Transformation and fusion of Streptococcus faecalis protoplasts

Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains.

Section: Methodssupporting

confidence: 87%

Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability

Chopin¹,

Chopin²,

Rouault³

et al. 1986

“…The formation of bacterial protoplasts and the regeneration of their cell walls are essential features in the initial steps of development of either a protoplast transformation or a fusion system for strain improvement. Specific conditions for protoplast formation have been developed in plant cells (10,23,25), basidiomycetes (6), fungi (22), and many bacteria (4,5,8,12,15,16,18,19,24,27), including aerotolerant anaerobes such as Clostridium spp. (1,11,14).…”

mentioning

confidence: 99%

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium

Chen¹,

Ohmiya²,

Shimizu³

1986

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcusfaecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. (i) The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. (ii) The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37°C. (iii) Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.

“…(12), Streptococcus spp. (6,17,25), and many other species have successfully been bred, it was suggested that the success of hybrid formation by protoplast fusion depends on the taxonomic proximity of the strains (13). Recent results obtained in this area of investigation, however, have shown that this may not be the case, as has been described for the interspecific protoplast fusion between Streptomycesfradiae and Streptomyces bikiensis (9,19) and between Streptococcus cremoris and Streptococcus lactis (18).…”

Section: Discussionmentioning

confidence: 99%

Intergeneric protoplast fusion between Fusobacterium varium and Enterococcus faecium for enhancing dehydrodivanillin degradation

Chen¹,

Ohmiya²,

Shimizu³

1987

Intergeneric protoplast fusion between Fusobacterium varium (Pcs Glu+) and Enterococcus faecium (Pcr Glu-) was performed under strictly anaerobic conditions to improve dehydrodivanillin (DDV) degradation. The fusion frequency obtained from the selective medium (Pc+ Glu-) was about 0.9 x 10-5 to 1.3 x 10-5. The seven fusants isolated were all gram-negative anaerobes with rod shapes like that of F. varium and with main phenotypical properties of cocci like those of E. faecium such as esculin and starch hydrolysis, milk clotting, and lactate production. Five fusants showed enhanced DDV degradation activities that were 2 to 4 times higher than those of parental strains. Genetic relatedness between a fusant (FE7) and the parents was estimated by DNA-DNA Southern blot hybridization with 32P-labeled chromosomal DNA fragments of F. varium and E. faecium as respective probes. The fusant FE7 presented a very high cross-hybridization with both probes, indicating a high DNA homology between the fusant and both parental strains. Almost all the fusants obtained here have stably kept the properties described above for about 2 years. These results suggest that intergeneric gene transfer takes place through protoplast fusion and that the fusants that were obtained are stable recombinants.