Influence of lactococcal plasmid on the specific growth rate of host cells

AIMS: To investigate a lactococcal plasmid responsible for a reduction in growth rate of its host cell. METHODS AND RESULTS:Lactococcus lactis subsp. lactis biovar. diacetylactis DRC1 carries a high number of plasmids. The DRC1 wild-type strain was found to grow more slowly than a plasmid-free derivative of DRC1. The plasmids extracted from DRC1 together with an indicator plasmid were cotransformed into the plasmid-free strain DRC1021. A 7.4-kb cryptic plasmid, designated pDR1-1, was found to significantly affect the maximum specific growth rate ( micro max) of the host cell. Polymerase chain reaction (PCR) analysis was carried out in order to detect the presence of pDR1-1 in the other L. lactis strains. The micro max of the single pDR1-1-positive strain was determined to be the same as that of DRC1. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that pDR1-1 (or a pDR1-1-like plasmid) is a critical factor in the reduction of the micro max of DRC1, and that its effect on the micro max is significantly greater than that of any other coexisting plasmid.     

Growth Kinetics of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> ssp. <Emphasis Type="Italic">diacetylactis</Emphasis> Harboring Different Plasmid Content

The effect of plasmid content on growth of Lactococcus lactis ssp. diacetylactis harboring different plasmids and on plasmid stability was studied. Strain DRC-2C is a plasmid Lac⁺- and Prt⁺-free strain. Strain DRC-2 utilizes lactose as carbohydrate and has proteinase activity. The plasmid-free strain DRC-2C exhibited none of these features. Plasmid-encoded properties were clearly identified. Results showed that plasmid content decreased bacterial growth in terms of the specific growth rate determined. Slightly lower specific growth rate and lactic acid production were observed in the strain of higher plasmid content owing to the plasmid presence, causing metabolic burden to the host cell. The plasmid profile results showed that the number of bands in the two strains before and after fermentation were the same. This indicated that the plasmids were stably maintained and unchanged during the fermentation. Received: 27 July 2002 / Accepted: 27 August 2002     

Optimized transformation by electroporation of Lactococcus lactis IL1403

Summary Rolling circle replication of plasmid pGKV21 in L. lactis was a more stable replication mode than the theta replication of plasmid pIL253. When the plasmid pGKV21 was used to develop and optimize transformation of L. lactis by electroporation, optimum transformation was obtained using dense suspension of late-exponential phase cells subjected to a high voltage (12.5 kV/cm) and a short pulse time (0.5 ms) in the presence of a plasmid DNA. With plasmid vector pGKV21, a transformation frequency of 2.2 × 10⁸ transformants per g of DNA was obtained.     

Effect of Plasmid Incompatibility on DNA Transfer to Streptococcus cremoris

Several Streptococcus cremoris strains were used in protoplast transformation and interspecific protoplast fusion experiments with Streptococcus lactis and Bacillus subtilis, with pGKV110, pGKV21, and ΔpAMβ1 as the marker plasmids. ΔpAMβ1 is a 15.9-kilobase nonconjugative, deletion derivative of pAMβ1, which is considerably larger than the pGKV plasmids (approximately 4.5 kilobases). In general, ΔpAMβ1 was transferred more efficiently than the pGKV plasmids. Using electroporation, we were able to demonstrate that failure of efficient transfer for the pGKV plasmids was, except for one case, caused by incompatibility of these plasmids with resident plasmids of the recipient strain.     

Identification of Nisin-Producing Strains by Nisin-Controlled Gene Expression System

A specific method to identify nisin-producing strains was developed based on Nisin-Controlled gene Expression (NICE) vector pSec:Nuc. The plasmid pSec:Nuc was transformed into non-nisin-producing strain Lactococcus lactis NZ9000, a host commonly used for the NICE system. The generating strain L. lactis NZ9000/pSec:Nuc could sense extracellular inducer nisin and efficiently secrete a reporter protein Nuc, the staphylococcal nuclease (Nuc) into the medium. Instead of using purified nisin, the culture supernatants of nisin-producing strains were also used as inducers. Therefore, the NICE system could be used to identify nisin-producing strains. With this principle, 4 among 56 lactococci strains isolated from raw milk were identified as nisin producers. The results were further confirmed by polymerase chain reaction amplification with their genomic DNA as templates, and nucleotide sequencing revealed that three of them produced nisin A, and the others produced nisin Z. Those results made it possible to isolate and identify nisin-producing strains specifically and rapidly using NICE system.     

