapid rate of acid production, a high sugar-to-lactate conversion efficiency, no detectable breakdown of proteins or lactic acid, and the ability to grow within a broad range of pH and temperature. When tested in laboratory silos using grass with a water-soluble carbohydrate content of 24 g/kg of aqueous extract, P. acidilactici G24 stimulated the natural Lactobacillus plantarum population and accelerated the rates of lactic acid production and pH decrease. After 6 days of fermentation, the inoculated silage exhibited a 12% decrease in ammonia nitrogen and an 11% increase in crude protein levels compared with uninoculated controls. The use of an L. plantarum inoculant at a rate of 10⁴ bacteria per g of grass in conjunction with P. acidilactici G24 produced no additional beneficial effect. Inoculation of grass with a water-soluble carbohydrate level of 8 g/kg of aqueous extract with P. acidilactici G24 led to no acceleration in the rate of L. plantarum growth or pH decrease. However, after 7 days of fermentation the inoculated silage had a 14% lower ammonia nitrogen protein content than did uninoculated controls. The results suggest that P. acidilactici G24 may be useful as a silage inoculant for crops with a sufficiently high water-soluble carbohydrate level.     

Characterization of a mutant of thiostrepton-producing Streptomyces azureus ATCC 14921 and its plasmids

A mutant, strain PK10, of Streptomyces azureus ATCC 14921 and its two plasmids were characterized and compared with another mutant, PK 100, and its plasmid. One PK 10 plasmid of 8.8 kb was identical to a pock-forming plasmid, pSA1.1, of PK100. The other olasmid which was found only in PK10 nd named pSA1.2 (size, 7.6 kb), was a non-pock forming derivative of pSA1.1 with deletions in two different regions (about 1.2 kb and 30 b long). The pcok-forming ability of strain PK10 on a plasmid-free strain was lower than that of strain PK100 which contained only pSA1.1. Strain PK10 had fewer copies of pSA1.1 than strain PK100, and had normal spore formation and thiostrepton production, which were depressed in the strain PK100. The pSA1.1 from both PK10 and PK100 amplified to 20 to 30 copies in the transformants and inhibited theri spore formation and thiostrepton production. Thus, the function of pSA1.1 appeared to be depressed by pSA1.2.     

Sequence analysis and characterization of two cryptic plasmids derived from Lactobacillus buchneri CD034

Lactobacillus buchneri is probably the most beneficial microorganism for efficient preservation of animal feed silages made from grass, maize and other plant material against aerobic spoilage. Its obligatory heterofermentative nature, acid resistance and robustness have drawn attention to this species for applications as silage starter culture as well as for genetic engineering. For the first time, two cryptic plasmids present in the same L. buchneri strain, L. buchneri CD034, were isolated, sequenced and characterized. The larger plasmid, designated pCD034-1 was found to be 3424 bp in length with a G + C content of 38.36%. The smaller plasmid, designated pCD034-2 was found to be 2707 bp in length with a G + C content of 38.60%. On both plasmids we predicted three open reading frames. On pCD034-1, ORF 1 encodes a putative replication protein which shares 99% identity with the RepA protein of a Lactobacillus plantarum derived pC194/pUB110-family plasmid. ORF 2 encodes a putative protein of unknown function. ORF 1 and ORF 2 of pCD034-2 correspond to RepA and RepB proteins similar to those of plasmid pLB4 from L. plantarum. ORF 3 of both plasmids encodes a putative mobilization protein similar to that of the pediococcal plasmid pF8801. Double strand origins, putative single strand origins and typical mobilization start signals were identified. Both plasmids were shown to be maintained at relatively high plasmid copy numbers. Two shuttle vectors carrying the origins of replication of pCD034-1 and pCD034-2 were constructed and used to successfully transform two other species isolated from the same environment. Hence, we consider the two novel L. buchneri plasmids a valuable resource for the generation of shuttle and expression vectors for LAB.     

Isolation and Characterization of a Plasmid fromLactobacillus fermentumConferring Erythromycin Resistance

Lactobacillus fermentumis a lactic acid bacterial species commonly found in the digestive tracts of pigs and rodents and also present in man. We characterized a 5.7-kb plasmid, pLEM3, conferring erythromycin resistance, which was isolated from a porcine strain ofL. fermentum.Plasmid pLEM3 established efficiently inL. fermentum,conferred high-level erythromycin resistance (MIC > 1 mg/ml), and was segregationally stable. A deletion derivative of pLEM3, called pLEM5, was constructed and found to be as genetically stable as the parent. A multiple cloning site was inserted into pLEM5, generating plasmid pLEM7. Nucleotide sequence determination of pLEM5 revealed similarities with known genes. The replicon itself is a member of the pC194 family of rolling circle plasmids. The region responsible for erythromycin resistance was 98.2% identical to theermgene of conjugative transposon Tn1545.     

