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

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.

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The two isozymes of rat intestinal alkaline phosphatase are products of two distinct genes

Physiological Genomics ◽

10.1152/physiolgenomics.2000.3.1.1 ◽

2000 ◽

Vol 3 (1) ◽

pp. 1-8 ◽

Cited By ~ 23

Author(s):

Q. XIE ◽

D. H. ALPERS

Keyword(s):

Alkaline Phosphatase ◽

Transcriptional Start Site ◽

In Vivo Studies ◽

Peroxisome Proliferator ◽

Start Site ◽

Intestinal Alkaline Phosphatase ◽

Promoter Regions ◽

Flanking Regions

Xie, Q., and D. H. Alpers. The two isozymes of rat intestinal alkaline phosphatase are products of two distinct genes. Physiol Genomics 3: 1–8, 2000.—Rat intestinal alkaline phosphatases (IAP-I and -II) differ in primary structure, substrate specificity, tissue localization, and response to fat feeding. This study identifies two distinct genes (∼5–6 kb) corresponding to each isozyme and containing 11 exons of nearly identical size. The exon-intron junctions are identical with those found in IAP genes from other species. The 1.7 and 1.2 bp of 5′ flanking regions isolated from each gene, respectively, contain Sp1 and gut-enriched Kruppel-like factor (GKLF) binding sites, but otherwise show little identity. There is a potential CAAT-box 14 bp 5′ to the transcriptional start site, 36 bp upstream from IAP-I, and a TATA-box 31 bp 5′ to the transcriptional start site, 55 bp upstream from IAP-II. Transfection of these promoter regions (linked to luciferase as a reporter gene) into a kidney cell line, COS-7, produced the differential response to oleic acid expected from in vivo studies, i.e., threefold increase using the 5′ flanking region of IAP-II, but not IAP-I. This response was not reproduced by 5,8,11,14-eicosatetraynoic acid (ETYA) or clofibrate, suggesting that peroxisome proliferator response elements are not involved. Isolation of the IAP-II gene will allow determination of the sequences responsible for dietary fat response in the enterocyte.

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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 ◽

10.1099/mic.0.064675-0 ◽

2014 ◽

Vol 160 (2) ◽

pp. 243-260 ◽

Cited By ~ 10

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.

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ScoC Regulates Bacilysin Production at the Transcription Level in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.01081-09 ◽

2009 ◽

Vol 191 (23) ◽

pp. 7367-7371 ◽

Cited By ~ 15

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.

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

Journal of Bacteriology ◽

10.1128/jb.00151-09 ◽

2009 ◽

Vol 191 (10) ◽

pp. 3273-3281 ◽

Cited By ~ 17

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.

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The Transcriptional Regulator RovS Controls the Attachment of Streptococcus agalactiae to Human Epithelial Cells and the Expression of Virulence Genes

Infection and Immunity ◽

10.1128/iai.00667-06 ◽

2006 ◽

Vol 74 (10) ◽

pp. 5625-5635 ◽

Cited By ~ 32

Author(s):

Ulrike M. Samen ◽

Bernhard J. Eikmanns ◽

Dieter J. Reinscheid

Keyword(s):

Gene Expression ◽

Streptococcus Agalactiae ◽

Bacterial Infections ◽

Virulence Genes ◽

Consensus Sequence ◽

Transcriptional Regulator ◽

Epithelial Cell Line ◽

Promoter Regions ◽

Mobility Shift ◽

Virulence Gene Expression

ABSTRACT Streptococcus agalactiae is part of the normal flora of the human gastrointestinal tract and also the leading cause of bacterial infections in human newborns and immunocompromised adults. The colonization and infection of different regions within the human host require a regulatory network in S. agalactiae that senses environmental stimuli and controls the formation of specific virulence factors. In the present study, we characterized an Rgg-like transcriptional regulator, designated RovS (regulator of virulence in Streptococcus agalactiae). Deletion of the rovS gene in the genome of S. agalactiae resulted in strain 6313 ΔrovS, which exhibited an increased attachment to immobilized fibrinogen and a significant increase in adherence to the eukaryotic lung epithelial cell line A549. Quantification of expression levels of known and putative S. agalactiae virulence genes by real-time PCR revealed that RovS influences the expression of fbsA, gbs0230, sodA, rogB, and the cyl operon. The altered gene expression in mutant 6313 ΔrovS was restored by plasmid-mediated expression of rovS, confirming the RovS deficiency as the cause for the observed changes in virulence gene expression in S. agalactiae. DNA electrophoretic mobility shift assays showed that RovS specifically binds to the promoter regions of fbsA, gbs0230, sodA, and the cyl operon, indicating that RovS directly regulates their expression. Deletion and mutation studies in the promoter region of fbsA, encoding the main fibrinogen receptor in S. agalactiae, identified a RovS DNA motif. Similar motifs were also found in the promoter regions of gbs0230, sodA, and the cyl operon, and alignments allowed us to propose a consensus sequence for the DNA-binding site of RovS.

