Conjugal transfer of group B streptococcal plasmids and comobilization of Escherichia coli-Streptococcus shuttle plasmids to Lactobacillus plantarum.

The antibiotic resistance group B streptococcal plasmids, pIP501 and pVA797, were conjugally transferred from Streptococcus faecalis to Lactobacillus plantarum. The Escherichia coli-Streptococcus shuttle plasmids, pVA838 and pSA3, were mobilized from S. sanguis to L. plantarum by pVA797 via cointegrate formation. pVA838 readily resolved from pVA797 and was present in L. plantarum as deletion derivatives. The pVA797::pSA3 cointegrate failed to resolve in L. plantarum.     

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.     

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.     

Use of the alr gene as a food-grade selection marker in lactic acid bacteria

Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Deltaalr) showed auxotrophy for D-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented D-alanine auxotrophy in the L. plantarum Deltaalr and L. lactis Deltaalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to D-cycloserine, a competitive inhibitor of Alr (600 and 200 micro g/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that D-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Deltaalr. The resulting strain could grow in the absence of D-alanine only when expression of the alr gene was induced with nisin.     

Plasmid profiles of ten strains of Lactobacillus plantarum

Abstract The presence of extrachromosomal DNA elements has been investigated in 10 strains of Lactobacillus plantarum .
8 of the strains contained from 1 to 6 plasmids of different M _r values spanning from 1.35 · 10⁶ to 15.4 · 10⁶.
6 of the strains are commonly used as starter cultures in dry sausage and all these strains contained plasmids. The remaining 4 strains were obtained from the American Type Culture Collection and only 2 of these strains were found to harbour plasmids.     

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.     

Growth and Survival of Genetically Manipulated Lactobacillus plantarum in Silage

The growth and persistence of two genetically manipulated forms of Lactobacillus plantarum NCDO (National Collection of Dairy Organisms) 1193 have been monitored in grass silage. Both recombinants contained pSA3, a shuttle vector for gram-positive organisms that encodes erythromycin resistance. In one of the recombinants, pSA3 was integrated onto the chromosome, whereas in the other, a pSA3 derivative designated pM25, which contains a Clostridium thermocellum cellulase gene cloned into pSA3, was maintained as an extrachromosomal element. This extrachromosomal element is a plasmid. Rifampin-resistant mutants were selected for the recombinants and the parent strain. When applied to minisilos at a rate of 10⁶ CFU/g of grass, both the recombinants and the parent strain proliferated to dominate the epiphytic microflora and induced an increase in the decline in pH compared with that of the noninoculated silos. The presence of extra genetic material did not appear to disadvantage the bacterium in comparison with the parent strain. The selective recovery of both strains by using rifampin and erythromycin was confirmed by Southern hybridization. Interestingly, the free plasmid (pM25) appeared more stable in silage than was expected from studies in MRS broth. The plasmid was retained by 85% of the rifampin-resistant L. plantarum colonies isolated from a day 30 silo. These data answer an important question by showing that genetically manipulated recombinants of L. plantarum can proliferate and compete with epiphytic lactic acid bacteria in silage.     

Instability of the plasmid carrying active penicillin acylase gene from Escherichia coli: conditions inducing insertional inactivation

Abstract The marker instability of the recombinant plasmid coding penicillin acylase production was investigated in Escherichia coli . In the absence of any selective pressure and under fully induced production of penicillin acylase the number of cells lacking plasmids increased during cultivation. Cells still harboring plasmids fully maintained their ability to synthesize the enzyme. However, when a selection for Tet^r was applied, the number of selected cells containing the actively synthesizing gene decreased. Changes occurring in plasmid DNA revealed high frequency of insertions affecting expression of penicillin acylase gene.     

Mobilization of CS fimbriae-associated plasmids of enterotoxigenic Escherichia coli of serotype O6: K15: H16 or H- into various wild-type hosts

