Production, regeneration and fusion of protoplasts in lactic streptococci

Gasson, M. J.

doi:10.1111/j.1574-6968.1980.tb05615.x

Cited by 73 publications

(18 citation statements)

References 7 publications

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“…1 × 10 9 cfu ml -l) were harvested during late exponential growth by centrifugation (12000 ×g, 30 min) at 4°C and washed twice in an equal volume of phosphate buffered saline (PBS). Washed bacteria were resuspended in 1.0% of the original culture volume of protoplasting buffer [11].…”

Section: Bacteriamentioning

confidence: 99%

Immune modification of the pathogenesis of Streptococcus uberis mastitis in the dairy cow

Hill¹

1994

FEMS Immunology and Medical Microbiology

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Two groups of 4 cows were vaccinated subcutaneously with live Streptococcus uberis strain 0140J or a surface extract derived from the same strain, at 14 days prior to the cessation of lactation (drying off) and at calving. Both groups also received an intramammary administration of the surface extract 7 days after drying off. A third group of unvaccinated animals acted as controls. Following intramammary challenge of two quarters per cow with the vaccine strain, all quarters on control cows and those vaccinated only with surface extract developed clinical mastitis. However, only 12.5% of challenged quarters on cows which were vaccinated with live bacteria developed clinical mastitis. In addition, the numbers of bacteria in the milk following challenge were 10(5) times higher from the control and extract vaccinated cows than those which received live vaccine. Serum levels of S. uberis specific IgG2 were elevated in the animals vaccinated with the live organism when compared to that of either extract-vaccinates or controls, whilst S. uberis specific levels of IgG1 and IgM were similar in all groups throughout the experiment. Specific antibody levels in milk were unaffected by vaccination. Despite increased levels of IgG2, no increase in opsonic activity was detected in any serum or milk samples. Peripheral blood lymphocytes from animals vaccinated with live organisms showed a considerable increase in proliferative response to S. uberis antigen in vitro when compared with lymphocytes from control and extract-vaccinated animals. These results suggest that neutrophils and specific opsonising antibody may not form the major defence against infection with S. uberis.

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Section: Bacteriamentioning

confidence: 99%

Immune modification of the pathogenesis of Streptococcus uberis mastitis in the dairy cow

Hill¹

1994

FEMS Immunology and Medical Microbiology

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“…Further purification of plasmid DNA by CsCl/ethidium bromide density-gradient centrifugation, and all subsequent manipulations of DNA in vitro and in E. coli, were performed as described by Maniatis et al (1982). L. lactis protoplasts were prepared from exponentially growing cultures as described previously (Gasson, 1980) and were transformed according to the method of Kondo & McKay (1984). Transformants were isolated on M17 agar plates containing 0.5 M-sucrose and 5 pg chloramphenicol ml-l using a top agar overlay.…”

Section: Media Reagents and Enzymesmentioning

confidence: 99%

Structure and Expression of the Lactococcus lactis Gene for Phospho- -galactosidase (lacG) in Escherichia coli and L. lactis

Vos

Gasson

1989

Microbiology

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The Lactococcus lactis subsp. lactis 712 lacG gene encoding phospho-beta-galactosidase was isolated from the lactose mini-plasmid pMG820 and cloned and expressed in Escherichia coli and L. lactis. The low phospho-beta-galactosidase activity in L. lactis transformed with high-copy-number plasmids containing the lacG gene contrasted with the high activity found in L. lactis containing the original, low-copy-number lactose plasmid pMG820, and indicated that the original lactose promoter was absent from the cloned DNA. In E. coli the phospho-beta-galactosidase could be overproduced using the strong inducible lambda PL promoter, which allowed a rapid purification of the active enzyme. The complete nucleotide sequence of the L. lactis lacG gene and its surrounding regions was determined. The deduced amino acid sequence was confirmed by comparison with the amino acid composition of the purified phospho-beta-galactosidase and its amino-terminal sequence. This also allowed the exact positioning of the lacG gene and identification of its characteristic Gram-positive translation initiation signals. The homologous expression data and the sequence organization of the L. lactis lacG gene indicate that the gene is organized into a large lactose operon which contains an intergenic promoter located in an inverted repeat immediately preceding the lacG gene. The organization and sequence of the L. lactis lacG gene were compared with those of the highly homologous lacG gene from Staphylococcus aureus. A remarkable bias for leucine codons was observed in the lacG genes of these two species. Heterogramic homology was observed between the deduced amino acid sequence of the L. lactis phospho-beta-galactosidase, that of the functionally analogous E. coli phospho-beta-glucosidase, and that of an Agrobacterium beta-glucosidase (cellobiase).

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“…Gasson (1980) first demonstrated protoplast fusion in lactococci. Since it is a relatively simple and unsophisticated technique and is neither strainnor species-specific, it has been considered as a viable approach to achieving gene exchange in lactic acid bacteria, particularly before the advent of electroporation, when other gene transfer methods were only applicable to a few strains.…”

Section: Protoplast Fusionmentioning

confidence: 99%

“…Protoplast fusion was employed as a gene transfer system for lactic streptococci prior to the introduction of a protoplast transformation system (Gasson, 1980). Intrageneric and intergeneric fusion of lactobacilli containing plasmids with selectable antibiotic resistance markers (Cocconcelli et al, 1986;Iwata et al, 1986) has been reported.…”

Section: Liposome-mediated Protoplast Transfectionfusion and Transfomentioning

confidence: 99%

Genetics and Biotechnology of Lactic Acid Bacteria

Gasson¹,

Vos²

1994

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Production, regeneration and fusion of protoplasts in lactic streptococci

Cited by 73 publications

References 7 publications

Immune modification of the pathogenesis of Streptococcus uberis mastitis in the dairy cow

Immune modification of the pathogenesis of Streptococcus uberis mastitis in the dairy cow

Structure and Expression of the Lactococcus lactis Gene for Phospho- -galactosidase (lacG) in Escherichia coli and L. lactis

Genetics and Biotechnology of Lactic Acid Bacteria

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