scholarly journals Identification and Characterization of a Novel Multidrug Resistance Operon, mdtRP (yusOP), of Bacillus subtilis

2009 ◽  
Vol 191 (10) ◽  
pp. 3273-3281 ◽  
Author(s):  
Ji-Yun Kim ◽  
Takashi Inaoka ◽  
Kazutaka Hirooka ◽  
Hiroshi Matsuoka ◽  
Makiko Murata ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Multidrug Resistance ◽  
Fusidic Acid ◽  
Novel Mutation ◽  
Sequencing Analysis ◽  
Efflux Transporter ◽  
Multidrug Efflux ◽  
Mobility Shift ◽  
Low Level ◽  
Gel Mobility Shift

ABSTRACT Using comparative genome sequencing analysis, we identified a novel mutation in Bacillus subtilis that confers a low level of resistance to fusidic acid. This mutation was located in the mdtR (formerly yusO) gene, which encodes a MarR-type transcriptional regulator, and conferred a low level of resistance to several antibiotics, including novobiocin, streptomycin, and actinomycin D. Transformation experiments showed that this mdtR mutation was responsible for multidrug resistance. Northern blot analysis revealed that the downstream gene mdtP (formerly yusP), which encodes a multidrug efflux transporter, is cotranscribed with mdtR as an operon. Disruption of the mdtP gene completely abolished the multidrug resistance phenotype observed in the mdtR mutant. DNase I footprinting and primer extension analyses demonstrated that the MdtR protein binds directly to the mdtRP promoter, thus leading to repression of its transcription. Moreover, gel mobility shift analysis indicated that an Arg83 → Lys or Ala67 → Thr substitution in MdtR significantly reduces binding affinity to DNA, resulting in derepression of mdtRP transcription. Low concentrations of fusidic acid induced the expression of mdtP, although the level of mdtP expression was much lower than that in the mdtR disruptant. These findings indicate that the MdtR protein is a repressor of the mdtRP operon and that the MdtP protein functions as a multidrug efflux transporter in B. subtilis.

scholarly journals ScoC Regulates Bacilysin Production at the Transcription Level in Bacillus subtilis

2009 ◽  
Vol 191 (23) ◽  
pp. 7367-7371 ◽  
Author(s):  
Takashi Inaoka ◽  
Guojun Wang ◽  
Kozo Ochi
Keyword(s):  
Bacillus Subtilis ◽  
Genetic Engineering ◽  
Genome Sequencing ◽  
Promoter Region ◽  
Sequencing Analysis ◽  
Transcription Level ◽  
Mobility Shift ◽  
State Regulator ◽  
Transition State Regulator ◽  
Gel Mobility Shift

ABSTRACT Bacillus subtilis mutants with high expression of the bacilysin operon ywfBCDEFG were isolated. Comparative genome sequencing analysis revealed that all of these mutants have a mutation in the scoC gene. The disruption of scoC by genetic engineering also resulted in increased expression of ywfBCDEFG. Primer extension and gel mobility shift analyses showed that the ScoC protein binds directly to the promoter region of ywfBCDEFG. Our results indicate that the transition state regulator ScoC, together with CodY and AbrB, negatively regulates bacilysin production in B. subtilis.

scholarly journals Bacillus subtilis LmrA Is a Repressor of the lmrAB and yxaGH Operons: Identification of Its Binding Site and Functional Analysis of lmrB and yxaGH

2004 ◽  
Vol 186 (17) ◽  
pp. 5640-5648 ◽  
Author(s):  
Ken-ichi Yoshida ◽  
Yo-hei Ohki ◽  
Makiko Murata ◽  
Masaki Kinehara ◽  
Hiroshi Matsuoka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Multidrug Resistance ◽  
Binding Site ◽  
Binding Sites ◽  
Promoter Region ◽  
Consensus Sequence ◽  
Efflux Transporter ◽  
Multidrug Efflux ◽  
Genome Wide

