scholarly journals Genetic and Biochemical Analysis of CodY-Binding Sites in Bacillus subtilis

2007 ◽  
Vol 190 (4) ◽  
pp. 1224-1236 ◽  
Author(s):  
Boris R. Belitsky ◽  
Abraham L. Sonenshein
Keyword(s):  
Bacillus Subtilis ◽  
Lactococcus Lactis ◽  
Binding Sites ◽  
Biochemical Analysis ◽  
Transcriptional Regulator ◽  
Promoter Regions ◽  
Dnase I Footprinting ◽  
Target Dna

ABSTRACT CodY is a global transcriptional regulator that is known to control directly the expression of at least two dozen operons in Bacillus subtilis, but the rules that govern the binding of CodY to its target DNA have been unclear. Using DNase I footprinting experiments, we identified CodY-binding sites upstream of the B. subtilis ylmA and yurP genes. The protected regions overlapped versions of a previously proposed CodY-binding consensus motif, AATTTTCWGAAAATT. Multiple single mutations were introduced into the CodY-binding sites of the ylmA, yurP, dppA, and ilvB genes. The mutations affected both the affinity of CodY for its binding sites in vitro and the expression in vivo of lacZ fusions that carry these mutations in their promoter regions. Our results show that versions of the AATTTTCWGAAAATT motif, first identified for Lactococcus lactis CodY, with up to five mismatches play an important role in the interaction of B. subtilis CodY with DNA.

scholarly journals Dual Regulation of the Bacillus subtilis Regulon Comprising the lmrAB and yxaGH Operons and yxaF Gene by Two Transcriptional Repressors, LmrA and YxaF, in Response to Flavonoids

2007 ◽  
Vol 189 (14) ◽  
pp. 5170-5182 ◽  
Author(s):  
Kazutake Hirooka ◽  
Satoshi Kunikane ◽  
Hiroshi Matsuoka ◽  
Ken-Ichi Yoshida ◽  
Kanako Kumamoto ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Multidrug Resistance ◽  
Promoter Region ◽  
Consensus Sequence ◽  
Transcriptional Repressors ◽  
Promoter Regions ◽  
In Vivo Experiments ◽  
Dnase I Footprinting

ABSTRACT Bacillus subtilis LmrA is known to be a repressor that regulates the lmrAB and yxaGH operons; lmrB and yxaG encode a multidrug resistance pump and quercetin 2,3-dioxygenase, respectively. DNase I footprinting analysis revealed that LmrA and YxaF, which are paralogous to each other, bind specifically to almost the same cis sequences, LmrA/YxaF boxes, located in the promoter regions of the lmrAB operon, the yxaF gene, and the yxaGH operon for their repression and containing a consensus sequence of AWTATAtagaNYGgTCTA, where W, Y, and N stand for A or T, C or T, and any base, respectively (three-out-of-four match [in lowercase type]). Gel retardation analysis indicated that out of the eight flavonoids tested, quercetin, fisetin, and catechin are most inhibitory for LmrA to DNA binding, whereas quercetin, fisetin, tamarixetin, and galangin are most inhibitory for YxaF. Also, YxaF bound most tightly to the tandem LmrA/YxaF boxes in the yxaGH promoter region. The lacZ fusion experiments essentially supported the above-mentioned in vitro results, except that galangin did not activate the lmrAB and yxaGH promoters, probably due to its poor incorporation into cells. Thus, the LmrA/YxaF regulon presumably comprising the lmrAB operon, the yxaF gene, and the yxaGH operon is induced in response to certain flavonoids. The in vivo experiments to examine the regulation of the synthesis of the reporter β-galactosidase and quercetin 2,3-dioxgenase as well as that of multidrug resistance suggested that LmrA represses the lmrAB and yxaGH operons but that YxaF represses yxaGH more preferentially.

scholarly journals Regulation of the Bacillus subtilis Divergent yetL and yetM Genes by a Transcriptional Repressor, YetL, in Response to Flavonoids

2009 ◽  
Vol 191 (11) ◽  
pp. 3685-3697 ◽  
Author(s):  
Kazutake Hirooka ◽  
Yusuke Danjo ◽  
Yuki Hanano ◽  
Satoshi Kunikane ◽  
Hiroshi Matsuoka ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Hydroxyl Group ◽  
Hydroxyl Groups ◽  
Promoter Regions ◽  
Gene Encoding ◽  
Gel Retardation ◽  
Dnase I Footprinting ◽  
Shine Dalgarno Sequence

