A modified protoplast-regeneration protocol facilitating the detection of cloned exoenzyme genes in Bacillus subtilis

Corfield, Valerie A.; Reid, Sharon J.; Bodmer, Jennifer; Thomson, Jennifer A.

doi:10.1016/0378-1119(84)90100-8

Cited by 21 publications

(4 citation statements)

References 17 publications

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“…For the construction of pCAGSp24, a 570-base-pair (bp) Sau3A fragment of pHZ202 containing the downstream promoter P3 was cloned into the BamHI site of the promoter probe plasmid pPL603bl, which is a derivative of pPL603 (16,58), lacks a late exponential promoter (11), and contains a short EcoRI linker containing a BamHI cloning site. After ligation and transformation in B. subtilis 1A46, recombinants were selected on DM3 agar supplemented with chloramphenicol (10 ,ugIml).…”

Section: Methodsmentioning

confidence: 99%

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Molecular analysis and regulation of the glnA gene of the gram-positive anaerobe Clostridium acetobutylicum

Janssen

Jones

et al. 1988

J Bacteriol

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The nucleotide sequence of a 2.0-kilobase DNA segment containing the Clostridium acetobutylicum ginA gene was determined. The upstream region of the glnA gene contained two putative extended promoter consensus sequences (Pi and P2), characteristic of gram-positive bacteria. A third putative extended gram-positive promoter consensus sequence (P3), oriented towards the ginA gene, was detected downstream of the structural gene. The sequences containing the proposed promoter regions Pi and P2 or P3 were shown to have promoter activity by subcloning into promoter probe vectors. The complete amino acid sequence (444 residues) of the C. acetobutylicum glutamine synthetase (GS) was deduced, and comparisons were made with the reported amino acid sequences of GS from other organisms. To determine whether the putative promoter P3 and a downstream region with an extensive stretch of inverted repeat sequences were involved in regulation of C. acetobutylicum glnA gene expression by nitrogen in Escherichia coli, deletion plasmids were constructed lacking P3 and various downstream sequences. Deletion of the putative promoter P3 and downstream inverted repeat sequences affected the regulation of GS and reduced the levels of GS approximately fivefold under nitrogen-limiting conditions but did not affect the repression of GS levels in cells grown under nitrogen-excess conditions.In the nitrogen metabolism of bacteria, glutamine synthetase (GS) (EC 6.3.1.2) plays a central role, as it catalyzes one of the main reactions by which ammonia is assimilated (54):

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Section: Methodsmentioning

confidence: 99%

“…A 570-bp Sau3A fragment (P24) harboring the putative P3 promoter sequence was subcloned into the B. subtilis promoter probe vector pPL603bl (11). All the chloramphenicol-resistant recombinants contained the P24 fragment in the same orientation adjacent to the chloramphenicol resistance gene (cat-86).…”

Section: Methodsmentioning

confidence: 99%

Molecular analysis and regulation of the glnA gene of the gram-positive anaerobe Clostridium acetobutylicum

Janssen

Jones

et al. 1988

J Bacteriol

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“…The use of high levels of X-S also allows the primary isolation of hyperproducers from natural sources; strains which produce low levels of amylase do not exhibit a halo at high X-S concentrations. Corfield et al (1984) reported that the addition of starch to hypertonic media enhances the regeneration of B. subtilis protoplasts. We have found this to be true for other Bacillus species, and have been routinely adding corn starch (2%) to the H C medium of Akamatsu & Sekiguchi (1981).…”

Section: Discussionmentioning

confidence: 99%

A note on the use of cross‐linked starch in microbiology with special reference to detecting amylase production

Modi¹,

Bhatt²,

Shaikh³

et al. 1986

Journal of Applied Bacteriology

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Anilogel-E, a cross-linked starch, can be used with distinct advantages where native starch or soluble starch are conventionally used, e.g. in scoring for amylolytic organisms, as an ingredient of fermentation media, and in enhancing protoplast regeneration. It is particularly useful for the direct visualization of amylase producing micro-organisms on solid media, making prior replication of colonies unnecessary.

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“…2% (w/v) glucose in PABS led to a somewhat higher regeneration, but was not suitable because it also caused a simultaneous increase in the number of osmotic shock-resistant cells ( > 103 cfu/ml). Supplementing SMM with 0.1-5.0% (w/v) bovine serum albumin (fraction V, Sigma) or 1 mM phenylmethylsulfonyl fluoride [6], or modifying DM3 to contain 1% (w/v) starch (Merck 1252) [21] or agar up to 2.5% (w/v) did not further improve regeneration. With the batches used, the degree of purity of the sodium-succinate (purum or pro analysis from Fluka, or pro analysis from Merck) did not significantly affect the regeneration frequency.…”

Section: Protoplast Regenerationmentioning

confidence: 99%

Transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA

Vehmaanperä

1988

FEMS Microbiology Letters

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A method for efficient polyethylene glycol (PEG)‐mediated transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA is described. The best conditions found for protoplast regeneration included using 0.45 M sucrose both during the cultivation of the cells and (as an osmotic stabilizer) during their treatment with lysozyme, whereas 0.25 M sodium‐succinate was added to the regeneration plates. Under these conditions about 5–10% of input cells regenerated. The highest transformation frequency with plasmid DNA was obtained with a PEG 6000 concentration of 22.5% (w/v). Transforming B. amyloliquefaciens strains with the plasmid pUB110 isolated from B. amyloliquefaciens resulted in 2–4 · 105 transformants/μg DNA, 100–1 000‐times as high as with DNA from Bacillus subtilis, suggesting a restriction barrier between the two species. Transformation of B. amyloliquefaciens with plasmids pC194 or pE194 cop‐6 gave poor yields and no restriction barrier could be demonstrated for these plasmids. However, by curing pC194 from one of the transformants, a mutant strain compatible to both the plasmids could be isolated, yielding 2–3·104 transformants/μg DNA. Both laboratory and industrial B. amyloliquefaciens strains could be transformed with the procedure.

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A modified protoplast-regeneration protocol facilitating the detection of cloned exoenzyme genes in Bacillus subtilis

Cited by 21 publications

References 17 publications

Molecular analysis and regulation of the glnA gene of the gram-positive anaerobe Clostridium acetobutylicum

Molecular analysis and regulation of the glnA gene of the gram-positive anaerobe Clostridium acetobutylicum

A note on the use of cross‐linked starch in microbiology with special reference to detecting amylase production

Transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA

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