Effect of citrate on growth of Lactococcus lactis subsp. lactis in milk

The effect of citrate on the growth of Lactococcus lactis subsp. lactis var. diacetylactis in milk has been investigated. Five strains of Lactococcus lactis subsp. lactis var. diacetylactis were compared to their citrate-negative variants, which lack the plasmid coding for citrate permease. In most cases, acidification kinetics and the final bacterial concentration of pure cultures of parental and variant strains did not differ significantly. Co-cultures of parental and variant strains, however, systematically tended towards the predominance of parental strains. Citrate metabolism is responsible for this change, since the predominance of citrate-positive strains was not observed in the absence of citrate. Continuous culture in milk enabled the difference in growth rates between the parental strain Lactococcus lactis subsp. lactis var. diacetylactis CDI1 and its citrate-negative variant to be quantified by following changes in the populations of the two co-cultured strains. At 26 °C, the growth rate of the parental strain was 7% higher than that of its citrate-negative variant. These results show that citrate metabolism slightly stimulates the growth of lactococci in milk. Received: 18 February 1997 / Received revision: 2 May 1997 / Accepted: 4 May 1997     

Linear plasmids pWR1A and pWR1B of the yeast Wingea robertsiae are associated with a killer phenotype

Wingea robertsiae CBS6693 (synonym Debaryomyces robertsiae) was previously reported to harbor two cryptic linear plasmids, designated pWR1A (8.3 kb) and pWR1B (14.6 kb). Reexamination of a putative plasmid encoded killer phenotype involved UV-curing as well as a highly sensitive toxin assay. Killer activities of concentrated culture supernatants prepared from both, a plasmid carrying and a cured plasmid-free strain, were examined in liquid media. Supernatants collected from plasmid carrying strains subjected to cultures of the plasmid-free derivative had clear concentration-dependent inhibitory effects, whereas plasmid harboring cells were not affected. Incubation at 65 degrees C for 10 min totally destroyed the toxin. Since supernatants prepared from the plasmid-free strain did not possess such killer activity and the presence of the plasmids confered resistance, toxin as well as immunity functions appear plasmid encoded. Beyond this, chitin affinity chromatography and Western blot analysis proved plasmid specific expression and secretion of a protein displaying similarities to the alpha-subunit of the Kluyveromyces lactis killer toxin. The assay applied in this study will most probably allow disclosure of other hidden killer phenomena, which may have escaped detection by conventionally applied plate assays.     

Adsorption of bisphenol A by lactic acid bacteria,Lactococcus, strains

Ten strains of the genus Lactococcus were examined for their ability to remove bisphenol A [2, 2-bis(4-hydroxyphenyl)propane; BPA], which is known as an endocrine disrupter. Nine strains of the lactococci tested could remove BPA from media during growth, although the removal ratio was below 9%. When BPA was incubated with lyophilized cells of lactococci for 1 h, the concentration of BPA in the media was decreased by 9–62%. Especially, the highest removal ratio of BPA was observed for Lactococcus lactis subsp. lactis 712. The lactococci could adsorb BPA but not degrade it, because the lactococci maintained the ability to remove BPA from the medium after autoclaving. When the lyophilized cells of L. lactis subsp. lactis 712 were also incubated with six analogues of BPA, they effectively adsorbed hydrophobic compounds such as 2, 2′-diphenylpropane and bisphenol A dimethylether. The BPA-adsorbing ability of lactococci could be due to the hydrophobic binding effect. The removal ratio of BPA by L. lactis subsp. lactis 712 was increased after treatment with sodium dodecyl sulfate and decreased after digestion with trypsin. These results suggest that the hydrophobic proteins on cell surface may be involved in the BPA-adsorbing ability of lactococci.     

Atypical citrate‐fermenting Lactococcus lactis strains isolated from dromedary’s milk

Aim: The aim was to isolate and characterize Lactococcus strains with new properties compared to those of usual Lactococcus dairy starters derived from cow’s milk. Methods and Results: Algerian dromedary’s milk was screened for proteolytic isolates able to grow rapidly on agar milk medium. PCR experiments revealed that 74 proteolytic isolates belonged to the genus Lactococcus and harboured the prtP gene encoding the lactococcal cell‐surface proteinase. Among these, 85% were able to ferment citrate (Cit⁺ phenotype) and were classified as Lactococcus lactis ssp. lactis biovar. diacetylactis. This classification was confirmed after sequencing of the 16S rDNA gene of five Cit⁺ isolates. In contrast to dairy lactococci described in the literature, several Cit⁺ isolates exhibited a tolerance to 50°C (Ther⁺) and alkaline pH. Two genetic approaches allowed to show the presence of four independent plasmids (so‐called pTher, pPrt, pLac, pCit) associated with the four respective phenotypes: Ther⁺, cell‐surface proteinase activity PrtP (PrtP⁺), lactose catabolism (Lac⁺) and citrate utilization (Cit⁺). Two types of pCit plasmid were amplified by inverse PCR: class 1 was characterized by a 9‐kb plasmid harbouring the expected lactococcal citQRP operon and class 2 by a 23‐kb plasmid harbouring the Leuconostoc cit cluster (citI‐CitMCDEFGRP). Conclusions: This work enlarges knowledge of the biovariety diacetylactis by far mainly limited to the citrate‐fermenting ability and suggests that the cit plasmid system of some lactococcal strains could have been acquired from another lactic acid bacteria (Leuconostoc spp.). Significance and Impact of the Study: This study reveals new potential dairy lactococci starters of the biovariety diacetylactis able to grow rapidly in milk at a higher temperature in addition to their casein, lactose and citrate‐utilizing abilities.     

