Isolation and characterization of a 2.2‐kb operon preceding the α‐amylase gene of <i>Bacillus amyloliquefaciens</i>

Kallio, Pauli T.; Ulmanen, Ismo; Palva, Ilkka

doi:10.1111/j.1432-1033.1986.tb09782.x

Cited by 6 publications

(3 citation statements)

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“…Deletion of the palindromic sequence had no effect on ox-amylase regulation when cloned in B. subtilis (16). Instead, the inverted repeat functioned as a transcription termination signal for a 2.2-kilobase (kb) operon located upstream of the cx-amylase gene (17).In this communication, we demonstrate that the inverted repeat in both amyRI and amyR2 is dispensable for otamylase regulation in B. siubtilis. Additionally, deletion of sequences 3' to the promoter in amyRI-cat-86 transcription fusions eliminated catabolite repression of chloramphenicol acetyltransferase (CAT) but had no effect on temporal activation at the onset of stationary phase.…”

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“…6). In B. amyloliquefaciens, the inverted repeat functions as a transcription terminator for a 2.2-kb operon upstream of the ao-amylase gene (17). Transcription terminates in a string of T's near the 3' end of the inverted repeat.…”

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Genetic analysis of the promoter region of the Bacillus subtilis alpha-amylase gene

Weickert

Chambliss

1989

J Bacteriol

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The amyR2 allele of the Bacillus subtilis ox-amylase cis-regulatory region enhances production of amylase and transcription of amyE, the structural gene, by two-to threefold over amyRi. The amylase gene bearing each of these alleles was cloned on plasmids of about 10 to 15 copies per chromosome. Transcription of the cloned amylase gene by each amyR allele was activated at the end of exponential growth and was subject to catabolite repression by glucose. The amount of amylase produced was roughly proportional to the copy number of the plasmid, and cells containing the amyR2-bearing plasmid, pAR2, produced two-to threefold more amylase than cells with the amyRi plasmid, pAMY10. Deletion of DNA 5' to the ot-amylase promoter, including deletion of the A+T-rich inverted repeat found in amyR1 and amyR2, had no effect on expression or transcription of a-amylase. Deletion of DNA 3' to the amyRI promoter did not impair temporal activation of chloramphenicol acetyltransferase in amyRl-cat-86 transcriptional fusions, but catabolite repression was abolished. When an 8-base-pair linker was inserted in pAMY10 at the same site from which the 3' deletion was made, amylase expression doubled and was repressed less by glucose. Both the deletion and the insertion disrupted four bases at the 3' end of the putative amylase operator region. Site-directed mutagenesis was used to change bases in the promoter-operator region of amyRl to their amyR2 counterparts. Either change alone increased amylase production twofold, but only the change at +7, next to the linker insertion of 3' deletion site, yielded the increased amylase activity in the presence of glucose that is characteristic of the amyR2 strain. The double mutant behaved most like strains carrying the amyR2 allele.The expression of Bacillus subtilis ot-amylase involves activation of synthesis at the end of exponential growth (30) and repression by easily metabolized carbon sources (11, 36; our unpublished results). This resembles regulation of the sporulation process (for a review, see reference 20). The catabolite control mechanisms governing a-amylase synthesis and sporulation differ at least partially (27), but the mechanism for temporal activation may be the same.The B. subtilis a-amylase gene, amyE, is regulated by the cis-acting region amyR (44,49). There are at least three alleles of the amyR locus: amyRi (from B. subtilis 168), amyR2 (from B. subtilis subsp. natto), and amyR3 (from B. subtilis subsp. amylosacchariticus) (48). amyR2 and amyR3 are amylase-hyperproducing alleles of amyRI (44,48,49). amyRI and amyR2 have been cloned and sequenced (45,46 (-7.3 kcal/mol) of the 29-base A+T-rich inverted repeat sequence found in amyRI. It has been suggested that the amyR2 inverted repeat is responsible for the hyperproduction phenotype (45). Deletion of this upstream palindromic sequence in an amyR2-bla fusion strain was reported to cause lower 1-lactamase production and reduced glucose catabolite repression (39).The B. amyloliquefaciens Q-amylase regulatory region contains a 4...

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Section: Discussionmentioning

confidence: 99%