Abstract CS fimbriae-associated plasmids of two enterotoxigenic Escherichia coli strains of serotype O6: K15: H16 or H- (biotypes A and F) with M _r values of 51 × 10⁶ and 72 × 10⁶, respectively, were mobilized into various alternative host bacteria. Expression of CS1 or CS2 fimbriae was obtained when either of the CS fimbriae-associated plasmids was introduced into CS Fim⁻, O6: K15: H16 or H- recipients with rhamnose-negative and rhamnose-positive fermentation phenotypes, respectively, whereas CS3 fimbriae were expressed irrespective of the biotype of the recipient. On transfer into a CS Fim⁻ variant of an enterotoxigenic O8: H9 strain and into two K-12 strains, a CS3-fimbriae-only phenotype was conferred by the presence of either of the plasmids. When a CS Fim⁻ variant of a Rha⁺ CS2-fimbriae-only strain of serotype O6: K15: H16 harboured either of the plasmids, both CS2 and CS3 fimbriae were expressed, indicating that the rare CS2-fimbriae-only wild-type phenotype is probably due to the presence of a defective plasmid in such strains. Mobilization of the 51 MDa CS fimbriae-associated plasmid into five non-enterotoxigenic Rha⁺ porcine isolates of E. coli with O6 serotypes other than O6: K15: H16 or H- yielded CS3-fimbriae-only transconjugants. Thus the correlation between a Rha⁺ fermentation phenotype and expression of CS2 fimbriae does not hold in general for O-group 6 strains.     

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.     

Water-relation parameters of giant and normal cells of Capsicum annuum pericarp

Abstract. Measurements of the water-relation parameters of the giant subepidermal cells (volume, V = 0.119 to 1.658 mm³; = 0.53±0.35 mm³, SD, n = 23) and the smaller mesocarp parenchyma cells ( V = 0.10 to 0.79×10⁻³ mm³; = 0.36±0.27×10⁻³ mm³, SD, n = 6) of the inner pericarp surface of Capsicum annuum L. were made using the Jülich pressure probe. The volumetric elastic modulus ɛ for the large cells was between 1.5 and 27 MPa for a pressure range of 0.09 to 0.41 MPa. For the small cells ɛ was 0.1 to 0.6 MPa for a pressure range of 0.22 to 0.39 MPa. The turgor pressure P , the half-time of water exchange T _1/2, and the hydraulic conductivity L _p were as follows, with SD and number of replicates: large cells, P = 0.27±0.06 MPa (23), T _1/2=2.7±2.2 s (46), L _p=5.8±3.7 pm s⁻¹ Pa⁻ (46); small cells, P = 0.33±0.07 MPa (6), T _1/2= 33±10s (12), L _p=0.21±0.07 pm s⁻¹ Pa⁻¹ (12). The determination of these basic water-relation parameters is considered as a prerequisite for future ecotoxicological and phytopathological studies. The differences between the large and the small cells are discussed in relation to a desirable biophysical definition of succulence. Further, for the large cells a pressure and volume dependence of ɛ was demonstrated.     

Dominance of Lactobacillus plantarum strains in grass silage as demonstrated by a novel competition assay

An assay was developed for assessing the competitive ability of potential Lactobacillus plantarum silage inoculants. This assay was based on the ability of the test inoculant to outcompete a standard strain ( Lact. plantarum DCU101) co-inoculated at the same rate of 5 × 10⁵ colony forming units g^-1 of grass. Total populations of Lact. plantarum were enumerated with a selective medium and Lact. plantarum DCU101 was identified with a strain-specific DNA probe. The DNA probe was based on a small (2.2 kb), cryptic, indigenous plasmid which was cloned into pAT153, a multicopy cloning vector. Seven Lact. plantarum strains, six of which were isolated from well-preserved grass silages, were used to inoculate laboratory scale silos, and variation in strain dominance was monitored over the 14 d ensilage period.     

Development of an amylolytic Lactobacillus plantarum silage strain expressing the Lactobacillus amylovorus alpha-amylase gene.

An amylolytic Lactobacillus plantarum silage strain with the starch-degrading ability displayed by Lactobacillus amylovorus was developed. An active fragment of the gene coding for alpha-amylase production in L. amylovorus was cloned and integrated into the chromosome of the competitive inoculant strain L. plantarum Lp80 at the cbh locus. The alpha-amylase gene fragment was also introduced into L. plantarum Lp80 on an autoreplicative plasmid. Both constructions were also performed in the laboratory strain L. plantarum NCIB8826. All four recombinant strains secreted levels of amylase ranging from 23 to 69 U/liter, compared with 47 U/liter for L. amylovorus. Secretion levels were higher in L. plantarum NCIB8826 than in L. plantarum Lp80 derivatives and were higher in recombinant strains containing autoreplicative plasmids than in the corresponding integrants. The L. plantarum Lp80 derivative containing the L. amylovorus alpha-amylase gene fragment integrated into the host chromosome secreted alpha-amylase to a level comparable to that of L. amylovorus and was stable over 50 generations of growth under nonselective conditions. It grew to a higher cell density than either the parent strain or L. amylovorus in MRS medium containing a mixture of starch and glucose as the fermentable carbohydrate source. This recombinant alpha-amylolytic L. plantarum strain would therefore seem to have considerable potential as a silage inoculant for crops such as alfalfa, in which water-soluble carbohydrate levels are frequently low but starch is present as an alternative carbohydrate source.     