Expression of citrate permease gene of plasmid pCM1 isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7 in Lactobacillus casei L-49-4

Recombinant vector pJLECit (8,232 bp) was constructed using citrate permease gene contained in the 3,919-bp fragment of plasmid pCM1 (8,280 bp) isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7, repA and ori from pLU1, and pMB1 ori and the erythromycin resistance gene from pJIR418. Lactobacillus casei L-49-4 (plasmid-free mutant of strain L-49) harboring the constructed pJLECit converted citrate into diacetyl/acetoin. Citrate uptake rate of resting cells was the highest at pH 5.5 and 10 mM citrate concentration. Diacetyl formation activity by the cell-free extracts of Lb. casei L-49-4 (pJLECit) grown in de Man–Rogosa–Sharpe (MRS) broth was higher than that of cells grown in MRS broth without citrate. On the other hand, diacetyl reductase activity of cells grown in MRS broth was lower than that of cells grown in MRS broth without citrate.     

Establishment of taxonomic status of new prospective bacteriocin-synthesizing <Emphasis Type="Italic">Lactococci</Emphasis> strains of various origins

The taxonomic status of new prospective bacteriocin-synthesizing strains of mesophilic lactococci isolated from raw milk and milk products from different regions of Russia and also of strain F-119, obtained by protoplast fusion of two related strains with low bacteriocin-synthesizing activity, was established by classical methods of identification. The values of antibiotic activity displayed by the strains toward a test microorganism Bacillus coagulans were up to 4650 IU/ml, which is significantly higher than in natural lactococci strains. In spite of some differences in morphology, ability to ferment carbohydrates, requirements for nutrients, and antibiotic suspectability, the strains were identified as Lactococcus lactis subsp. lactis. The new strains differed from the classic nisin-producing strain L. lactis subsp. lactis MGU by a remarkably broad spectrum of bactericidal and fungicidal activity. Study of 16S rRNA gene sequences of new natural strains, fusants F-119 and another one obtained earlier, F-116, and their parental strains in comparison with reference strains confirmed the new strains’ taxonomic status as Lactococcus lactis subsp. lactis. The nucleotide sequences of 16S rRNA genes were deposited with GenBank under accession numbers EF100777-EF114305.     

Prebiotic-non-digestible oligosaccharides preference of probiotic bifidobacteria and antimicrobial activity against Clostridium difficile

Bifidobacterium breve 46, Bifidobacterium lactis 8:8 and Bifidobacterium longum 6:18 and three reference strains B. breve CCUG 24611, B. lactis JCM 10602, and Bifidobacterium pseudocatenulatum JCM 1200 were examined for acid and bile tolerance, prebiotic utilization and antimicrobial activity against four Clostridium difficile (CD) strains including the hypervirulent strain, PCR ribotype NAP1/027. B. lactis 8:8 and B. lactis JCM 10602 exhibited a high tolerance in MRSC broth with pH 2.5 for 30 min. B. breve 46 and B. lactis 8:8 remained 100% viable in MRSC broth with 5% porcine bile after 4 h. All six strains showed a high prebiotic degrading ability (prebiotic score) with galactooligosaccharides (GOS), isomaltooligosaccharides (IMOS) and lactulose as carbon sources and moderate degradation of fructooligosaccharides (FOS). Xylooligosaccharides (XOS) was metabolized to a greater extent by B. lactis 8:8, B. lactis JCM 10602, B. pseudocatenulatum JCM 1200 and B. longum 6:18 (prebiotic score >50%). All strains exhibited extracellular antimicrobial activity (AMA) against four CD strains including the CD NAP1/027. AMA of B. breve 46, B. lactis 8:8 and B. lactis JCM 10602 strains was mainly ascribed to a combined action of organic acids and heat stable, protease sensitive antimicrobial peptides when cells were grown in MRSC broth with glucose and by acids when grown with five different prebiotic-non-digestible oligosaccharides (NDOs). None of C. difficile strains degraded five prebiotic-NDOs. Whole cells of B. breve 46 and B. lactis 8:8 and their supernatants inhibited the growth and toxin production of the CD NAP1/027 strain.     

