Genetic transformation of Streptococcus thermophilus by electroporation

Somkuti, George A.; Steinberg, Dennis H.

doi:10.1016/0300-9084(88)90095-8

Cited by 100 publications

(58 citation statements)

References 20 publications

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“…The resulting vector pSTKOC was transformed into freshly prepared E. coli competent cells by a standard heat-shock method (Sambrook et al, 1989) and propagated at 37 ºC. Plasmid was recovered from E. coli by alkaline lysis followed by CsCl/ethidium bromide ultracentrifugation (Stougaard & Molin, 1981) and introduced by electrotransformation into S. thermophilus ST110 according to a previously described protocol (Somkuti & Steinberg, 1988). Chromosomal integration of the vector was accomplished by a two-step homologous-recombination protocol (Renye & Somkuti, 2012).…”

Section: Conditions For Evaluating Synthetic Qsipmentioning

confidence: 99%

Effect of a BlpC-Based Quorum-Sensing Induction Peptide on Bacteriocin Production in Streptococcus thermophilus

Somkuti¹,

Renye²

2014

JFR

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Bacteriocin synthesis in some Streptococcus thermophilus strains is under the control of a complex blp locus but bacteriocin is produced only when a quorum-sensing regulatory mechanism is activated by the protein product of the blpC component. To demonstrate the regulatory effect of BlpC in S. thermophilus ST110 (NRRL-B59671), which naturally produces bacteriocin, the effect of the 30mer quorum-sensing induction peptide (QSIP) embedded in BlpC was tested in a knockout mutant in which the blpC gene was eliminated and was devoid of antimicrobial activity. Between concentrations of 30 and 250 ng/ml, the addition of synthetic QSIP to cultures at several points of the growth curve resulted in the accumulation of up to 3,200 units/ml of bacteriocin after 8 h of growth at 37 ºC. Addition of QSIP to the culture in late log phase (OD 660 ≥ 1.0) when the medium pH is already 4.8 or lower, failed to trigger bacteriocin production. We used synthetic QSIP to survey its impact on 35 strains of S. thermophilus that do not display bacteriocin activity by agar diffusion assays. The addition of QSIP (250 ng/ml) to S. thermophilus cultures in the early or mid-log phase induced bacteriocin production in two strains that could be re-classified as bacteriocin producers. The results demonstrated the involvement of a quorum-sensing regulatory mechanism in bacteriocin synthesis in S. thermophilus ST110 and also demonstrated the utility of the 30 mer QSIP in discovering bacteriocins with potentially novel antimicrobial spectra by enhancing bacteriocin production in other strains of S. thermophilus that ordinarily display a bacteriocin-negative phenotype.

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Section: Conditions For Evaluating Synthetic Qsipmentioning

confidence: 99%

Effect of a BlpC-Based Quorum-Sensing Induction Peptide on Bacteriocin Production in Streptococcus thermophilus

Somkuti¹,

Renye²

2014

JFR

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“…Electroporation is also applied to the transformation of bacterial microorganisms. First example was the stimulation of transformation of Bacillus cereus protoplasts by electric field pulses (Shivarova et al 1983); more recently the electroporation technique was successfully applied to transform Streptococcus thermophilus (Somkuti and Steinberg 1987), Streptomyces lividans protoplast (Mac Nell 1987), Lactobacillus casei (Chassy and Flickinger 1987), Campylobacterjejuni (Miller et al 1988), different strains of Lactic streptococci (Powell et al 1988) Enterococcus faecalis protoplasts, Pseudomonas putida and Escherichia coli (Fiedler and Wirth;Taketo 1988). Although Corynebacteria osmotically sensitive cells can be transformed by the PEG technique, there is actually no method available to transform intact cells.…”

Section: Introductionmentioning

confidence: 99%

Electrotransformation of intact and osmotically sensitive cells of Corynebacterium glutamicum

Wolf

Pühler

Neumann

1989

Appl Microbiol Biotechnol

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Summary. Intact and osmotically sensitive cells ofCorynebacterium glutamicum can be efficiently transformed by electroporation. This was shown by using the plasmid vector pUL-330 (5.2 kb), containing the kanamycin resistance gene of transposon Tn5. The following electric parameters yielded efficient transformation. For intact cells: one exponentially decaying field pulse E = Eo exp ( -t/re) with time constants re = 450-500 ~ts and with initial field intensities of Eo= 35-40 kV cm-l; prepulse temperature 20 ° C. Cell regeneration (survival) was 100%-80%. Transformation efficiency can be increased by an additional freeze and thaw cycle of the cells, prior to electropotation. Lysozyme treated cells (osmotically sensitive) were transformed with three successive pulses of Eo = 25-30 kV cm-1. Cell regeneration under these conditions was found to be 20-30%. The optimum yield of transformants/~tg plasmid-DNA was 3 × 103 for intact cells, 2 × 104 for intact cells which were frozen and thawed twice and 7 x 104 for osmotically sensitive cells if the cell suspension was pulsed at a cell density of 1-3 x 108/ml and at a DNA concentration of 0.2 Ixg/ ml up to _< 2 Ixg/ml. The data obtained for osmotically sensitive cells suggest that the temperature increase accompanying the electric field pulse enhances colony formation and transformation efficiency if the initial prepulse temperature is _> 20 ° C, although regeneration of electroporated C. 91utamicum cells starts to decrease at temperatures > 20 ° C.

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“…Several gene transfer techniques such as conjugation (12,37), transfection (27), and transformation (28,45) have been reported for S. thenmophilus. These techniques enable the examination and use of already existing bacterial plasmids as cloning vectors as well as a beginning in designing new vector systems based on cryptic, homogenic plasmids (13,15,26,44,46).…”

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Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus

et al. 1993

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Several pGEM5-and pUC19-derived plasmids containing a selectable erythromycin resistance marker were integrated into the chromosome of Streptococcus thermophilw at the loci of the lactose-metabolizing genes.Integration occurred via homologous recombination and resulted in cointegrates between plasmid and genome, flanked by the homologous DNA used for integration. Selective pressure on the plasmid-located erythromycin resistance gene resulted in multiple amplifications of the integrated plasmid. Release of this selective pressure, however, gave way to homologous resolution of the cointegrate structures. By integration and subsequent resolution, we were able to replace the chromosomal lacZ gene with a modified copy carrying an in vitro-generated deletion. In the same way, we integrated a promoterless chloramphenicol acetyltransferase (cat) gene between the chromosomal lacS and lacZ genes of the lactose operon. The inserted cat gene became a functional part of the operon and was expressed and regulated accordingly. Selective pressure on the essential lacS and lacZ genes under normal growth conditions in milk ensures the maintenance and expression of the integrated gene. As there are only minimal repeated DNA sequences (an NdeI site) flanking the inserted cat gene, it was stably maintained even in the absence of lactose, i.e., when grown on sucrose or glucose. The methodology represents a stable system in which to express and regulate foreign genes in S. thermophilus, which could qualify in the future for an application with food.

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Genetic transformation of Streptococcus thermophilus by electroporation

Cited by 100 publications

References 20 publications

Effect of a BlpC-Based Quorum-Sensing Induction Peptide on Bacteriocin Production in Streptococcus thermophilus

Effect of a BlpC-Based Quorum-Sensing Induction Peptide on Bacteriocin Production in Streptococcus thermophilus

Electrotransformation of intact and osmotically sensitive cells of Corynebacterium glutamicum

Directed genomic integration, gene replacement, and integrative gene expression in Streptococcus thermophilus

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