ABSTRACT The Bacillus subtilis lmrAB operon is involved in multidrug resistance. LmrA is a repressor of its own operon, while LmrB acts as a multidrug efflux transporter. LmrA was produced in Escherichia coli cells and was shown to bind to the lmr promoter region, in which an LmrA-binding site was identified. Genome-wide screening involving DNA microarray analysis allowed us to conclude that LmrA also repressed yxaGH, which was not likely to contribute to the multidrug resistance. LmrA bound to a putative yxaGH promoter region, in which two tandem LmrA-binding sites were identified. The LmrA regulon was thus determined to comprise lmrAB and yxaGH. All three LmrA-binding sites contained an 18-bp consensus sequence, TAGACCRKTCWMTATAWT, which could play an important role in LmrA binding.

scholarly journals Drug-Induced Conformational Changes in Multidrug Efflux Transporter AcrB from Haemophilus influenzae

2007 ◽  
Vol 189 (15) ◽  
pp. 5550-5558 ◽  
Author(s):  
Vishakha Dastidar ◽  
Weimin Mao ◽  
Olga Lomovskaya ◽  
Helen I. Zgurskaya
Keyword(s):  
Multidrug Resistance ◽  
Haemophilus Influenzae ◽  
Conformational Changes ◽  
Efflux Transporter ◽  
Multidrug Efflux ◽  
Drug Induced ◽  
Gram Negative ◽  
Membrane Fusion Protein ◽  
Outer Membrane Channel

ABSTRACT In gram-negative bacteria, transporters belonging to the resistance-nodulation-cell division (RND) superfamily of proteins are responsible for intrinsic multidrug resistance. Haemophilus influenzae, a gram-negative pathogen causing respiratory diseases in humans and animals, constitutively produces the multidrug efflux transporter AcrB (AcrBHI). Similar to other RND transporters AcrBHI associates with AcrAHI, the periplasmic membrane fusion protein, and the outer membrane channel TolCHI. Here, we report that AcrABHI confers multidrug resistance when expressed in Escherichia coli and requires for its activity the E. coli TolC (TolCEC) protein. To investigate the intracellular dynamics of AcrABHI, single cysteine mutations were constructed in AcrBHI in positions previously identified as important for substrate recognition. The accessibility of these strategically positioned cysteines to the hydrophilic thiol-reactive fluorophore fluorescein-5-maleimide (FM) was studied in vivo in the presence of various substrates of AcrABHI and in the presence or absence of AcrAHI and TolCEC. We report that the reactivity of specific cysteines with FM is affected by the presence of some but not all substrates. Our results suggest that substrates induce conformational changes in AcrBHI.

scholarly journals In Bacillus subtilis LutR is part of the global complex regulatory network governing the adaptation to the transition from exponential growth to stationary phase

Microbiology ◽  
2014 ◽  
Vol 160 (2) ◽  
pp. 243-260 ◽  
Author(s):  
Öykü İrigül-Sönmez ◽  
Türkan E. Köroğlu ◽  
Büşra Öztürk ◽  
Ákos T. Kovács ◽  
Oscar P. Kuipers ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Exponential Growth ◽  
Dna Microarrays ◽  
Antibiotic Production ◽  
Transcriptional Profiling ◽  
Mobility Shift ◽  
Carbohydrate Utilization ◽  
Altered Expression ◽  
Gel Mobility Shift

The lutR gene, encoding a product resembling a GntR-family transcriptional regulator, has previously been identified as a gene required for the production of the dipeptide antibiotic bacilysin in Bacillus subtilis. To understand the broader regulatory roles of LutR in B. subtilis, we studied the genome-wide effects of a lutR null mutation by combining transcriptional profiling studies using DNA microarrays, reverse transcription quantitative PCR, lacZ fusion analyses and gel mobility shift assays. We report that 65 transcriptional units corresponding to 23 mono-cistronic units and 42 operons show altered expression levels in lutR mutant cells, as compared with lutR + wild-type cells in early stationary phase. Among these, 11 single genes and 25 operons are likely to be under direct control of LutR. The products of these genes are involved in a variety of physiological processes associated with the onset of stationary phase in B. subtilis, including degradative enzyme production, antibiotic production and resistance, carbohydrate utilization and transport, nitrogen metabolism, phosphate uptake, fatty acid and phospholipid biosynthesis, protein synthesis and translocation, cell-wall metabolism, energy production, transfer of mobile genetic elements, induction of phage-related genes, sporulation, delay of sporulation and cannibalism, and biofilm formation. Furthermore, an electrophoretic mobility shift assay performed in the presence of both SinR and LutR revealed a close overlap between the LutR and SinR targets. Our data also revealed a significant overlap with the AbrB regulon. Together, these findings reveal that LutR is part of the global complex, interconnected regulatory systems governing adaptation of bacteria to the transition from exponential growth to stationary phase.