ABSTRACT DNA microarray analysis revealed that transcription of the Bacillus subtilis yetM gene encoding a putative flavin adenine dinucleotide-dependent monooxygenase was triggered by certain flavonoids during culture and was derepressed by disruption of the yetL gene in the opposite orientation situated immediately upstream of yetM, which encodes a putative MarR family transcriptional regulator. In vitro analyses, including DNase I footprinting and gel retardation analysis, indicated that YetL binds specifically to corresponding single sites in the divergent yetL and yetM promoter regions, with higher affinity to the yetM region; the former region overlaps the Shine-Dalgarno sequence of yetL, and the latter region contains a perfect 18-bp palindromic sequence (TAGTTAGGCGCCTAACTA). In vitro gel retardation and in vivo lacZ fusion analyses indicated that some flavonoids (kaempferol, apigenin, and luteolin) effectively inhibit YetL binding to the yetM cis sequence, but quercetin, galangin, and chrysin do not inhibit this binding, implying that the 4-hydroxyl group on the B-ring of the flavone structure is indispensable for this inhibition and that the coexistence of the 3-hydroxyl groups on the B- and C-rings does not allow antagonism of YetL.

scholarly journals CfaD-Dependent Expression of a Novel Extracytoplasmic Protein from Enterotoxigenic Escherichia coli

2007 ◽  
Vol 189 (14) ◽  
pp. 5060-5067 ◽  
Author(s):  
M. Carolina Pilonieta ◽  
Maria D. Bodero ◽  
George P. Munson
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Secretory Pathway ◽  
Dnase I ◽  
Enterotoxigenic Escherichia Coli ◽  
Watery Diarrhea ◽  
Dnase I Footprinting ◽  
Virulence Regulator

ABSTRACT H10407 is a strain of enterotoxigenic Escherichia coli (ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promoter cfaAp. One site extends from positions −23 to −56, and the other extends from positions −73 to −103 (numbering relative to the transcription start site of cfaAp). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene, cexE. In vitro DNase I footprinting confirmed that both sites are genuine binding sites, and cexEp::lacZ reporters demonstrated that CfaD is required for the expression of cexE in vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC.

scholarly journals AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions

2015 ◽  
Vol 197 (24) ◽  
pp. 3788-3796 ◽  
Author(s):  
Takayuki Kuge ◽  
Haruhiko Teramoto ◽  
Masayuki Inui
Keyword(s):  
Corynebacterium Glutamicum ◽  
Binding Sites ◽  
Large Scale ◽  
Transcriptional Regulator ◽  
Scale Production ◽  
Primary Role ◽  
Promoter Regions ◽  
Independent Manner ◽  
Content Type

ABSTRACTInCorynebacterium glutamicumATCC 31831, a LacI-type transcriptional regulator AraR, represses the expression ofl-arabinose catabolism (araBDA), uptake (araE), and the regulator (araR) genes clustered on the chromosome. AraR binds to three sites: one (BSB) between the divergent operons (araBDAandgalM-araR) and two (BSE1and BSE2) upstream ofaraE.l-Arabinose acts as an inducer of the AraR-mediated regulation. Here, we examined the roles of these AraR-binding sites in the expression of the AraR regulon. BSBmutation resulted in derepression of botharaBDAandgalM-araRoperons. The effects of BSE1and/or BSE2mutation onaraEexpression revealed that the two sites independently function as theciselements, but BSE1plays the primary role. However, AraR was shown to bind to these sites with almost the same affinityin vitro. Taken together, the expression ofaraBDAandaraEis strongly repressed by binding of AraR to a single site immediately downstream of the respective transcriptional start sites, whereas the binding site overlapping the −10 or −35 region of thegalM-araRandaraEpromoters is less effective in repression. Furthermore, downregulation ofaraBDAandaraEdependent onl-arabinose catabolism observed in the BSBmutant and the AraR-independentaraRpromoter identified withingalM-araRadd complexity to regulation of the AraR regulon derepressed byl-arabinose.IMPORTANCECorynebacterium glutamicumhas a long history as an industrial workhorse for large-scale production of amino acids. An important aspect of industrial microorganisms is the utilization of the broad range of sugars for cell growth and production process. MostC. glutamicumstrains are unable to use a pentose sugarl-arabinose as a carbon source. However, genes forl-arabinose utilization and its regulation have been recently identified inC. glutamicumATCC 31831. This study elucidates the roles of the multiple binding sites of the transcriptional repressor AraR in the derepression byl-arabinose and thereby highlights the complex regulatory feedback loops in combination withl-arabinose catabolism-dependent repression of the AraR regulon in an AraR-independent manner.