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

Journal of Bacteriology ◽

10.1128/jb.187.19.6659-6667.2005 ◽

2005 ◽

Vol 187 (19) ◽

pp. 6659-6667 ◽

Cited By ~ 45

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.

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Cis- andtrans-acting factors influence expression of thenorM-encoded efflux pump ofNeisseria gonorrhoeaeand levels of gonococcal susceptibility to substrate antimicrobials

10.1101/308031 ◽

2018 ◽

Author(s):

Corinne E. Rouquette-Loughlin ◽

Vijaya Dhulipala ◽

Jennifer L. Reimche ◽

Erica Raterman ◽

Afrin A. Begum ◽

...

Keyword(s):

Quaternary Ammonium ◽

Efflux Pump ◽

Transcriptional Start Site ◽

Transcriptional Regulator ◽

Start Site ◽

Peptidoglycan Synthesis ◽

Regulatory Systems ◽

Expression Of Genes ◽

Extension Analysis ◽

Ammonium Compounds

ABSTRACTThe gonococcal NorM efflux pump exports substrates with a cationic moiety including quaternary ammonium compounds such as berberine (BE) and ethidium bromide (EB) as well as antibiotics such as ciprofloxacin and solithromycin. ThenorMgene is part of a four gene operon that is transcribed from a promoter containing a polynucleotide tract of 6 or 7 thymidines (Ts) between the -10 and -35 hexamers; the majority of gonococcal strains analyzed herein contained a T-6 sequence. Primer extension analysis showed that regardless of the length of the poly-T tract, the same transcriptional start site (TSS) was used for expression ofnorM. Interestingly, the T-6 tract correlated with a higher level of bothnorMexpression and gonococcal resistance to NorM substrates BE and EB. Analysis of expression of genes downstream ofnorMshowed that the product of thetetR-like gene has the capacity to activate expression ofnorMas well asmurB, which encodes an acetylenolpyroylglucosamine reductase predicted to be involved in the early steps of peptidoglycan synthesis. Moreover, loss of the TetR-like transcriptional regulator modestly increased gonococcal susceptibility to NorM substrates EB and BE. We conclude that bothcis- andtrans-acting regulatory systems can regulate expression of thenorMoperon and influence levels of gonococcal susceptibility to antimicrobials exported by NorM.

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Identification of a novelcis-acting element for fibroblast-specific transcription of theFSP1 gene

AJP Renal Physiology ◽

10.1152/ajprenal.1998.275.2.f306 ◽

1998 ◽

Vol 275 (2) ◽

pp. F306-F314 ◽

Cited By ~ 6

Author(s):

Hirokazu Okada ◽

Theodore M. Danoff ◽

Andreas Fischer ◽

Jesus M. Lopez-Guisa ◽

Frank Strutz ◽

...

Keyword(s):

S100 Protein ◽

Luciferase Reporter ◽

Flanking Sequence ◽

Promoter Regions ◽

Mobility Shift ◽

Cis Acting ◽

First Intron ◽

Nuclear Extracts ◽

Ef Hands ◽

Gel Mobility Shift

The FSP1 gene encodes a filament-binding S100 protein with paired EF hands that is specifically expressed in fibroblasts. This led us to look for cis-acting elements in the FSP1 promoter that might engage nuclear transcription factors unique to fibroblasts. The first exon of FSP1 is noncoding, therefore, a series of luciferase reporter minigenes were created containing varying lengths of 5′-flanking sequence, the first intron, and the noncoding region of the second exon. A position and promoter-dependent proximal element between −187 and −88 bp was shown to be active in fibroblasts but not in epithelium. Sequence in the first intron from +777 to +964 had an enhancing effect that was not cell type specific. Hsv TK reporter constructs driven by this promoter/intron cassette in transgenic mice were coexpressed appropriately with FSP1 in tissue fibroblasts. Gel mobility shift competitor assays identified a novel domain, FTS-1 (fibroblast transcription site-1; TTGAT from −177 to −173 bp), that specifically interacts with nuclear extracts from fibroblasts. The necessity of this binding site was confirmed by site-specific mutagenesis. Database searches also turned up putative FTS-1 sites in the early promoter regions of other fibroblast expressed proteins, including the α1 and α2(I), and α1(III) collagens and the αSM-actin gene. We hypothesize that the selective engagement of FTS-1 elements may contribute to the mesenchymal phenotype of fibroblasts and perhaps other dedifferentiated cells.