Growth kinetics on oligo- and polysaccharides and promising features of three antioxidative potential probiotic strains

Aims: To determine the antioxidative activity, glutathione production, acid and bile tolerance and carbohydrate preferences of Lactobacillus plantarum LP 1, Streptococcus thermophilus Z 57 and Bifidobacterium lactis B 933. Methods and Results: The intact bacteria exhibited antioxidative capacity against linolenic acid and ascorbate oxidation. The antioxidative activity of cell-free extracts was determined by chemiluminescent assay and agreed with total glutathione content. Superoxide dismutase was negligible in all the strains. Bile and gastric juice resistance was tested in vitro to estimate the transit tolerance in the upper gastrointestinal tract. Bifidobacterium lactis B 933 and L. plantarum LP 1 were more acid tolerant than S. thermophilus Z 57. All the strains were resistant to bile. Among 13 indigestible carbohydrates, galacto-oligosaccharides and fructo-oligosaccharides were utilized by all the strains and did not affect survival in human gastric juice. Conclusions: These potential probiotic strains exhibited antioxidative properties and good viability in gastric juice and bile may indicate tolerance to the transit through the upper gastrointestinal tract. Galacto-oligosaccharides and fructo-oligosaccharides are the most appropriate prebiotics to be used in effective synbiotic formulations. Significance and Impact of the Study: These results outline promising strains with antioxidative properties. Carbohydrate preferences can be exploited in order to develop synbiotic products.     

Transfer of plasmids pBR322 and pBR325 in wastewater from laboratory strains of Escherichia coli to bacteria indigenous to the waste disposal system.

Laboratory strains of Escherichia coli containing plasmid pBR325 (or pBR322) were coincubated with a mobilizer strain of E. coli (containing the conjugative plasmid R100-1) and a recipient strain of bacteria. Bacterial strains isolated from raw wastewater or a plasmid-free E. coli laboratory strain served as recipients. Transfer of the pBR plasmid into the recipient strain occurred during a 25-h coincubation in either L broth or sterilized wastewater; transfer frequencies were several orders of magnitude lower in wastewater. After the coincubation, recipients exhibited both plasmid-encoded phenotypic characteristics and an altered plasmid profile, as shown by agarose gel electrophoresis of purified plasmid DNA.     

Transfer of plasmids pBR322 and pBR325 in wastewater from laboratory strains of Escherichia coli to bacteria indigenous to the waste disposal system

Laboratory strains of Escherichia coli containing plasmid pBR325 (or pBR322) were coincubated with a mobilizer strain of E. coli (containing the conjugative plasmid R100-1) and a recipient strain of bacteria. Bacterial strains isolated from raw wastewater or a plasmid-free E. coli laboratory strain served as recipients. Transfer of the pBR plasmid into the recipient strain occurred during a 25-h coincubation in either L broth or sterilized wastewater; transfer frequencies were several orders of magnitude lower in wastewater. After the coincubation, recipients exhibited both plasmid-encoded phenotypic characteristics and an altered plasmid profile, as shown by agarose gel electrophoresis of purified plasmid DNA.     

Loss of Plasmid-Mediated Oligopeptide Transport System in Lactococci: Another Reason for Slow Milk Coagulation

Fast milk-coagulating (Fmc⁺) strains of lactococci are known to segregate slow milk-coagulating (Fmc⁻) variants, which has been attributed to loss of proteinase (Prt) activity encoded by plasmid DNA. It was found that the Fmc⁻phenotype could also be due to loss of a plasmid encoding an oligopeptide permease (Opp) system. InLactococcus lactissubsp.lactis(L. lactis) C2O, lactose metabolism (Lac) and Prt were linked to pJK550 and the Opp system to pJK430. InLactococcus lactissubsp.cremorisSK11, known to possess Prt on a 78-kb plasmid, DNA sequence analysis of a 7.4-kb region from the Lac plasmid, pSK11L, revealed that it possessed the Opp system. The Lac plasmid inL. lactisC2 encoded both the Prt and Opp systems. Fmc⁻derivatives ofL. lactisC2 were missing theprtgenes and had Opp integrated into the chromosome, possibly due to transposition events. Growth studies showed the Opp systems were functional and, in combination with Prt, produced the Fmc⁺phenotype.     

