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 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 A specialized flavone biosynthetic pathway has evolved in the medicinal plant,Scutellaria baicalensis

2016 ◽  
Vol 2 (4) ◽  
pp. e1501780 ◽  
Author(s):  
Qing Zhao ◽  
Yang Zhang ◽  
Gang Wang ◽  
Lionel Hill ◽  
Jing-Ke Weng ◽  
...  
Keyword(s):  
Herbal Remedy ◽  
Wide Spectrum ◽  
Human Tumor ◽  
Scutellaria Baicalensis ◽  
Hydroxyl Group ◽  
Mouse Tumor ◽  
Gene Encoding ◽  
Beneficial Effects

Wogonin and baicalein are bioactive flavones in the popular Chinese herbal remedy Huang-Qin (Scutellaria baicalensisGeorgi). These specialized flavones lack a 4′-hydroxyl group on the B ring (4′-deoxyflavones) and induce apoptosis in a wide spectrum of human tumor cells in vitro and inhibit tumor growth in vivo in different mouse tumor models. Root-specific flavones (RSFs) fromScutellariahave a variety of reported additional beneficial effects including antioxidant and antiviral properties. We describe the characterization of a new pathway for the synthesis of these compounds, in which pinocembrin (a 4′-deoxyflavanone) serves as a key intermediate. Although two genes encoding flavone synthase II (FNSII) are expressed in the roots ofS.baicalensis, FNSII-1 has broad specificity for flavanones as substrates, whereas FNSII-2 is specific for pinocembrin. FNSII-2 is responsible for the synthesis of 4′-deoxyRSFs, such as chrysin and wogonin, wogonoside, baicalein, and baicalin, which are synthesized from chrysin. A gene encoding a cinnamic acid–specific coenzyme A ligase (SbCLL-7), which is highly expressed in roots, is required for the synthesis of RSFs by FNSII-2, as demonstrated by gene silencing. A specific isoform of chalcone synthase (SbCHS-2) that is highly expressed in roots producing RSFs is also required for the synthesis of chrysin. Our studies reveal a recently evolved pathway for biosynthesis of specific, bioactive 4′-deoxyflavones in the roots ofS.baicalensis.

scholarly journals Improvement of α-Amylase Production by Modulation of Ribosomal Component Protein S12 in Bacillus subtilis 168

2006 ◽  
Vol 72 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Kazuhiko Kurosawa ◽  
Takeshi Hosaka ◽  
Norimasa Tamehiro ◽  
Takashi Inaoka ◽  
Kozo Ochi
Keyword(s):  
Bacillus Subtilis ◽  
Antibiotic Production ◽  
Point Mutations ◽  
Protease Production ◽  
Gene Encoding ◽  
Translational Activity ◽  
Amylase Production ◽  
Ribosomal Protein S12

ABSTRACT The capacity of ribosomal modification to improve antibiotic production by Streptomyces spp. has already been demonstrated. Here we show that introduction of mutations that produce streptomycin resistance (str) also enhances α-amylase (and protease) production by a strain of Bacillus subtilis as estimated by measuring the enzyme activity. The str mutations are point mutations within rpsL, the gene encoding the ribosomal protein S12. In vivo as well as in vitro poly(U)-directed cell-free translation systems showed that among the various rpsL mutations K56R (which corresponds to position 42 in E. coli) was particularly effective at enhancing α-amylase production. Cells harboring the K56R mutant ribosome exhibited enhanced translational activity during the stationary phase of cell growth. In addition, the K56R mutant ribosome exhibited increased 70S complex stability in the presence of low Mg2+ concentrations. We therefore conclude that the observed increase in protein synthesis activity by the K56R mutant ribosome reflects increased stability of the 70S complex and is responsible for the increase in α-amylase production seen in the affected strain.

scholarly journals Identification of a Mutation in the Bacillus subtilis S-Adenosylmethionine Synthetase Gene That Results in Derepression of S-Box Gene Expression

2006 ◽  
Vol 188 (10) ◽  
pp. 3674-3681 ◽  
Author(s):  
Brooke A. McDaniel ◽  
Frank J. Grundy ◽  
Vineeta P. Kurlekar ◽  
Jerneja Tomsic ◽  
Tina M. Henkin
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Rna Structure ◽  
Synthetase Activity ◽  
Gene Encoding ◽  
Sam Synthetase ◽  
Transcription In Vitro ◽  
Adenosylmethionine Synthetase

ABSTRACT Genes in the S-box family are regulated by binding of S-adenosylmethionine (SAM) to the 5′ region of the mRNA of the regulated gene. SAM binding was previously shown to promote a rearrangement of the RNA structure that results in premature termination of transcription in vitro and repression of expression of the downstream coding sequence. The S-box RNA element therefore acts as a SAM-binding riboswitch in vitro. In an effort to identify factors other than SAM that could be involved in the S-box regulatory mechanism in vivo, we searched for trans-acting mutations in Bacillus subtilis that act to disrupt repression of S-box gene expression during growth under conditions where SAM pools are elevated. We identified a single mutant that proved to have one nucleotide substitution in the metK gene, encoding SAM synthetase. This mutation, designated metK10, resulted in a 15-fold decrease in SAM synthetase activity and a 4-fold decrease in SAM concentration in vivo. The metK10 mutation specifically affected S-box gene expression, and the increase in expression under repressing conditions was dependent on the presence of a functional transcriptional antiterminator element. The observation that the mutation identified in this search affects SAM production supports the model that the S-box RNAs directly monitor SAM in vivo, without a requirement for additional factors.

