New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments

Sullivan, Mark A.; Yasbin, Ronald E.; Young, Frank E.

doi:10.1016/0378-1119(84)90161-6

Cited by 256 publications

(141 citation statements)

References 12 publications

Supporting

Mentioning

139

Contrasting

Unclassified

Order By: Relevance

“…Plasmid pDML995 is a derivative of pMK4 (17) in which the B. licheniformis 749/I divergeon (blaP-blaI-blaR1) is inserted. Plasmid pCIP152 is a pLEX derivative plasmid (Novagen) allowing the BlaI-WT overexpression (16).…”

Section: Methodsmentioning

confidence: 99%

Dimerization and DNA Binding Properties of theBacillus licheniformis 749/I BlaI Repressor

Filée

Vreuls

Herman

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

In the absence of penicillin, the ␤-lactamase encoding gene blaP of Bacillus licheniformis 749/I is negatively regulated by the transcriptional repressor BlaI. Three palindromic operator regions are recognized by BlaI: two in the blaP promoter (OP1 and OP2) and one (OP3) in the promoter of the blaI-blaR1 operon. In this study, the dissociation constant of the purified BlaI dimer was estimated at 25 M by equilibrium ultracentrifugation. Quantitative Western blot analysis indicates that the intracellular concentrations of BlaI in B. licheniformis 749/I and Bacillus subtilis transformed by a multicopy plasmid harboring the ␤-lactamase locus (blaP-blaIblaR1) were lower than (1.9 M) or in the same range as (75 M) the dissociation constant, respectively. This suggests that BlaI is partially dimeric in the cytoplasm of these strains and interacts in vivo with its operators as a preformed dimer. This hypothesis is supported by band shift assays on an operator containing a randomized half-operator sequence. The global dissociation constants of the operator-BlaI dimer complexes were measured by band shift assays and estimated as K dOP1 ‫؍‬ 1.7 ؎ 0.5 10 The blaP gene encodes the class A ␤-lactamase of Bacillus licheniformis 749/I. In the absence of ␤-lactams antibiotics, the BlaI repressor prevents the transcription of the blaP gene (1-3). Two additional genes, blaR1 and blaR2, are also involved in the induction of the ␤-lactamase synthesis (4, 5). The blaP, blaI, and blaR1 genes are clustered in a divergeon (bla divergeon) in which blaI and blaR1 form an operon (Fig. 1). The blaR1 gene encodes a transmembrane protein that acts as penicillin receptor (6 -8), and blaR2 is not identified yet and is not linked to the bla divergeon. The BlaR2 protein is essential for the inactivation of BlaI. In Staphylococcus aureus, the blaZ and mecA genes, encoding respectively a ␤-lactamase and the low affinity penicillinbinding protein 2Ј, are regulated by similar elements (9, 10).The blaI gene encodes a 128-residue protein characterized by two functional and separate domains (11). The DNA-binding domain, a helix-turn-helix recognition motif, is located in the N-terminal region, and the dimerization domain is in the Cterminal region. After deletion of the C-terminal domain, the repressor becomes unable to dimerize and to form a stable complex with its operators. DNase footprinting experiments and filter binding reactions revealed the presence of three regulatory regions that are recognized specifically by BlaI (11). These operators present a 23-bp-long dyad symmetry and show the deduced 5Ј-AAAGTATTACATATGTAACNTTT-3Ј consensus sequence (Fig. 1).In the presence of ␤-lactam antibiotics, the C-terminal domain of BlaR1 is acylated (6, 7). This event triggers the activation of the putative cytoplasmic metalloprotease motif of BlaR1 by self-proteolysis (8, 12). In S. aureus, the ␤-lactamase induction correlates with BlaI proteolysis between residues Asn 101 and Phe 102 , and it is postulated that the activated form of BlaR1 proteolyses BlaI (12-...

show abstract

Section: Methodsmentioning

confidence: 99%

Dimerization and DNA Binding Properties of theBacillus licheniformis 749/I BlaI Repressor

Filée

Vreuls

Herman

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Escherichia coli strain DH5␣ was used for plasmid constructions, whereas E. coli strain K38/pGP1-2 (11) was used to overproduce wild type and mutant TRAP proteins. The 1.1-kilobase EcoRI-HindIII fragment containing the wild type or mutant mtrB alleles were subcloned into pMK3 (12). These plasmids were used to transform the TRAPdeficient B. subtilis strain CYBS306 (13).…”

Section: Methodsmentioning

confidence: 99%

Effects of Mutations in the l-Tryptophan Binding Pocket of the trp RNA-binding Attenuation Protein ofBacillus subtilis

