Dennis H. Steinberg

Genetic transformation of Streptococcus thermophilus by electroporation

A rapid and convenient electroporation procedure was developed for the genetic transformation of intact cells of Streptococcus thermophilus with various species of plasmid DNA. Transformation frequency was influenced by the capacitance and voltage selected for electric pulsing, the pH and composition of the electroporation medium and the molecular mass of the transforming DNA. Electroporation is a simple and effective technique to introduce plasmid DNA into S. thermophilus and useful in the development of recombinant DNA technology for this important industrial microorganism.

Distribution and analysis of plasmids inStreptococcus thermophilus

SummaryIn a survey of 35 strains ofStreptococcus thermophilus, 13 strains were found to harbor plasmid DNA. Most of these strains contained plasmid species varying in size from 2.2 to 7.15 kilobases. Only three strains had more than one plasmid species. Each of the nine distinct types of plasmid DNAs identified had two or more unique recognition sites for restriction endonucleases. The characteristics of the indigenous cryptic plasmids ofS. thermophilus may allow their development as cloning vectors useful in the genetic engineering of this species and other streptococci that are important in food production

Permeabilization of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus with ethanol

Abstract.Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus cultures were treated with ethanol and tested for viability and β-galactosidase activity. Exposure of the biomass of test cultures to 30%–55% ethanol (vol/vol) caused a 100% loss of viability and up to 15-fold increase in measurable β-galactosidase activity in both streptococci and lactobacilli. Ethanol-treated cell suspensions could be stored for up to 6 months without loss of enzyme activity. The nonviable permeabilized biomass of the more active S. thermophilus was used to achieve up to 80% hydrolysis of lactose in aqueous solutions and non-fat milk.

Permeabilization of Streptococcus thermophilus and the expression of beta-galactosidase

Studies were carried out to determine the efficacy of several permeabilizing agents in inducing high-level expression of β-galactosidase in Streptococcus thermophilus. Sodium dodecyl sulfate, Triton X-100, sodium deoxycholate, and one commercial bile acid preparation were effective as membrane destabilizing agents allowing lactose influx and hydrolysis by cytoplasmic β-galactosidase in treated cells. Cells exposed to Oxgall or Triton X-100 displayed 15 times higher levels of β-galactosidase activity than control cells. Detergent treatment also induced extensive cell death or significant injury to cell populations resulting in long delays before resumption of growth. A permeabilized suspension of S. thermophilus corresponding to 108 cfu ml−1 released 87% of glucose available in a 5% lactose solution within 10 min at 50°;C.

Expression of Streptomyces sp. cholesterol oxidase in Lactobacillus casei

SummaryThree strains of Lactobacillus casei were electrotransformed with pNCO937, an 8.1-kb recombinant plasmid carrier of a Streptomyces sp. cholesterol oxidase gene. Transformation frequency was generally low and strain-dependent, ranging from 6 to 40 transformants/μg DNA. L. casei transformants stably maintained pNCO937 with no indication of deletion mutational events. Transformants produced active cholesterol oxidase and sonicated cells formed 4-cholesten-3-one from both free and lipoprotein-bound cholesterol. L. casei shows promise as a host suitable for studying heterologous gene expression in lactobacilli.

Distribution of Plasmid-Borne Stress Protein Genes in Streptococcus thermophilus and Other Lactic Acid Bacteria

Abstract. The presence of heat stress protein genes (hsp) was tested by Southern hybridization analysis in total DNA extracts from species of the genus Streptococcus (47 strains), Lactobacillus (34 strains), Lactococcus (24 strains), and Leuconostoc (5 strains). The biotinylated hsp16.4 probe prepared from an ORF2 fragment of pER341 (2.8 kb) tested positively with restricted DNA extracts of seven Streptococcus thermophilus strains and a single strain of Lactococcus lactis subsp. cremoris. In all positive S. thermophilus strains, the hsp was located on plasmids ranging from ca. 2.8 kb to 11 kb in size, while hsp was present in a 7.5-kb plasmid in Lactococcus lactis subsp. cremoris. Southern blots with a rep probe showed that all hsp16.4+ plasmids in S. thermophilus strains also shared homology with the replication function (rep) of pER341, suggesting the common origin of these plasmids.