scholarly journals The H2O2 Stress-Responsive Regulator PerR Positively Regulates srfA Expression in Bacillus subtilis

2005 ◽  
Vol 187 (19) ◽  
pp. 6659-6667 ◽  
Author(s):  
Kentaro Hayashi ◽  
Taku Ohsawa ◽  
Kazuo Kobayashi ◽  
Naotake Ogasawara ◽  
Mitsuo Ogura
Keyword(s):  
Bacillus Subtilis ◽  
Promoter Region ◽  
Feeding Assay ◽  
Mobility Shift ◽  
Genetic Competence ◽  
Upstream Promoter ◽  
Upstream Promoter Region ◽  
Fusion Analysis ◽  
Gel Mobility Shift

ABSTRACT srfA is an operon required for the synthesis of surfactin and the development of genetic competence in Bacillus subtilis. We observed that the expression of srfA is downregulated upon treatment with H2O2. Thus, we examined the involvement of several oxidative stress-responsive transcription factors in srfA expression. Our DNA microarray analysis revealed that the H2O2 stress-responsive regulator PerR is required for srfA expression. This was confirmed by lacZ fusion analysis. A ComX feeding assay and epistatic analyses revealed that the role of PerR in srfA expression is independent of other known regulators of srfA expression, namely, comQXP, rapC, and spx. Gel mobility shift and footprint assays revealed that PerR binds directly to two tandemly arranged noncanonical PerR boxes located in the upstream promoter region of srfA. A transcriptional srfA-lacZ fusion lacking both PerR boxes showed diminished and PerR-independent expression, indicating that the PerR boxes we identified function as positive cis elements for srfA transcription.

scholarly journals Bacillus subtilis 168 Contains Two Differentially Regulated Genes Encoding l-Asparaginase

2002 ◽  
Vol 184 (8) ◽  
pp. 2148-2154 ◽  
Author(s):  
Susan H. Fisher ◽  
Lewis V. Wray
Keyword(s):  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Mutational Analysis ◽  
Limited Growth ◽  
Mobility Shift ◽  
Regulatory Factors ◽  
Bacillus Subtilis 168 ◽  
Dnase I Footprinting ◽  
Genes Encoding ◽  
Gel Mobility Shift

ABSTRACT Expression of the two Bacillus subtilis genes encoding l-asparaginase is controlled by independent regulatory factors. The ansZ gene (formerly yccC) was shown by mutational analysis to encode a functional l-asparaginase, the expression of which is activated during nitrogen-limited growth by the TnrA transcription factor. Gel mobility shift and DNase I footprinting experiments indicate that TnrA regulates ansZ expression by binding to a DNA site located upstream of the ansZ promoter. The expression of the ansA gene, which encodes the second l-asparaginase, was found to be induced by asparagine. The ansA repressor, AnsR, was shown to negatively regulate its own expression.

scholarly journals Bacillus subtilis Aconitase Is Required for Efficient Late-Sporulation Gene Expression

2006 ◽  
Vol 188 (17) ◽  
pp. 6396-6405 ◽  
Author(s):  
Alisa W. Serio ◽  
Kieran B. Pechter ◽  
Abraham L. Sonenshein
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Activation ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Binding Activity ◽  
High Catalytic Activity ◽  
Spore Coat ◽  
Mobility Shift ◽  
Gel Mobility Shift

ABSTRACT Bacillus subtilis aconitase, encoded by the citB gene, is homologous to the bifunctional eukaryotic protein IRP-1 (iron regulatory protein 1). Like IRP-1, B. subtilis aconitase is both an enzyme and an RNA binding protein. In an attempt to separate the two activities of aconitase, the C-terminal region of the B. subtilis citB gene product was mutagenized. The resulting strain had high catalytic activity but was defective in sporulation. The defect was at a late stage of sporulation, specifically affecting expression of σK-dependent genes, many of which are important for spore coat assembly and require transcriptional activation by GerE. Accumulation of gerE mRNA and GerE protein was delayed in the aconitase mutant strain. Pure B. subtilis aconitase bound to the 3′ untranslated region of gerE mRNA in in vitro gel mobility shift assays, strongly suggesting that aconitase RNA binding activity may stabilize gerE mRNA in order to allow efficient GerE synthesis and proper timing of spore coat assembly.

