Bruce M. Chassy

Transformation of bacteria by electroporation

Abstract The introduction of DNA into bacteria by transformation is an essential step in the construction of recombinant strains. Recently, electroporation, or electropermeabilization, in which a brief high voltage electric discharge is used to render cells permeable to DNA, has revolutionized the transformation of bacteria. The technique is fast, simple, reproducible, frequently gives very high transformation frequency and appears to be applicable to a wide range of bacterial types previously thought untransformable. The technique can also offer advantages for transformable bacteria such as Escherichia coli .

A gentle method for the lysis of oral streptococci

Abstract A number of strains of oral streptococci, previously thought to be virtually lysozyme resistant, have been successfully lysed by a new procedure. The bacteria are grown in Brain-Heart Infusion supplemented with D,L-threonine which inhibits cell-wall cross linking. Spheroplasts are formed by treatment with lysozyme in 0.01 M Tris-HCl pH 8.2–12% polyethylene glycol. The spheroplasts are harvested by centrifugation and lysed by the addition of 1% sodium dodecyl sulfate. High MW DNA and plasmids have been isolated from cells treated in this manner.

Strategies for the development of bacterial transformation systems

An effective transformation system is a prerequisite for facile genetic manipulation of bacteria. Bacteria may be naturally competent for transformation or may be treated with various agents, such as Tris buffers or divalent metal ions, to induce competence. Transformation can also be accomplished by electroporation, or by fusion of protoplasts with PEG in the presence of transforming DNA. Unfortunately, the mechanism by which cells become permeable to DNA and the process by which DNA enters the cells is frequently unknown. In order to establish a transformation system for an untransformable bacterium, recipient strains and transforming DNA must be carefully selected. Since it is impossible to predict in advance which method of transformation will be successful with a particular bacterial strain, several techniques are usually evaluated. This review describes a number of factors that appear to be critical for developing a transformation system and presents a strategy for experimentation with novel bacteria.

Cyclic Nucleotide Phosphodiesterase in Dictyostelium discoideum: Interconversion of Two Enzyme Forms

An extracellular cyclic nucleotide phosphodiesterase was isolated from either growing cultures or aggregating amoebas of Dictyostelium discoideum. The enzyme is released in a form with a low Michaelis constant (15 micromolar) and spontaneously undergoes a slow conversion to a less active form with a high Michaelis constant (2 mnillimolar). Inactivation was prevented or reversed by use of Clelands reagent, dithiothreitol. The two enzyme forms may be part of a mechanism for control of concentration of cyclic adenosine monophosphate.

Nucleotide sequence of the β-d-phosphogalactoside galactohydrolase gene of Lactobacillus casei: comparison to analogous pbg genes of other Gram-positive organisms

Lactose metabolism in Lactobacillus casei occurs via phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and subsequent cleavage of lactose-6-phosphate by beta-D-phosphogalactoside galactohydrolase (P-beta Gal). The genes for lactose uptake and P-beta Gal have been shown to be plasmid-associated in L. casei 64H [Chassy et al., Curr. Microbiol. 1 (1978) 141-144]. The cloned P-beta Gal-coding gene (pbg) previously described [Lee et al., J. Bacteriol. 152 (1982) 1138-1146] was subcloned on a 2.9-kb KpnI-Bg/II fragment isolated from pLZ605. Sequence analysis of this fragment revealed an open reading frame of 1422 bp capable of coding for a protein product containing 474 amino acids and having an Mr of 53,989. The L. casei protein showed a high degree of homology to the proteins whose sequence was deduced from the nucleotide sequence of the pbg genes of Staphylococcus aureus and Streptococcus lactis. Because of the significant homologies observed, as reflected in amino acid content as well as predicted structural characteristics of the three proteins, we suggest a common origin for the P-beta Gals of these three organisms.

