Characterisation and Molecular Cloning of an Erythromycin Resistance Plasmid of Lactococcus lactis Isolated from Chicken Cecum

ABSTRACT Sixteen exopolysaccharide (EPS)-producing Lactococcus lactis strains were analyzed for the chemical compositions of their EPSs and the locations, sequences, and organization of theeps genes involved in EPS biosynthesis. This allowed the grouping of these strains into three major groups, representatives of which were studied in detail. Previously, we have characterized theeps gene cluster of strain NIZO B40 (group I) and determined the function of three of its glycosyltransferase (GTF) genes. Fragments of the eps gene clusters of strains NIZO B35 (group II) and NIZO B891 (group III) were cloned, and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase involved in the second step of repeating-unit synthesis. The NIZO B40 priming glucosyltransferase gene epsD was replaced with an erythromycin resistance gene, and this resulted in loss of EPS production. This epsD deletion was complemented with priming GTF genes from gram-positive organisms with known function and substrate specificity. Although no EPS production was found with priming galactosyltransferase genes from L. lactis orStreptococcus thermophilus, complementation with priming glucosyltransferase genes involved in L. lactis EPS andStreptococcus pneumoniae capsule biosynthesis could completely restore or even increase EPS production in L. lactis.

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Post-transformational rearrangement of an in vitro reconstructed group-A streptococcal erythromycin resistance plasmid

Plasmid ◽

10.1016/0147-619x(79)90058-1 ◽

1979 ◽

Vol 2 (4) ◽

pp. 605-616 ◽

Cited By ~ 39

Author(s):

Detlev Behnke ◽

Horst Malke ◽

Manfred Hartmann ◽

Friedrich Walter

Keyword(s):

Erythromycin Resistance ◽

Resistance Plasmid ◽

Group A

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A Plasmid Selection System in Lactococcus lactis and Its Use for Gene Expression in L. lactis and Human Kidney Fibroblasts

Applied and Environmental Microbiology ◽

10.1128/aem.68.10.5051-5056.2002 ◽

2002 ◽

Vol 68 (10) ◽

pp. 5051-5056 ◽

Cited By ~ 20

Author(s):

Jacob Glenting ◽

Søren M. Madsen ◽

Astrid Vrang ◽

Anders Fomsgaard ◽

Hans Israelsen

Keyword(s):

Lactococcus Lactis ◽

Fluorescent Protein ◽

Human Kidney ◽

Heterologous Proteins ◽

Selection System ◽

Erythromycin Resistance ◽

Homoserine Dehydrogenase ◽

Mammalian Expression ◽

Genes Encoding ◽

Green Fluorescent

ABSTRACT We report the development of a nonantibiotic and nonpathogenic host-plasmid selection system based on lactococcal genes and threonine complementation. We constructed an auxotrophic Lactococcus lactis MG1363Δthr strain which carries deletions in two genes encoding threonine biosynthetic enzymes. To achieve plasmid-borne complementation, we then constructed the minimal cloning vector, pJAG5, based on the genes encoding homoserine dehydrogenase-homoserine kinase (the hom-thrB operon) as a selective marker. Using strain MG1363Δthr, selection and maintenance of cells carrying pJAG5 were obtained in threonine-free defined media. Compared to the commonly used selection system based on erythromycin resistance, the designed complementation system offers a competitive and stable plasmid selection system for the production of heterologous proteins in L. lactis. The potential of pJAG5 to deliver genes for expression in eukaryotes was evaluated by insertion of a mammalian expression unit encoding a modified green fluorescent protein. The successful delivery and expression of genes in human kidney fibroblasts indicated the potential of the designed nonantibiotic host-plasmid system for use in genetic immunization.

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