RESUMO
A commercial grass silage starter strain of Lactobacillus plantarum was transformed by high-frequency electroporation with plasmids containing an alpha-amylase gene from Bacillus stearothermophilus and an endoglucanase gene from Clostridium thermocellum. Both genes were expressed from their native regulatory signals, and active enzymes were found in the supernatant. However, the segregational stability of the transforming plasmids was rather low. Therefore, the transforming genes were inserted in the L. plantarum chromosome by means of single homologous recombination. In the majority of the transformants, this led to extremely stable segregation and expression of the transforming genes, without generating secondary mutations in the host. Increased selective pressure led to tandem amplification of the transforming DNA. The transformed strains demonstrated the ability of L. plantarum to express heterologous gene products; they can be used to detect the inoculum in silage ecology studies; and they demonstrate the feasibility of engineering truly cellulolytic silage starter bacteria.
Assuntos
Celulase/genética , Cromossomos Bacterianos/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Lactobacillus/enzimologia , alfa-Amilases/genética , Celulase/biossíntese , Celulase/metabolismo , Clonagem Molecular , DNA Bacteriano/genética , Escherichia coli/genética , Amplificação de Genes , Lactobacillus/genética , Fenótipo , Plasmídeos , Recombinação Genética , Mapeamento por Restrição , Transformação Bacteriana , alfa-Amilases/biossínteseRESUMO
Two plasmids, pLAB1000 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of Lactobacillus hilgardii. Both plasmids were cloned in Escherichia coli and characterized through restriction mapping. A 1.6-kb XbaI-SacI fragment of pLAB1000 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtilis. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLAB1000 plasmid. These could stably be maintained in Lactobacillus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLAB1000 have been observed in different strains of the related species L. plantarum.