scholarly journals Binding and transcriptional activation of non-flagellar genes by the Escherichia coli flagellar master regulator FlhD2C2

Microbiology ◽  
2005 ◽  
Vol 151 (6) ◽  
pp. 1779-1788 ◽  
Author(s):  
Graham P. Stafford ◽  
Tomoo Ogi ◽  
Colin Hughes
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Class Ii ◽  
Promoter Regions ◽  
E Coli ◽  
Flagellar Genes

The gene hierarchy directing biogenesis of peritrichous flagella on the surface of Escherichia coli and other enterobacteria is controlled by the heterotetrameric master transcriptional regulator FlhD2C2. To assess the extent to which FlhD2C2 directly activates promoters of a wider regulon, a computational screen of the E. coli genome was used to search for gene-proximal DNA sequences similar to the 42–44 bp inverted repeat FlhD2C2 binding consensus. This identified the binding sequences upstream of all eight flagella class II operons, and also putative novel FlhD2C2 binding sites in the promoter regions of 39 non-flagellar genes. Nine representative non-flagellar promoter regions were all bound in vitro by active reconstituted FlhD2C2 over the K D range 38–356 nM, and of the nine corresponding chromosomal promoter–lacZ fusions, those of the four genes b1904, b2446, wzz fepE and gltI showed up to 50-fold dependence on FlhD2C2 in vivo. In comparison, four representative flagella class II promoters bound FlhD2C2 in the K D range 12–43 nM and were upregulated in vivo 30- to 990-fold. The FlhD2C2-binding sites of the four regulated non-flagellar genes overlap by 1 or 2 bp the predicted −35 motif of the FlhD2C2-activated σ 70 promoters, as is the case with FlhD2C2-dependent class II flagellar promoters. The data indicate a wider FlhD2C2 regulon, in which non-flagellar genes are bound and activated directly, albeit less strongly, by the same mechanism as that regulating the flagella gene hierarchy.

scholarly journals Binding Site Recognition by Rns, a Virulence Regulator in the AraC Family

1999 ◽  
Vol 181 (7) ◽  
pp. 2110-2117 ◽  
Author(s):  
George P. Munson ◽  
June R. Scott
Keyword(s):  
Binding Sites ◽  
Shigella Flexneri ◽  
Transcriptional Level ◽  
Regulatory Cascade ◽  
Dnase I Footprinting ◽  
Nucleotide Interactions ◽  
Virulence Regulator ◽  
Arac Family

ABSTRACT The expression of CS1 pili by enterotoxigenic strains ofEscherichia coli is regulated at the transcriptional level and requires the virulence regulator Rns, a member of the AraC family of regulatory proteins. Rns binds at two separate sites upstream of Pcoo (the promoter of CS1 pilin genes), which were identified in vitro with an MBP::Rns fusion protein in gel mobility and DNase I footprinting assays. At each site, Rns recognizes asymmetric nucleotide sequences in two regions of the major groove and binds along one face of the DNA helix. Both binding sites are required for activation of Pcoo in vivo, because mutagenesis of either site significantly reduced the level of expression from this promoter. Thus, Rns regulates the expression of CS1 pilin genes directly, not via a regulatory cascade. Analysis of Rns-nucleotide interactions at each site suggests that binding sites for Rns and related virulence regulators are not easily identified because they do not bind palindromic or repeated sequences. A strategy to identify asymmetric binding sites is presented and applied to locate potential binding sites upstream of other genes that Rns can activate, including those encoding the CS2 and CFA/I pili of enterotoxigenic E. coli and the global regulator virB of Shigella flexneri.

scholarly journals Transcriptional Repression Mediated by LysR-Type Regulator CatR Bound at Multiple Binding Sites