Yakhnin

Trimble²,

Chiaro

et al. 2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

The Bacillus subtilis tryptophan biosynthetic genes are regulated by the trp RNA-binding attenuation protein (TRAP). Cooperative binding of L-tryptophan activates TRAP so that it can bind to RNA. The crystal structure revealed that L-tryptophan forms nine hydrogen bonds with various amino acid residues of TRAP. We performed site-directed mutagenesis to determine the importance of several of these hydrogen bonds in TRAP activation. We tested both alanine substitutions as well as substitutions more closely related to the natural amino acid at appropriate positions. Tryptophan binding mutations were identified in vivo having unchanged, reduced, or completely eliminated repression activity. Several of the in vivo defective TRAP mutants exhibited reduced affinity for tryptophan in vitro but did not interfere with RNA binding at saturating tryptophan concentrations. However, a 10-fold decrease in TRAP affinity for tryptophan led to an almost complete loss of regulation, whereas increased TRAP affinity for tryptophan had little or no effect on the in vivo regulatory activity of TRAP. One hydrogen bond was found to be dispensable for TRAP activity, whereas two others appear to be essential for TRAP function. Another mutant protein exhibited tryptophan-independent RNA binding activity. We also found that trp leader RNA increases the affinity of TRAP for tryptophan.Expression of the Bacillus subtilis tryptophan biosynthetic (trp) genes is negatively regulated in response to tryptophan by TRAP, 1 the trp RNA-binding attenuation protein, which is the product of the mtrB gene (reviewed in 1). TRAP-mediated regulation of the trp genes includes a transcription attenuation and two translation control mechanisms. The untranslated trp operon leader transcript contains inverted repeats that allow folding of the transcript to form three RNA secondary structures. Two of these structures, the antiterminator and terminator, overlap by four nucleotides and therefore are mutually exclusive ( Fig. 1) (2-5). The TRAP binding target in the trp leader transcript consists of 11 (G/U)AG repeats, six of which are present within the antiterminator (6). When activated by tryptophan, TRAP binds to the 11 triplet repeats, which prevents formation of the antiterminator structure. This allows formation of the overlapping terminator, which promotes transcription termination upstream of the trp structural genes. In the absence of TRAP binding, the antiterminator structure forms, which results in transcription of the entire operon (7,8). An additional RNA secondary structure forms at the extreme 5Ј-end of the B. subtilis trp leader transcript. Disruption of the 5Ј-stem loop results in a substantial reduction in trp operon regulation in vivo and reduced affinity of TRAP for trp leader RNA in vitro (9). TRAP-5Ј-stem loop RNA interaction reduces the number of (G/U)AG repeats required for stable TRAP-trp leader RNA association. Thus it appears that the 5Ј-structure increases the rate of TRAP-trp leader RNA association, promoting the likelihood that tryptop...

show abstract

“…Chromosomal DNA from this strain was digested with HindIII and ligated at high concentration to the large HindIII fragment of pMK4 (Sullivan et al, 1984) containing a B. subtilis origin of replication and the cat gene. The ligation mixture was used to transform B. subtilis 168 and Spc R Cm R transformants were selected.…”

Section: Dna Manipulations and Pcrmentioning

confidence: 99%

Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT

Stülke

Martin‐Verstraete

Zagorec³

et al. 1997

Molecular Microbiology

165

187

View full text Add to dashboard Cite

SummaryGlucose is the preferred carbon and energy source of Bacillus subtilis. It is transported into the cell by the glucose-specific phosphoenolpyruvate:sugar phosphotransferase system (PTS) encoded by the ptsGHI locus. We show here that these three genes (ptsG, ptsH, and ptsI ) form an operon, the expression of which is inducible by glucose. In addition, ptsH and ptsI form a constitutive ptsHI operon. The promoter of the ptsGHI operon was mapped and expression from this promoter was found to be constitutive. Deletion mapping of the promoter region revealed the presence of a transcriptional terminator as a regulatory element between the promoter and coding region of the ptsG gene. Mutations within the ptsG gene were characterized and their consequences on the expression of ptsG studied. The results suggest that expression of the ptsGHI operon is subject to negative autoregulation by the glucose permease, which is the ptsG gene product. A regulatory gene located upstream of the ptsGHI operon, termed glcT, was also identified. The GlcT protein is a novel member of the BglG family of transcriptional antiterminators and is essential for the expression of the ptsGHI operon. A deletion of the terminator alleviates the need for GlcT. The activity of GlcT is negatively regulated by the glucose permease.

show abstract

New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments

Cited by 256 publications

References 12 publications

Dimerization and DNA Binding Properties of theBacillus licheniformis 749/I BlaI Repressor

Dimerization and DNA Binding Properties of theBacillus licheniformis 749/I BlaI Repressor

Effects of Mutations in the l-Tryptophan Binding Pocket of the trp RNA-binding Attenuation Protein ofBacillus subtilis

Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT

Contact Info

Product

Resources

About