Electrotransformation of Lactobacillus manihotivorans LMG 18010T and LMG 18011

An electroporation procedure was developed for the genetic transformation of intact cells of Lactobacillus manihotivorans , a new Lactobacillus species isolated from cassava sour starch fermentation in Colombia. Transformation efficiency of Lact. manihotivorans strains LMG 18010^T and LMG 18011 was measured and compared with electroporability of Lact. plantarum strains NCIMB 8299 and LMG 9211, by investigating the effect of changes in various parameters. For Lact. manihotivorans strain LMG 18010^T, the composition of the culture medium, such as the type of peptone and the presence of Tween-80, was found to be the most critical parameter, as well as the aeration conditions of the culture; better electroporation was obtained without air. The presence of MgCl₂ in the recovery medium was favourable to regeneration of electroporated cells. Plasmid-curing of the cells did not improve their electroporability. Transformants were obtained with Lact. manihotivorans strain LMG 18010^T and the plasmids pLZ12 and pGK13, whereas Lact. manihotivorans strain LMG 18011 was transformable with plasmids pLP825 and pLZ12, with different electroporation conditions.     

Mutants of Acanthamoeba castellanii Resistant to Erythromycin, Chloramphenicol, and Oligomycin

SYNOPSIS. Cell lines of Acanthamoeba castellanii resistant to erythromycin (Ery^R), chloramphenicol (Cap^R), and oligomycin (Oli^R) have been isolated. These may be the first such mutants for A. castellanii. These mutants have been phenotypically stable for 2 years, surviving storage and vegetative multiplication in the absence of drugs. Resistance was specific for each drug, but double mutants (e.g. Ery^RCap^R) were obtained by stepwise selection. Mutant frequencies were determined in multiwell plates; <10 colony forming units (CFU/10⁵ amebas were observed in wild-type populations 12 days after incubation in 500 μg Ery/ml, 2.5 mg Cap/ml, or 15 μg Oli/ml. After 30 days, averages of 100 CFU/10⁵ amebas were observed in Ery and Cap, whereas, frequencies for Oli remained unchanged. Frequencies for Ery^R and Cap^R were consistent with rates of recovery from these drugs in batch cultures. We were unable to obtain spontaneous mutants resistant to cycloheximide, emetine, 5-fluorodeoxyuridine, or ethidium bromide. Ery^R, Cap^R and Oli^R could be mitochondrial mutants.     

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.     

Plasmid formation and its relation to the formation of spontaneously developing pocks in Streptomyces azureus ATCC 14921

Streptomyces azureus ATCC 14921 harboured a plasmid pSA1 together with its chromosomal integrated sequence (pSA1_int). The att P site on the plasmid was located at ca 170 bp Bam HI- Sph I fragment by site-specific integration. The free form was generated from the integrated sequence during the development of its host mycelia in the solid culture, but not in the liquid culture. The free form seemed to elicit the formation of spontaneously developing pocks on its host mycelia in the solid culture.     

Development of a minimal growth medium for Lactobacillus plantarum

Aim:  A medium with minimal requirements for the growth of Lactobacillus plantarum WCFS was developed. The composition of the minimal medium was compared to a genome-scale metabolic model of L. plantarum .
Methods and Results:  By repetitive single omission experiments, two minimal media were developed: PMM5 (true minimal medium) and PMM7 [a pseudominimal medium, supporting proper biomass formation of 350 mg l⁻¹ dry weight (DW)]. The specific growth rate of L. plantarum on PMM7 was found to be 50% and 63% lower when compared to growth on established growth media (chemically defined medium and MRS, respectively). Using a genome-scale metabolic model of L. plantarum , it was predicted that PMM5 and PMM7 would not support the growth of L. plantarum . This is because the biosynthesis of para- aminobenzoic acid ( p ABA) was predicted to be essential for growth. The discrepancy in simulated growth and experimental growth on PMM7 was further investigated for p ABA; a molecule which plays an important role in folate production. The growth performance and folate production were determined on PMM7 in the presence and absence of p ABA. It was found that a 12 000-fold reduction in folate pools exerted no influence on formation of biomass or growth rate of L. plantarum cultures when grown in the absence of p ABA.
Conclusion:  Largely reduced folate production pools do not have an effect on the growth of L. plantarum , showing that L. plantarum makes folate in a large excess.
Significance and Impact of the study:  These experiments illustrate the importance of combining genome-scale metabolic models with growth experiments on minimal media.     