scholarly journals Identification of the Actinobacillus pleuropneumoniae Leucine-Responsive Regulatory Protein and Its Involvement in the Regulation of In Vivo-Induced Genes

2006 ◽  
Vol 75 (1) ◽  
pp. 91-103 ◽  
Author(s):  
Trevor K. Wagner ◽  
Martha H. Mulks
Keyword(s):  
Regulatory Protein ◽  
Bacterial Pathogen ◽  
Actinobacillus Pleuropneumoniae ◽  
Promoter Regions ◽  
Host Animal ◽  
Gene Encoding ◽  
Serotype 1 ◽  
Branched Chain

ABSTRACT Actinobacillus pleuropneumoniae is a gram-negative bacterial pathogen that causes a severe hemorrhagic pneumonia in swine. We have previously shown that the limitation of branched-chain amino acids (BCAAs) is a cue that induces the expression of a subset of A. pleuropneumoniae genes identified as specifically induced during infection of the natural host animal by using an in vivo expression technology screen. Leucine-responsive regulatory protein (Lrp) is a global regulator and has been shown in Escherichia coli to regulate many genes, including genes involved in BCAA biosynthesis. We hypothesized that A. pleuropneumoniae contains a regulator similar to Lrp and that this protein is involved in the regulation of a subset of genes important during infection and recently shown to have increased expression in the absence of BCAAs. We report the identification of an A. pleuropneumoniae serotype 1 gene encoding a protein with similarity to amino acid sequence and functional domains of other reported Lrp proteins. We further show that purified A. pleuropneumoniae His6-Lrp binds in vitro to the A. pleuropneumoniae promoter regions for ilvI, antisense cps1AB, lrp, and nqr. A genetically defined A. pleuropneumoniae lrp mutant was constructed using an allelic replacement and sucrose counterselection method. Analysis of expression from the ilvI and antisense cps1AB promoters in wild-type, lrp mutant, and complemented lrp mutant strains indicated that Lrp is required for induction of expression of ilvI under BCAA limitation.

scholarly journals Metabolism of apigenin and related compounds in the rat. Metabolite formation in vivo and by the intestinal microflora in vitro

1972 ◽  
Vol 128 (4) ◽  
pp. 901-911 ◽  
Author(s):  
L. A. Griffiths ◽  
G. E. Smith
Keyword(s):  
Oral Administration ◽  
Intestinal Microflora ◽  
Biochanin A ◽  
Hydroxyl Group ◽  
Hydroxyl Groups ◽  
Flavonoid Compounds ◽  
Ring Fission ◽  
Micro Organisms

1. The metabolism of a group of flavonoid compounds related in structure to apigenin (4′,5,7-trihydroxyflavone) and including apigenin, apiin, naringin, phlorrhizin, acacetin, kaempferol, robinin, chrysin, tectochrysin and 4′,7-dihydroxyflavone, was studied both in vivo after oral administration to the rat, and in vitro in cultures of micro-organisms derived from the intestine of the rat. 2. The rat intestinal microflora is capable of effecting degradation of flavonoid compounds to metabolites observed in the urine after oral administration of the specific flavonoid. 3. All compounds possessing free 5- and 7-hydroxyl groups in the A ring and a free 4′-hydroxyl group in the B ring gave rise to ring-fission products, which included 4′-hydroxyphenylacyl derivatives. 4. On anaerobic incubation in a thioglycollate medium, intestinal micro-organisms can effect flavonoid-ring fission, cleavage of glycosidic bonds and the reduction of double bonds in the side chains of certain metabolites. 5. Two flavonoids (chrysin and tectochrysin) undergo hydroxylation in the 4′-position in vivo but not during incubation with the intestinal microflora in vitro. 6. Observations on the metabolism of other compounds substituted in the 4′-position, e.g. epiafzelechin, pelargonin and the isoflavones, genistein, biochanin A, daidzein and formononetin, by the intestinal microflora of the rat are also reported.

scholarly journals Characterization of ResDE-Dependent fnr Transcription in Bacillus subtilis

2006 ◽  
Vol 189 (5) ◽  
pp. 1745-1755 ◽  
Author(s):  
Hao Geng ◽  
Yi Zhu ◽  
Karl Mullen ◽  
Cole S. Zuber ◽  
Michiko M. Nakano
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Transcription Initiation ◽  
Sequence Similarity ◽  
Anaerobic Respiration ◽  
Amino Acid Residues ◽  
Promoter Regions ◽  
Α Subunit ◽  
Dnase I Footprinting

