pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis

1998 ◽  
Vol 44 (12) ◽  
pp. 1186-1192
Author(s):  
Guy Daxhelet ◽  
Philippe Gilot ◽  
Etienne Nyssen ◽  
Philippe Hoet
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Initiation Site ◽  
Chloramphenicol Acetyltransferase ◽  
Ribosome Binding Site ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Ribosome Binding

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B. subtilis resistance to high chloramphenicol concentrations. CAT expression peaked at the early postexponential phase of B. subtilis growth. The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.Key words: chloramphenicol acetyltransferase, Bacillus subtilis, postexponential gene expression, plasmid pUB110, ribosome-binding site, transcriptional promoter.

scholarly journals Np4A alarmones function in bacteria as precursors to RNA caps

2020 ◽  
Vol 117 (7) ◽  
pp. 3560-3567 ◽  
Author(s):  
Daniel J. Luciano ◽  
Joel G. Belasco
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Rna Degradation ◽  
Initiation Site ◽  
Bacterial Cells ◽  
E Coli ◽  
N Incorporation ◽  
Nascent Transcripts

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5′ end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

E. coli lactose operon ribosome binding site

Nature ◽  
1974 ◽  
Vol 249 (5458) ◽  
pp. 647-649 ◽  
Author(s):  
NANCY MAIZELS
Keyword(s):  
Binding Site ◽  
Ribosome Binding Site ◽  
Lactose Operon ◽  
E Coli ◽  
Ribosome Binding

Molecular analysis of mutatedLactobacillus acidophiluspromoter-like sequence P15

2000 ◽  
Vol 46 (10) ◽  
pp. 938-945 ◽  
Author(s):  
Slavica Arsenijevic ◽  
Ljubisa Topisirovic
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Molecular Analysis ◽  
Lactobacillus Acidophilus ◽  
Promoter Activity ◽  
Lactobacillus Reuteri ◽  
Promoter Sequence ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Promoter Probe

The promoter-like sequence P15 that was previously cloned from the chromosome of Lactobacillus acidophilus ATCC 4356 is active in Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus acidophilus, and Escherichia coli, but not in Lactococcus lactis. N-methyl-N-nitroso-N-guanidine (MNNG) mutagenesis of P15 was used to select for a promoter active in L. lactis MG1363. Molecular analysis of the mutated promoter (designated P16) revealed a 90 bp deletion and a T[Formula: see text]A transversion. This deletion, in combination with the addition to the transversion, created a promoter with putative -35 and -10 hexamers identical to the consensus promoter sequence found in E. coli and Bacillus subtilis vegetative promoters. The activity of P16 was measured by its ability to promote chloramphenicol resistance in different bacteria when inserted in the promoter-probe plasmid pBV5030 (designated pLA16). The MIC of chloramphenicol in L. lactis, L. reuteri, L. plantarum, E. coli, and L. acidophilus harbouring pLA16 were 30, 170, 180, >500, and 3 µg/mL, respectively. This represents an increase in promoter activity compared to P15 in L. reuteri of 3-fold, in L. plantarum of 9-fold, and in E. coli of at least 2.5-fold, but a decrease in L. acidophilus of 7-fold.Key words: Lactobacillus acidophilus, promoter-like sequence, mutagenesis.

scholarly journals A Novel Bacillus subtilis Gene,antE, Temporally Regulated and Convergent to and Overlapping dnaE

1999 ◽  
Vol 181 (1) ◽  
pp. 353-356 ◽  
Author(s):  
Lin-Fa Wang ◽  
Sung-Soo Park ◽  
Roy H. Doi
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Binding Site ◽  
Ribosome Binding Site ◽  
Small Protein ◽  
Ribosome Binding ◽  
Untranslated Sequence ◽  
Partial Homology ◽  
Subtilis Gene

ABSTRACT A Bacillus subtilis promoter, Px, that functions in a convergent manner with the sigA operon promoter P3 has been found in the sigA operon. Promoter Px is turned on at the same time as promoter P3 during early sporulation. The transcript from promoter Px codes for a small protein with partial homology to the OmpR protein from Escherichia coli and also carries an untranslated sequence at its 3′ end that is complementary to the 5′ end of the P3 transcript, which codes for the ribosome binding site ofdnaE. The gene controlled by Px has been calledantE. The expression of antE does not require ςB, ςE, or ςH. Px was transcribed in vitro by the ςA holoenzyme and is the seventh promoter to be recognized in the ςA operon. A possible role for the antE gene during early sporulation is proposed.

scholarly journals Citrate synthase from Mycobacterium smegmatis. Cloning, sequence determination and expression in Escherichia coli

1991 ◽  
Vol 278 (1) ◽  
pp. 225-234 ◽  
Author(s):  
M David ◽  
S Lubinsky-Mink ◽  
A Ben-Zvi ◽  
M Suissa ◽  
S Ulitzur ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Mycobacterium Smegmatis ◽  
Citrate Synthase ◽  
Coli Strain ◽  
Ribosome Binding Site ◽  
E Coli ◽  
Ribosome Binding ◽  
Lac Promoter

A Mycobacterium smegmatis PstI library was constructed by cloning these fragments downstream from the lac promoter of the expression vector pHG171. Three identically sized clones were isolated by complementation of an Escherichia coli strain (chi 2338) deficient in citrate synthase. One insert (pBL265) was used in hybridization experiments with DNA from E. coli and M. smegmatis and it was demonstrated that the clones were indeed from M. smegmatis. The transcription of the M. smegmatis citrate synthase gene in E. coli relied upon the lac promoter. In translation experiments performed in vitro pBL265 gave rise to a novel protein of about 42 kDa. This band was not seen in ‘opposite-orientation’ subclones. Various subclones in which the 5′-end was shortened nevertheless complement E. coli chi 2338 and produce the 42 kDa protein. This demonstrates that the M. smegmatis citrate synthase gene uses its own ribosome-binding site in E. coli. The relevant 1.8 kb of the 2.8 kb insert was sequenced. A consensus E. coli ribosome-binding site was found centred precisely 10 bp upstream of the methionine codon. Other interesting features revealed by the sequence are discussed. Citrate synthase activity was assayed in vitro and the mycobacterial enzyme was found to be similar to those of the Gram-positive bacteria.

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