Improved homo <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid fermentation from xylose by abolishment of the phosphoketolase pathway and enhancement of the pentose phosphate pathway in genetically modified xylose-assimilating <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

In order to achieve efficient homo L-lactic acid fermentation from xylose, we first carried out addition of xylose assimilation ability to Lactococcus lactis IL 1403 by introducing a plasmid carrying the xylRAB genes from L. lactis IO-1 (pXylRAB). Then modification of xylose assimilation pathway was carried out. L. lactis has two pathways for xylose assimilation called the phosphoketolase pathway (PK pathway) that produces both lactic acid and acetic acid and the pentose phosphate pathway (PP pathway) that produces only lactic acid as a final product. Thus a mutant strain that disrupted its phosphokeolase gene (ptk) was constructed. The Δptk mutant harboring pXylRAB lacked the PK pathway and produced predominantly lactic acid from xylose via the PP pathway, although its fermentation rate slightly decreased. Further introduction of the transketolase gene (tkt) to disrupted ptk locus led restoration of fermentation rate and this was attributed to enhancement of the PP pathway. As a result, ptk::tkt strain harboring pXylRAB produced 50.1 g/l of L-lactic acid from xylose with a high optical purity of 99.6% and a high yield of 1.58 (moles per mole xylose consumed) that is close to theoretical value of 1.67 from xylose.     

Characterization of a plasmid involved with cointegrate formation and lactose metabolism in Lactococcus lactis subsp. lactis OZS1

A 55 kilobase (kb) plasmid (pOZS550) in the non-clumping Lactococcus lactis subsp. lactis strain OZS1 carrying genes for lactose metabolism was characterised. A mobilizable cointegrate plasmid which is formed between pOZS550 and pOZS448 carries the necessary information for conjugation and transfer. Cointegrate formation was found to involve an insertional element located on pOZS550. The insertion sequence was found to be identical to ISS1 located on pSK08 in the clumping L. lactis subsp. lactis strain ML3. Restriction maps of pOZS550 and pSK08 were similar suggesting a close ancestral relationship, although pSK08, in addition to the lactose metabolism genes, expressed genes for proteinase activity and cell clumping, which were not expressed by pOZS550, and carried two copies of ISS1 compared to one on pOZS550. Furthermore, hybridization of the 18 base pair inverted repeat, of the insertion sequence, with various L. lactis subsp. lactis strains and two L. lactis subsp. cremoris strains showed moderate to strong hybridization to one plasmid in each organism.     

Growth kinetics of Lactococcus lactis ssp diacetylactis harboring different plasmid content

The effect of plasmid content on growth of Lactococcus lactis ssp. diacetylactis harboring different plasmids and on plasmid stability was studied. Strain DRC-2C is a plasmid Lac(+)- and Prt(+)-free strain. Strain DRC-2 utilizes lactose as carbohydrate and has proteinase activity. The plasmid-free strain DRC-2C exhibited none of these features. Plasmid-encoded properties were clearly identified. Results showed that plasmid content decreased bacterial growth in terms of the specific growth rate determined. Slightly lower specific growth rate and lactic acid production were observed in the strain of higher plasmid content owing to the plasmid presence, causing metabolic burden to the host cell. The plasmid profile results showed that the number of bands in the two strains before and after fermentation were the same. This indicated that the plasmids were stably maintained and unchanged during the fermentation.     

Overexpression of the cat-86 Gene Is Associated with Thermosensitivity in Bacillus subtilis

Bacillus subtilis harboring the cat-86 constitutive plasmid pPL708C2 with an ochre mutation at the 9th codon (terc 9) was sensitive to chloramphenicol (Cm^s) and exhibited relative thermostability when heated at 47°C. Reversion to chloramphenicol resistance (Cm^r) occurred at a frequency of 5.4 × 10⁻⁸. All of the plasmid Cm^r revertants tested were thermosensitive. Similarly, wild-type pPL708C2 present in B. subtilis also rendered the bacterium thermosensitive. When a nonsense mutation is introduced at codon 141, however, this terc 141 variant of pPL708C2 failed to thermosensitize B. subtilis. Another variant of pPL708C2 that produces intact yet catalytically inactive CAT-86 has both His-16 and His-17 at the active site replaced by Pro. Nevertheless, cells of B. subtilis carrying this variant were thermosensitive. Plasmid-free and pPL708C2-bearing strains did not exhibit differences in major heat shock proteins. Electron micrographs revealed a threefold increase of inclusion bodies present in a strain harboring pPL708C2 when compared with those in an isogenic plasmid-free strain. Received: 26 July 1999 / Accepted: 30 August 1999