Modification of in vitro metabolism of T-2 toxin by esterase inhibitors.

A shuttle vector that can replicate in both Streptococcus spp. and Escherichia coli has been constructed by joining the E. coli plasmid pACYC184 (chloramphenicol and tetracycline resistance) to the streptococcal plasmid pGB305 (erythromycin resistance). The resulting chimeric plasmid is designated pSA3 (chloramphenicol, erythromycin, and tetracycline resistance) and has seven unique restriction sites: EcoRI, EcoRV, BamHI, SalI, XbaI, NruI, and SphI. Molecular cloning into the EcoRI or EcoRV site results in inactivation of chloramphenicol resistance, and cloning into the BamHI, SalI, or SphI site results in inactivation of tetracycline resistance in E. coli. pSA3 was transformed and was stable in Streptococcus sanguis and Streptococcus mutans in the presence of erythromycin. We have used pSA3 to construct a library of the S. mutans GS5 genome in E. coli, and expression of surface antigens in this heterologous host has been confirmed with S. mutans antiserum. A previously cloned determinant that specifies streptokinase was subcloned into pSA3, and this recombinant plasmid was stable in the presence of a selective pressure and expressed streptokinase activity in E. coli, S. sanguis (Challis), and S. mutans.     

Recombinant Lactobacillus plantarum expressing and secreting heterologous oxalate decarboxylase prevents renal calcium oxalate stone deposition in experimental rats

Background

Calcium oxalate (CaOx) is the major constituent of about 75% of all urinary stone and the secondary hyperoxaluria is a primary risk factor. Current treatment options for the patients with hyperoxaluria and CaOx stone diseases are limited. Oxalate degrading bacteria might have beneficial effects on urinary oxalate excretion resulting from decreased intestinal oxalate concentration and absorption. Thus, the aim of the present study is to examine the in vivo oxalate degrading ability of genetically engineered Lactobacillus plantarum (L. plantarum) that constitutively expressing and secreting heterologous oxalate decarboxylase (OxdC) for prevention of CaOx stone formation in rats. The recombinants strain of L. plantarum that constitutively secreting (WCFS1OxdC) and non-secreting (NC8OxdC) OxdC has been developed by using expression vector pSIP401. The in vivo oxalate degradation ability for this recombinants strain was carried out in a male wistar albino rats. The group I control; groups II, III, IV and V rats were fed with 5% potassium oxalate diet and 14^th day onwards group II, III, IV and V were received esophageal gavage of L. plantarum WCFS1, WCFS1OxdC and NC8OxdC respectively for 2-week period. The urinary and serum biochemistry and histopathology of the kidney were carried out. The experimental data were analyzed using one-way ANOVA followed by Duncan’s multiple-range test.

Results

Recombinants L. plantarum constitutively express and secretes the functional OxdC and could degrade the oxalate up to 70–77% under in vitro. The recombinant bacterial treated rats in groups IV and V showed significant reduction of urinary oxalate, calcium, uric acid, creatinine and serum uric acid, BUN/creatinine ratio compared to group II and III rats (P < 0.05). Oxalate levels in kidney homogenate of groups IV and V were showed significant reduction than group II and III rats (P < 0.05). Microscopic observations revealed a high score (4+) of CaOx crystal in kidneys of groups II and III, whereas no crystal in group IV and a lower score (1+) in group V.

Conclusion

The present results indicate that artificial colonization of recombinant strain, WCFS1OxdC and NC8OxdC, capable of reduce urinary oxalate excretion and CaOx crystal deposition by increased intestinal oxalate degradation.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0086-y) contains supplementary material, which is available to authorized users.     

Cloning and analysis of bile salt hydrolase genes from Lactobacillus plantarum CGMCC No. 8198

Genes coding for bile salt hydrolase of Lactobacillus plantarum CGMCC 8198, a novel probiotic strain isolated from silage, were identified, analyzed and cloned. L. plantarum strongly resisted the inhibitory effects of bile salts and also decreased serum cholesterol levels by 20 % in mice with hypercholesterolemia. Using RT-PCR analysis, bsh2, bsh3 and bsh4 were upregulated by bile salts in a dose-dependent manner. All three bsh genes had high similarity with those of other Lactobacillus strains. All three recombinant BSHs had high activities for the hydrolysis of glycodeoxycholic acids and taurodeoxycholic acids.     