Transformation of Neurospora crassa with recombinant plasmids containing the cloned glutamate dehydrogenase (am) gene: evidence for autonomous replication of the transforming plasmid.

We have characterized Neurospora crassa transformants obtained with plasmid pJR2, which consists of the Neurospora glutamate dehydrogenase (am) gene cloned in pUC8 and an am132 host strain which contains a deletion encompassing the cloned fragment. Every one of 33 transformants tested showed extreme meiotic instability: less than 1 or 2% am+ progeny were obtained in initial or successive backcrosses between am+ transformants and am132 or in intercrosses between am+ progeny. Furthermore, am+ progeny from backcrosses gave a high proportion of auxotrophic (am) mitotic segregants during vegetative growth. These results indicate that the am+ character is not stably integrated into chromosomal DNA in any of the transformants tested. Nuclear DNAs from six transformants were analyzed by Southern hybridization. All six transformants contained sequences homologous to pJR2. Four showed restriction fragments expected for unmodified pJR2, but most showed additional bands. Southern blots of undigested DNAs showed that the plasmid sequences are present predominantly in high-molecular-weight form (larger than 20 kilobases). Southern blots showed that auxotrophic (am) progeny from a backcross to am132 had lost restriction bands corresponding to free plasmid but retained additional bands, apparently integrated into chromosomal DNA in a nonfunctional manner. Considered together, these results are most reasonably interpreted as follows: recombinant plasmids containing the am+ gene can replicate autonomously in N. crassa, the free plasmids are present in oligomeric or modified form or both, and plasmid sequences also integrate at multiple sites in the deletion host but in a nonfunctional manner. An alternate interpretation--that tandem repeats of the plasmid are integrated into chromosomal DNA but eliminated during meiosis--cannot be completely excluded. However, stable integration of the am gene can be obtained under a variety of other conditions, viz., using the am gene cloned in a phage lambda vector (J. A. Kinsey and J. A. Rambosek, Mol. Cell. Biol. 4:117-122, 1984), using derivatives of pJR2, or using pJR2 to transform a frameshift mutant.     

Characterization and species recognition of Lactobacillus plantarum strains by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene

Twenty-one strains, labelled Lactobacillus plantarum or Lact. plantarum -like, and isolated from different natural sources, were characterized by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene using Hin dIII and Eco RI cleaved chromosomal DNA, together with Lact. plantarum ATCC 14917^T, Lact. pentosus ATCC 8041^T, Lact. plantarum ATCC 10776 and Lact. plantarum ATCC 8014. The fermentation patterns on API 50CH were recorded at 30°C and 37°C for all strains. The phenotypes were heterogeneous, and the ability to ferment 17 of the 49 carbohydrates varied. The fermentation of some carbohydrates, for example D-raffinose and D-arabitol, was temperature-dependent. Strains having identical API profiles were separated by the plasmid profile. All strains but one (affiliated to Lact. casei ) had identical 16S ribosomal DNA sequences ( Lact. plantarum/Lact. pentosus ). The RFLP study resulted in identical ribopatterns for 17 of the strains, including the type strain of Lact. plantarum (pattern A1). Four strains had related fragment patterns to that of Lact. plantarum sensu stricto; three of these strains had more than 60% DNA: DNA homology to the type strain of Lact. plantarum , and one had less than 50% DNA: DNA homology to Lact. plantarum ATCC 14917^T. Two strains had fragment patterns similar to the type strain of Lact. pentosus , and they had more than 80% DNA: DNA homology to Lact. pentosus ATCC 8041^T. One of the Lact. pentosus strains shared one band with the A1 pattern. The ribopatterns of Lact. plantarum were homogeneous (identical for 85% of the strains), irrespective of phenotype and source of isolation. RFLP of the 16S rRNA genes using Eco RI and Hin dIII might be used for species recognition of Lact. plantarum , but seems less suitable for strain typing.