1998 ◽  
Vol 180 (9) ◽  
pp. 2367-2372 ◽  
Author(s):  
Sudha A. Chugani ◽  
Matthew R. Parsek ◽  
A. M. Chakrabarty
Keyword(s):  
Binding Site ◽  
Structural Gene ◽  
Binding Sites ◽  
Transcriptional Start Site ◽  
Dnase I ◽  
Site Directed Mutagenesis ◽  
Dnase I Footprinting ◽  
Gel Shift

ABSTRACT The catBCA operon of Pseudomonas putidaencodes enzymes involved in the catabolism of benzoate. Transcription of this operon requires the LysR-type transcriptional regulator CatR and an inducer molecule, cis,cis-muconate. Previous gel shift assays and DNase I footprinting have demonstrated that CatR occupies two adjacent sites proximal to thecatBCA promoter in the presence of the inducer. We report the presence of an additional binding site for CatR downstream of thecatBCA promoter within the catB structural gene. This site, called the internal binding site (IBS), extends from +162 to +193 with respect to the catB transcriptional start site and lies within the catB open reading frame. Gel shift analysis and DNase I footprinting determined that CatR binds to this site with low affinity. CatR binds cooperatively with higher affinity to the IBS in the presence of the two upstream binding sites. Parallel in vivo and in vitro studies were conducted to determine the role of the internal binding site. We measured β-galactosidase activity ofcatB-lacZ transcriptional fusions in vivo. Our results suggest a probable cis-acting repressor function for the internal binding site. Site-directed mutagenesis of the IBS verified this finding. The location of the IBS within the catBstructural gene, the cooperativity observed in footprinting studies, and phasing studies suggest that the IBS likely participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA.

scholarly journals Engineering of a Bacillus subtilis Strain with Adjustable Levels of Intracellular Biotin for Secretory Production of Functional Streptavidin

2002 ◽  
Vol 68 (3) ◽  
pp. 1102-1108 ◽  
Author(s):  
Sau-Ching Wu ◽  
Sui-Lam Wong
Keyword(s):  
Bacillus Subtilis ◽  
Binding Sites ◽  
Culture Medium ◽  
Regulatory Region ◽  
Subtilis Strain ◽  
Biotin Deficiency ◽  
Poor Growth ◽  
Biotin Binding

ABSTRACT Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.

scholarly journals YrxA Is the Transcriptional Regulator That Represses De Novo NAD Biosynthesis in Bacillus subtilis

2005 ◽  
Vol 187 (20) ◽  
pp. 7155-7160 ◽  
Author(s):  
Paola Rossolillo ◽  
Ilaria Marinoni ◽  
Elisa Galli ◽  
Anna Colosimo ◽  
Alessandra M. Albertini
Keyword(s):  
Bacillus Subtilis ◽  
Nicotinic Acid ◽  
Promoter Region ◽  
De Novo ◽  
Transcriptional Regulator ◽  
Transcription Repression ◽  
De Novo Biosynthesis ◽  
Nad Biosynthesis

ABSTRACT The first genetic, in vivo, and in vitro evidences that YrxA is the regulator of NAD de novo biosynthesis in Bacillus subtilis are hereby reported. The protein is essential to the transcription repression of the divergent operons nadBCA and nifS-yrxA in the presence of nicotinic acid and binds to their shared operator-promoter region.

scholarly journals Acetolactate Synthase from Bacillus subtilis Serves as a 2-Ketoisovalerate Decarboxylase for Isobutanol Biosynthesis in Escherichia coli

2009 ◽  
Vol 75 (19) ◽  
pp. 6306-6311 ◽  
Author(s):  
Shota Atsumi ◽  
Zhen Li ◽  
James C. Liao
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Bacillus Subtilis ◽  
Lactococcus Lactis ◽  
Acetolactate Synthase ◽  
Side Chains ◽  
Decarboxylase Activity ◽  
Small Side

ABSTRACTA pathway toward isobutanol production previously constructed inEscherichia coliinvolves 2-ketoacid decarboxylase (Kdc) fromLactococcus lactisthat decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde. Here, we showed that a strain lacking Kdc is still capable of producing isobutanol. We found that acetolactate synthase fromBacillus subtilis(AlsS), which originally catalyzes the condensation of two molecules of pyruvate to form 2-acetolactate, is able to catalyze the decarboxylation of KIV like Kdc both in vivo and in vitro. Mutational studies revealed that the replacement of Q487 with amino acids with small side chains (Ala, Ser, and Gly) diminished only the decarboxylase activity but maintained the synthase activity.

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