ABSTRACT The ResD-ResE signal transduction system is required for transcription of genes involved in aerobic and anaerobic respiration in Bacillus subtilis. Phosphorylated ResD (ResD∼P) interacts with target DNA to activate transcription. A strong sequence similarity was detected in promoter regions of some ResD-controlled genes including fnr and resA. Single-base substitutions in the fnr and resA promoters were performed to determine a ResD-binding sequence. DNase I footprinting analysis indicated that ResD∼P itself does not bind to fnr, but interaction of ResD∼P with the C-terminal domain of the α subunit (αCTD) of RNA polymerase (RNAP) facilitates cooperative binding of ResD∼P and RNAP, thereby increasing fnr transcription initiation. Consistent with this result, amino acid substitutions in αCTD, such as Y263A, K267A, A269I, or N290A, sharply reduced fnr transcription in vivo, and the K267A αCTD protein, unlike the wild-type protein, did not increase ResD∼P binding to the fnr promoter. Amino acid residues of αCTD required for ResD-dependent fnr transcription, with the exception of N290, which may interact with DNA, constitute a distinct surface, suggesting that these residues likely interact with ResD∼P.

scholarly journals Functional Analysis of Bacillus subtilis Genes Involved in the Biosynthesis of 4-Thiouridine in tRNA

2012 ◽  
Vol 194 (18) ◽  
pp. 4933-4940 ◽  
Author(s):  
Lauren J. Rajakovich ◽  
John Tomlinson ◽  
Patricia C. Dos Santos
Keyword(s):  
Functional Analysis ◽  
Bacillus Subtilis ◽  
Gene Encoding ◽  
Cysteine Desulfurase ◽  
Content Type ◽  
Sulfur Transfer ◽  
Essential Enzyme ◽  
Sulfur Trafficking

ABSTRACTThiI has been identified as an essential enzyme involved in the biosynthesis of thiamine and the tRNA thionucleoside modification, 4-thiouridine. InEscherichia coliandSalmonella enterica, ThiI acts as a sulfurtransferase, receiving the sulfur donated from the cysteine desulfurase IscS and transferring it to the target molecule or additional sulfur carrier proteins. However, inBacillus subtilisand most species from theFirmicutesphylum, ThiI lacks the rhodanese domain that contains the site responsible for the sulfurtransferase activity. The lack of the gene encoding for a canonical IscS cysteine desulfurase and the presence of a short sequence of ThiI in these bacteria pointed to mechanistic differences involving sulfur trafficking reactions in both biosynthetic pathways. Here, we have carried out functional analysis ofB. subtilisthiIand the adjacent gene,nifZ, encoding for a cysteine desulfurase. Gene inactivation experiments inB. subtilisindicate the requirement of ThiI and NifZ for the biosynthesis of 4-thiouridine, but not thiamine.In vitrosynthesis of 4-thiouridine by ThiI and NifZ, along with labeling experiments, suggests the occurrence of an alternate transient site for sulfur transfer, thus obviating the need for a rhodanese domain.In vivocomplementation studies inE. coliIscS- or ThiI-deficient strains provide further support for specific interactions between NifZ and ThiI. These results are compatible with the proposal thatB. subtilisNifZ and ThiI utilize mechanistically distinct and mutually specific sulfur transfer reactions.

scholarly journals Growth Phase and Metal-Dependent Transcriptional Regulation of the fecA Genes in Helicobacter pylori

2009 ◽  
Vol 191 (11) ◽  
pp. 3717-3725 ◽  
Author(s):  
Alberto Danielli ◽  
Simona Romagnoli ◽  
Davide Roncarati ◽  
Lorenzo Costantino ◽  
Isabel Delany ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Growth Phase ◽  
Transcriptional Control ◽  
Exponential Phase ◽  
Metal Availability ◽  
Promoter Regions ◽  
Nickel Iron ◽  
Dnase I Footprinting

ABSTRACT Balancing metal uptake is essential for maintaining a proper intracellular metal concentration. Here, we report the transcriptional control exerted by the two metal-responsive regulators of Helicobacter pylori, Fur (iron-dependent ferric uptake regulator) and NikR (nickel-responsive regulator), on the three copies of the fecA genes present in this species. By monitoring the patterns of transcription throughout growth and in response to nickel, iron, and a metal chelator, we found that the expression of the three fecA genes is temporally regulated, responds to metals in different ways, and is selectively controlled by either one of the two regulators. fecA1 is expressed at a constant level throughout growth, and its expression is iron sensitive; the expression of fecA2 is mainly off, with minor expression coming up in late exponential phase. In contrast, the expression of fecA3 is maximal in early exponential phase, gradually decreases with time, and is repressed by nickel. The direct roles of Fur and NikR were studied both in vitro, by mapping the binding sites of each regulator on the promoter regions via DNase I footprinting analysis, and in vivo, by using primer extension analyses of the fecA transcripts in fur and nikR deletion strains. Overall, the results show that the expression of each fecA gene is finely tuned in response to metal availability, as well as during the bacterial growth phase, suggesting specific and dedicated functions for the three distinct FecA homologues.

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