scholarly journals Identification of Bacillus subtilis CysL, a Regulator of the cysJI Operon, Which Encodes Sulfite Reductase

2002 ◽  
Vol 184 (17) ◽  
pp. 4681-4689 ◽  
Author(s):  
Isabelle Guillouard ◽  
Sandrine Auger ◽  
Marie-Françoise Hullo ◽  
Farid Chetouani ◽  
Antoine Danchin ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Start Site ◽  
Transcriptional Regulator ◽  
Sulfite Reductase ◽  
Sulfur Source ◽  
Start Site ◽  
Cysteine Biosynthesis ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Gel Mobility Shift

ABSTRACT The way in which the genes involved in cysteine biosynthesis are regulated is poorly characterized in Bacillus subtilis. We showed that CysL (formerly YwfK), a LysR-type transcriptional regulator, activates the transcription of the cysJI operon, which encodes sulfite reductase. We demonstrated that a cysL mutant and a cysJI mutant have similar phenotypes. Both are unable to grow using sulfate or sulfite as the sulfur source. The level of expression of the cysJI operon is higher in the presence of sulfate, sulfite, or thiosulfate than in the presence of cysteine. Conversely, the transcription of the cysH and cysK genes is not regulated by these sulfur sources. In the presence of thiosulfate, the expression of the cysJI operon was reduced 11-fold, whereas the expression of the cysH and cysK genes was increased, in a cysL mutant. A cis-acting DNA sequence located upstream of the transcriptional start site of the cysJI operon (positions −76 to −70) was shown to be necessary for sulfur source- and CysL-dependent regulation. CysL also negatively regulates its own transcription, a common characteristic of the LysR-type regulators. Gel mobility shift assays and DNase I footprint experiments showed that the CysL protein specifically binds to cysJ and cysL promoter regions. This is the first report of a regulator of some of the genes involved in cysteine biosynthesis in B. subtilis.

scholarly journals Regulation of the Bacillus subtilis ytmI Operon, Involved in Sulfur Metabolism

2005 ◽  
Vol 187 (17) ◽  
pp. 6019-6030 ◽  
Author(s):  
Pierre Burguière ◽  
Juliette Fert ◽  
Isabelle Guillouard ◽  
Sandrine Auger ◽  
Antoine Danchin ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Promoter Region ◽  
Sulfur Metabolism ◽  
Point Mutations ◽  
Constitutive Expression ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Mobility Shift ◽  
Common Motif ◽  
Gel Mobility Shift

ABSTRACT The YtlI regulator of Bacillus subtilis activates the transcription of the ytmI operon encoding an l-cystine ABC transporter, a riboflavin kinase, and proteins of unknown function. The expression of the ytlI gene and the ytmI operon was high with methionine and reduced with sulfate. Using deletions and site-directed mutagenesis, a cis-acting DNA sequence important for YtlI-dependent regulation was identified upstream from the −35 box of ytmI. Gel mobility shift assays confirmed that YtlI specifically interacted with this sequence. The replacement of the sulfur-regulated ytlI promoter by the xylA promoter led to constitutive expression of a ytmI′-lacZ fusion in a ytlI mutant, suggesting that the repression of ytmI expression by sulfate was mainly at the level of YtlI synthesis. We further showed that the YrzC regulator negatively controlled ytlI expression while this repressor also acted on ytmI expression via YtlI. The cascade of regulation observed in B. subtilis is conserved in Listeria spp. Both a YtlI-like regulator and a ytmI-type operon are present in Listeria spp. Indeed, the Lmo2352 protein from Listeria monocytogenes was able to replace YtlI for the activation of ytmI expression and a lmo2352′-lacZ fusion was repressed in the presence of sulfate via YrzC in B. subtilis. A common motif, AT(A/T)ATTCCTAT, was found in the promoter region of the ytlI and lmo2352 genes. Deletion of part of this motif or the introduction of point mutations in this sequence confirmed its involvement in ytlI regulation.

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