Evidence for plasmid-associated lactose metabolism inLactobacillus casei subsp.casei

Lac variants ofLactobacillus casei subsp.casei DR1002 (formerly 64H) have been produced using acriflavin, ethidium bromide, mitomycin C, or combinations of these agents. Two successive transfers in the presence of acriflavin and mitomycin C or ethidium bromide and mitomycin C resulted in nearly a 100% loss of lactose fermentation. Cesium chloride-ethidium bromide isopycnic gradient ultracentrifugal analysis of purified lysates demonstrated that the 23-mdal plasmid (pDR101) found inL. casei DR1002 was consistently absent in Lac− clones. We concluded that, as in lactic streptococci, lactose metablism is a plasmid-mediated train inL. casei DR1002.

Molecular cloning and nucleotide sequence of the factor IIIlac gene of Lactobacillus casei

The lactose-specific factor III (FIIIlac of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Lactobacillus casei and purified to homogeneity by conventional protein purification methods. Its apparent native Mr, estimated from steric exclusion chromatography (approx. 39 kDa), and subunit Mr, estimated from sodium dodecyl sulfate-polyacrylamide gels, indicated that it exists as a trimer of identical subunits of 13 kDa. The gene for FIII L. casei lac was cloned into Escherichia coli using the vector pUC18. The coding sequences were contained on an 860-bp BglII-HindIII DNA fragment of the L. casei lactose plasmid, pLZ64. A protein identical in properties to FIII L. casei lac was isolated from clones of E. coli carrying this DNA insert. The nucleotide sequence of the FIII L. casei lac gene was determined by the dideoxy chain-termination technique. The 336-bp open reading frame for FIII L. casei lac was followed by a stem-loop structure, analogous to a Rho-independent terminator. We concluded that the FIII L. casei lac was the terminal gene in what appears to be an operon comprised of the lactose-PTS-P-beta Gal-coding genes. Comparison of the deduced amino acid sequence of FIII L. caseilac with the amino acid sequence of FIII S. aureus lac (derived from peptide sequencing) demonstrated a high degree of homology (49 identical residues and 21 conservative exchanges out of 103 total aa residues). The FIII L. casei lac lacked his82, previously identified as the phosphorylation site of FIII S. aureus. lac His80 was proposed to be the site of histidyl phosphorylation of FIII L. casei lac.

Prospects for improving economically significant Lactobacillus strains by ‘genetic technology’

Abstract Members of the genus Lactobacillus are used in a number of economically significant food and industrial fermentations. New developments in molecular cloning and genetic exchange systems may lead to improved starter cultures that have new and unusual properties.

Initial characterization of sucrose-6-phosphate hydrolase from Streptococcusmutans and its apparent identity with intracellular invertase

Abstract An intracellular enzyme catalyzing the hydrolysis of sucrose-6-phosphate to glucose-6-phosphate and fructose has been identified in extracts of Streptococcus mutans 6715-10. The preparation was purified chromatographically and found to have an apparent molecular weight of 42,000. The enzyme has as a Km for sucrose-6-phosphate of 0.21 mM, a pH optimum of 7.1, is quite stable and requires no added cofactors or metal ions. Sucrose is a competitive inhibitor of sucrose-6-phosphate hydrolysis (Ki = 8. 12 mM). A previously described intracellular invertase copurifies with the enzyme and could not be separated from it by disc gel electrophoresis. It is concluded that intracellular invertase is a sucrose-6-phosphate hydrolase with a low catalytic activity for hydrolysis of sucrose.

Extracellular invertase in Streptococcus mutans and Streptococcus salivarius

Summary Cultures of representative oral streptococci were fractionated and assayed for glycosyltransferases and invertase. Considerable extracellular invertase activity was found in five Streptococcus mutans strains and one Streptococcus salivarius strain. For example, in cultures of S. mutans LM-7, grown with glucose as the energy source, the invertase activity in the medium was ten times greater than the activity found associated with the cells. The invertase was characterized as a β-fructofuranosidase (EC 3.2.1.26). In the cases studied, the extra- and intracellular invertases are always present, but their levels vary independently and depend on the carbohydrate in the medium.