Cloning of a tyrosinase gene in Streptococcus thermophilus

SummaryThe streptococcal cloning vector pIL253 (4.96-kbp, Emr) was used to introduce the Streptomyces antibioticus tyrosinase (mel) gene (1.56-kbp) into S. thermophilus, an important microbe in dairy fermentations. Electrotransformants of S. thermophilus ST128 contained 6.51-kbp recombinant plasmids which probed positively in Southern hybridizations with the biotin-labeled mel fragment. Western blots of cell extracts resolved by SDS-PAGE showed the presence of a ca. 31-kDa band thus confirming the synthesis of tyrosinase protein by genetic transformants.

Sensitivity of Streptococcus thermophilus to chemical permeabilization

Abstract.Streptococcus thermophilus cultures were treated with conjugated and unconjugated bile salts and tested for β-galactosidase activity. Na-deoxycholate and chenodeoxycholate were more efficient permeabilizing agents than cholate, and all three bile salts were superior to their corresponding glyco- and tauro-conjugates. Treatment with sodium dodecyl sulfate resulted in the highest measurable β-galactosidase levels in permeabilized cells, whereas response to Triton X-100 was variable and strain dependent. Na-deoxycholate, chenodeoxycholate, and sodium dodecyl sulfate caused cell injury and arrested culture growth for 4 h or longer. The nongrowing permeabilized biomass of S. thermophilus was used to hydrolyze lactose in aqueous solutions and milk.

Construction and characterization of shuttle plasmids for lactic acid bacteria and Escherichia coli

The chimeric plasmid pBN183 was first constructed in Escherichia coli by ligating the BamHI-digested E. coli plasmid pBR322 and a Bg/II-linearized streptococcal plasmid, pNZ18. The pBN183 transformed E. coli to ApR at a frequency of (8.2 +/- 1.2) x 10(5) colony forming units (CFU)/microgram DNA. Electrotransformation of Streptococcus thermophilus with pBN183 yielded CmR, ApS clones at a frequency of (2.6 +/- 0.3) x 10(1) CFU/microgram DNA. Plasmid screening with pBN183-transformed S. thermophilus clones revealed that ca. 70% of these transformants contained deleted plasmids. Plasmid pBN183A, a pBN183 deletion mutant lacking one copy of a tandemly arranged, highly homologous DNA sequence, was isolated for further study. It transformed E. coli to ApR and S. thermophilus to CmR with frequencies of (4.8 +/- 0.1) x 10(5) and (8.1 +/- 0.2) x 10(2) CFU/microgram DNA, respectively. Screening of S. thermophilus transformants did not show the presence of deleted plasmids. Based on the structure of pBN183A, a new shuttle plasmid, pDBN183, was constructed from pBN183 by removal of the small (1.2 kb) Sa/I fragment. Transformation frequencies of pDBN183 were (5.0 +/- 1.3) x 10(5) and (4.6 +/- 0.2) x 10(2) CFU/microgram DNA with E. coli and S. thermophilus, respectively. In contrast to the parent pBN183, only 17% of the pDBN183-transformed S. thermophilus contained deleted plasmids. Plasmid copy numbers of the three vectors in E. coli were estimated at 17-18 per chromosome. The three plasmids conferred ApR and CmR to E. coli, but only CmR to S. thermophilus. The insertion of a Streptomyces cholesterol oxidase gene (choA) into pDBN183 did not affect the plasmids stability in Lactobacillus casei, but resulted in deletion of the recombinant DNA in S. thermophilus.

Electrotransformation ofStreptococcus sanguis Challis

Plasmid DNA was introduced into noncompetent cells ofStreptococcus sanguis Challis by an electrotransformation technique. The procedure was simple and rapid, did not require elaborate pretreatment of cells, and yielded transformant colonies in 24 h. The maximum transformation efficiency attained was 2.1×104 transformants per μg of pVA736. Molecular rearrangements and deletions were not detected in plasmid DNA isolated from transformants.