Gene encoding a replication initiator protein and replication origin of conjugative plasmid pSA1.1 of Streptomyces cyaneus ATCC 14921

pSA1.1 is a 9.1-kb multicopy plasmid originally isolated from Streptomyces cyaneus (formerly S. azureus) ATCC 14921. This plasmid accumulates single-stranded DNA in S. lividans and is therefore considered to replicate by a rolling-circle replication. In the present work, the rep gene encoding the replication initiator protein and the replication origin ori of pSA1.1 were determined. The rep and ori are located on separate regions. The Rep protein of pSA1.1 belongs to superfamily I which includes A proteins of phages. Nucleotide sequence of the surrounding putative nicking site of pSA1.1 shows good agreement with those of the pC194 group plasmids and phages. The direction of replication was also determined.     

High level expression of a heterologous protein in Lactobacillus plantarum and its effect on the persistence of the recombinant strain in silage

Summary This paper reports the high level expression of the Staphylococcus aureus cat gene in Lactobacillus plantarum using the expression vector pMTL500F. When the recombinant strain of L. plantarum was grown in pure culture, CAT contributed 1.4% of the total soluble cell protein. The recombinant strain of L. plantarum continued to express a high level of CAT when inoculated into silage, the heterologous protein constituting up to 1.75% of the total soluble cell protein. The recombinant L. plantarum strain was still able to survive and proliferate when inoculated into silage, despite its additional metabolic load.     

Biological Role of Xanthomonadin Pigments in Xanthomonas campestris pv. Campestris

Previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenic Xanthomonas bacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. We used a Xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. Although xanthomonadin mutant strains were comparable to the parent strain for survival when exposed to UV light; after their exposure to the photosensitizer toluidine blue and visible light, survival was greatly reduced. Chromosomally restored mutant strains were completely restored for survival in these conditions. Likewise, epiphytic survival of a xanthomonadin mutant strain was greatly reduced in conditions of high light intensity, whereas a chromosomally restored mutant strain was comparable to the parent strain for epiphytic survival. These results are discussed with respect to previous results, and a model for epiphytic survival of X. campestris pv. campestris is presented.     

Influence of nitrogen fertilization on tropical‐grass silage assessed by ensiling process monitoring using chemical and microbial community analyses

Aims: Utilization of silage in livestock farming is expected to increase in developing countries in the tropical and subtropical parts of the world. The aim of this study was to investigate the influence of nitrogen fertilization on the chemical composition of herbage, ensiling process and silage quality, and to contribute to the improvement of tropical‐grass silage preparation. Methods and Results: Guinea grass grown under two different nitrogen‐fertilizer application conditions [1·5 kg N a^?1 (high‐N) and 0·5 kg N a^?1 (low‐N)] was packed in plastic bags, and its ensiling process was investigated by chemical and microbial‐community analyses. Relatively well‐preserved silage was obtained from high‐N herbage, which accumulated a high nitrate concentration. Denaturing gradient gel electrophoresis analysis revealed that Lactobacillus plantarum dominated throughout the ensiling of high‐N herbage and in the early phase of that of low‐N herbage. In low‐N silages prepared from ammonium sulfate‐ and urea‐fertilized herbage, Lact. plantarum was replaced by clostridia after 40 and 15 days of ensiling, respectively. Conclusions: Nitrate content of herbage is an important factor that influences silage quality, and careful fertilization management can facilitate stable and successful fermentation of tropical‐grass silage without any pretreatment. Significance and Impact of the Study: The positive effect of nitrate on the ensiling process of tropical‐grass was proved by microbial‐community analysis.     

Vector-free cloning of a bacterial endo-1,4-β-glucanase in Lactobacillus plantarum and its effect on the acidifying activity in silage: Use of recombinant cellulolytic Lactobacillus plantarum as silage inoculant

In this research, the advantage of use of cellulolytic recombinant Lactobacillus plantarum as microbial inoculants for alfalfa silage fermentation was evaluated. To such purpose, two L. plantarum strains, one (L. plantarum Lp80) currently commercialised and the other (L. plantarum B41) suitable as silage microbial additive, were genetically modified by integration of celA gene, encoding an alkaline endo-1,4--glucanase from Bacillus sp., in the chromosome, by means of a vector-free cloning technique. The heterologous gene was cloned in two fashions: preceded by two promoters (AC1 modification) or in translational coupling with a partial upstream ORF (AC2 modification). Therefore two different genetically modified organisms (GMOs) per each wild-type (WT), producing 43–59 U/l cellulase in 16 h, were examined. Thirty-five micro-ensiling experiments were carried out by inoculating the WT or the derived GMOs. L. plantarum B41AC1 cellulolytic clone exhibited significantly increased acidification capacity in silage samples incubated at 37°C. No advantage of use was evident for the other GMOs.     

Expression of a Clostridium thermocellum endoglucanase gene in Lactobacillus plantarum

Recombinant plasmid pM25 containing the celE gene of Clostridium thermocellum, which codes for an enzymatically active endoglucanase, was transformed into Lactobacillus plantarum by electroporation. Strains harboring pM25 expressed thermostable endoglucanase, which was found predominantly in the culture medium. Two other plasmids, pGK12 and pSA3, were transformed into L. plantarum, and the stability of each plasmid was evaluated.     