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Transcriptional regulation of the macs1-fadD1 operon encoding two acyl-CoA synthases involved in the physiological differentiation of Streptomyces coelicolor

Microbiology ◽

10.1099/mic.0.28553-0 ◽

2006 ◽

Vol 152 (5) ◽

pp. 1427-1439 ◽

Cited By ~ 5

Author(s):

Ana Arabolaza ◽

Claudia Banchio ◽

Hugo Gramajo

Keyword(s):

Tandem Repeats ◽

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Transcriptional Start Site ◽

Clear Correlation ◽

Mobility Shift ◽

Central Sequence ◽

Dnase I Footprinting ◽

Chain Acyl ◽

Gel Mobility Shift

The long-chain acyl-CoA synthase (ACS) FadD1 plays an important role in timing the levels of antibiotic production in Streptomyces coelicolor. fadD1 and macs1, encoding a putative medium-chain ACS, are part of a two-gene operon, whose expression is induced during the stationary phase of growth. Here it is reported that transcription of the macs1-fadD1 operon is positively regulated by AcsR, a LuxR-type transcriptional regulator. In an acsR mutant, expression of the macs1-fadD1 genes loses its normal up-regulation and the mutant becomes deficient in antibiotic production, in a clear correlation with the phenotype shown by a fadD1 null mutant. The absence of macs1-fadD1 induction in the acsR mutant was restored by complementation with a wild-type copy of the acsR gene, showing a strict link between AcsR and induction of the macs1-fadD1 operon. Gel mobility shift assays and DNase I footprinting indicated that AcsR binds to specific sequences about +162 nucleotides downstream of the macs1 transcriptional start site. In the putative operator sequence three almost identical direct tandem repeats of seven nucleotides were identified where the central sequence is essential for AcsR recognition and binding. Transcriptional fusions of the divergent pacsR and pmacs1 promoters indicated that AcsR does not regulate its own transcription, and that it binds to the operator region to control exclusively the growth-phase induction of the macs1-fadD1 operon.

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ArnR, a Novel Transcriptional Regulator, Represses Expression of the narKGHJI Operon in Corynebacterium glutamicum

Journal of Bacteriology ◽

10.1128/jb.01801-07 ◽

2008 ◽

Vol 190 (9) ◽

pp. 3264-3273 ◽

Cited By ~ 28

Author(s):

Taku Nishimura ◽

Haruhiko Teramoto ◽

Alain A. Vertès ◽

Masayuki Inui ◽

Hideaki Yukawa

Keyword(s):

Nitrate Reductase ◽

Corynebacterium Glutamicum ◽

Consensus Sequence ◽

Transcriptional Regulator ◽

Anaerobic Growth ◽

Mrna Levels ◽

Promoter Regions ◽

Mobility Shift ◽

Terminal Electron Acceptor ◽

Aerobic Conditions

ABSTRACT The narKGHJI operon that comprises putative nitrate/nitrite transporter (narK) and nitrate reductase (narGHJI) genes is required for the anaerobic growth of Corynebacterium glutamicum with nitrate as a terminal electron acceptor. In this study, we identified a gene, arnR, which encodes a transcriptional regulator that represses the expression of the narKGHJI operon in C. glutamicum cells under aerobic conditions. Disruption of arnR induced nitrate reductase activities of C. glutamicum cells and increased narKGHJI mRNA levels under aerobic growth conditions. DNA microarray analyses revealed that besides the narKGHJI operon, the hmp gene, which encodes flavohemoglobin, is negatively regulated by ArnR under aerobic conditions. Promoter-reporter assays indicated that arnR gene expression was positively autoregulated by its gene product, ArnR, under both aerobic and anaerobic conditions. Electrophoretic mobility shift assay experiments showed that purified hexahistidyl-tagged ArnR protein specifically binds to promoter regions of the narKGHJI operon and the hmp and arnR genes. A consensus sequence, TA(A/T)TTAA(A/T)TA, found in the promoter regions of these genes was demonstrated to be involved in the binding of ArnR. Effects on LacZ activity by deletion of the ArnR binding sites within the promoter regions fused to the reporter gene were consistent with the view that the expression of the narKGHJI operon is repressed by the ArnR protein under aerobic conditions, whereas the expression of the arnR gene is autoinduced by ArnR.

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