Electroporation has been used to introduce DNA into both eucaryotic and procaryotic cells. Since current methods for the transfection and transformation of lactobacilli using protoplasts are slow, inefficient, and inconsistent, we evaluated electroporation as a simple alternative. In initial experiments, PL‐1 phage DNA (40 kb) was transfected into untreated cells of Lactobacillus casei by electroporation. Approximately 50% of the CFU survived pulses of 5000 V/cm at 25 μF; conditions found to be optimal for the transfection process. The β‐galactosidase‐encoding plasmid, pLZ15 (28 kb), and the vectors pSA3 (Emr), pLP825 (Cmr) and pNZ12 (Cmr) transformed L. casei at efficiencies of 1.1–8.5 × 104 transformants/μg DNA. Plasmid DNA isolated from pNZ12 containing transformants was used to retransform L. casei and was indistinguishable from authentic preparations of pNZ12 by gel electrophoretic analysis.
The 1.3-kilobase-pair insertion element ISLJ, originally isolated from Lactobacillus casei S-1, was found to have an extremely restricted host range. By DNA-DNA hybridizations performed with Southern transfers by using a cloned internal fragment of ISL1 as a molecular probe, it was found that only 3 of 19 L. casei strains examined contained sequences that hybridized to the ISLJ probe. In two of these, the hybridizing sequences were found on lactose plasmids. No homologous sequences were detected in a survey of 14 other Lactobacillus strains (9 species) and 15 strains of other bacteria (8 genera, 12 species).Although insertion sequence (IS) elements are varied, common, and widely distributed among gram-negative bacteria (4), only four IS-like elements in gram-positive bacteria have been identified and sequenced (1,8,14,17 Salmonella (6). The host range, distribution, and frequency of gram-positive IS elements are at present unknown. We report here the results of a survey of 48 bacterial strains, representative of several genera in addition to Lactobacillus, for sequences hybridizing to ISLI (Table 1).DNA was isolated from L. casei S-1 and C-9 by the methods described by . DNA isolation from other Lactobacillus strains, Streptococcus spp., Staphylococcus aureus, and Actinomyces viscosus was by published procedures (2, 7). The procedure used to prepare DNA from all other strains was essentially that described by Marmur (10
The goal of this study was to investigate the likelihood of developing useful transformation systems for coryneform bacteria. Two species of coryneform bacteria, Brevibacterium lactofermentum and Corynebacterium lilium, were transformed with chimeras constructed from pUB110 and a cryptic coryneform plasmid (pGX1901). C. lilium protoplasts were also efficiently transfected with phage CS1 DNA. High transformation and transfection frequencies were obtained after only 2 min of lysozyme treatment of lysozyme-sensitive mutants. A series of experiments was also conducted to determine whether DNA from other species of important industrial microbes from the genus Bacillus could be expressed in coryneform bacteria. Evidence of restriction of Bacillus subtilis DNA by B. lactofermentum was observed but could be overcome. A Bacillus amyloliquefaciens a-amylase gene (amyEBamp) was subcloned onto a plasmid able to replicate in B. lactofermentum. B. lactofermentum transformants for this plasmid expressed amylase activity and produced material cross-reactive to amylase antibody. Coryneform bacteria are a diverse group of gram-positive bacteria widely distributed in nature. Many nonpathogenic soil isolates are important in industrial fermentations of amino acids such as glutamic acid (6, 21) and nucleotides (12) and in the conversion of steroids (16). Extensive studies have centered on the biochemical analysis of their amino acid biosynthetic pathways (10), but until recently little work was published on genetic transfer systems in coryneform bacteria. The isolation of endogenous plasmids and subsequent construction of hybrid cloning vectors carrying antibiotic resistance determinants (5, 13, 16, 17, 22) and the development of protoplast transfection and transformation (5, 13, 17, 18, 22) have provided valuable tools for the manipulation of biochemical pathways in coryneform bacteria. The three groups that have reported the transformation of coryneform bacteria have all used chimeras of coryneform cryptic plasmids and drug resistance plasmids from either Ba(cillhs siubtilis or Esclherichia coli (5, 16, 17, 22). This report describes the construction and characterization of useful chimeric cloning vehicles based on a 4.45-kilobase cryptic plasmid (pGX1901) isolated from Br-eviba('rtil,n lacutofrrrmentumn ATCC 13869. Plasmid pGX1901 is similar to cryptic plasmids in other coryneform bacteria (9, 17, 22) and was sequenced by our colleagues at Genex Corp. (D. Filpula, J. Nagle, and P. Carter, manuscript in preparation). These chimeras and DNA from a lytic phage, CS1 (W. McClements and D. Lineberger, unpublished data), were used to optimize conditions for protoplast transformation * Corresponding author.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.