Properties of the Probiotic Strain Lactobacillus plantarum 8-RA-3 Grown in a Biofilm by Solid Substrate Cultivation Method

The biofilm formation took place in 48?h within the solid substrate cultivation of Lactobacillus plantarum 8-RA-3 strain on the wheat bran saturated with the MRS medium. The drying of the bran fermented by lactobacilli resulted in a decrease in the number of colony-forming units (CFU) from 23.0?×?10⁸ to 6.9?×?10⁵?CFU/g in daily samples and to less than 10⁴?CFU/g in 2- and 3-day samples. However, according to the fluorescence-based live/dead assay data, more than 40?% of the non-cultured bacteria were viable. As a result of mice kept on a diet with the introduction of bran fermented by Lact. plantarum 8-RA-3 for 72?h into the fodder, a recovery of normal level of intestinal lactobacilli, inhibited by administration of antibiotic was noted. The strain genetically identical to the Lact. plantarum 8-RA-3 was isolated from the feces of these mice. The results indicate that solid substrate cultivated Lact. plantarum 8-RA-3 strain formed a biofilm. Once dried and transferred into a non-cultured state, biofilm cells retained its viability and biological activity.     

Expression of a Clostridium thermocellum endoglucanase gene in Lactobacillus plantarum.

Recombinant plasmid pM25 containing the celE gene of Clostridium thermocellum, which codes for an enzymatically active endoglucanase, was transformed into Lactobacillus plantarum by electroporation. Strains harboring pM25 expressed thermostable endoglucanase, which was found predominantly in the culture medium. Two other plasmids, pGK12 and pSA3, were transformed into L. plantarum, and the stability of each plasmid was evaluated.     

Characterization,Identification and Application of Lactic Acid Bacteria Isolated from Forage Paddy Rice Silage

There has been growing interest to develop forage rice as a new feed resource for livestock. This study was to characterize the natural population of lactic acid bacteria (LAB) and select potentially excellent strains for paddy rice silage preparation in China. One hundred and twenty-six strains were isolated and screened from paddy rice silage prepared using a small-scale fermentation system, and ninety-nine of these isolates were considered to be LAB based on their Gram-positive and catalase-negative morphology and the production of most of their metabolic products as lactic acid. These isolates were divided into eight groups (A-H) on the basis of their morphological and biochemical characteristics. The Group A to H strains were identified as Lactobacillus (L.) plantarum subsp. plantarum (species ratio: 8.1%), L. casei (5.1%), Leuconostoc (Ln.) pseudomesenteroides (11.1%), Pediococcus (P.) pentosaceus (24.2%), Enterococcus (E.) mundtii (12.1%), Lactococcus (Lc.) garvieae (15.2%), E. faecium (9.1%) and Lc. lactis subsp. lactis (15.2%) based on sequence analyses of their 16S rRNA and recA genes. P. pentosaceus was the most abundant member of the LAB population in the paddy rice silage. A selected strain, namely L. casei R 465, was found to be able to grow under low pH conditions and to improve the silage quality with low pH and a relatively high content of lactic acid. This study demonstrated that forage paddy rice silage contains abundant LAB species and its silage can be well preserved by inoculation with LAB, and that strain R 465 can be a potentially excellent inoculant for paddy rice silage.     

Characterization and transferability of Clostridium perfringens plasmids.

Two strains of Clostridium perfringens resistant to clindamycin (Cl), chloramphenicol (Cm), erythromycin (Em), and tetracycline (Tc) were isolated in France in 1974 and 1975. For one of these strains, curing experiments and molecular characterization of the extrachromosomal DNA clearly demonstrate the existence of two plasmids, plP401 (54 kilobases) and plP402 (63 kilobases), which, respectively, code for Tc Cm and Em Cl resistance. With mixed cultures, the Tc Cm plasmid is transferable to sensitive strains of C. perfringens; a segregation of these markers is frequently observed during mating experiments. In contrast, the transfer of the naturally occurring plasmid Em Cl does not occur at a significant rate. In performing transfer experiments in axenic mice, we obtained a Cl^r Em^r Tc^r transcipient whose chromosomal properties are those of a hybrid. When used in mating as a parental strain, this strain promotes chromosomal gene exchange. The role of the plasmid in this phenomenon is discussed, these transcipients being generally Cl^r Em^r Tc^r. The plasmid transfer is not limited to antibiotic resistance plasmids, the transferability of a bacteriocinogenic plasmid, plP404, harbored by C. perfringens BP6K-N5 being shown also. The transfer mechanism remains to be proved; it might be a conjugation process, a cell-to-cell contact being necessary for the transfer.