Characterization of a Mannose Utilization System in Bacillus subtilis

ABSTRACT The mannose operon of Bacillus subtilis consists of three genes, manP, manA, and yjdF, which are responsible for the transport and utilization of mannose. Upstream and in the same orientation as the mannose operon a regulatory gene, manR, codes for a transcription activator of the mannose operon, as shown in this study. Both mannose operon transcription and manR transcription are inducible by mannose. The presence of mannose resulted in a 4- to 7-fold increase in expression of lacZ from the manP promoter (PmanP ) and in a 3-fold increase in expression of lacZ from the manR promoter (PmanR ). The transcription start sites of manPA-yjdF and manR were determined to be a single A residue and a single G residue, respectively, preceded by −10 and −35 boxes resembling a vegetative σA promoter structure. Through deletion analysis the target sequences of ManR upstream of PmanP and PmanR were identified between bp −80 and −35 with respect to the transcriptional start site of both promoters. Deletion of manP (mannose transporter) resulted in constitutive expression from both the PmanP and PmanR promoters, indicating that the phosphotransferase system (PTS) component EIIMan has a negative effect on regulation of the mannose operon and manR. Moreover, both PmanP and PmanR are subject to carbon catabolite repression (CCR). By constructing protein sequence alignments a DNA binding motif at the N-terminal end, two PTS regulation domains (PRDs), and an EIIA- and EIIB-like domain were identified in the ManR sequence, indicating that ManR is a PRD-containing transcription activator. Like findings for other PRD regulators, the phosphoenolpyruvate (PEP)-dependent phosphorylation by the histidine protein HPr via His15 plays an essential role in transcriptional activation of PmanP and PmanR . Phosphorylation of Ser46 of HPr or of the homologous Crh protein by HPr kinase and formation of a repressor complex with CcpA are parts of the B. subtilis CCR system. Only in the double mutant with an HPr Ser46Ala mutation and a crh knockout mutation was CCR strongly reduced. In contrast, PmanR and PmanP were not inducible in a ccpA deletion mutant.

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The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development

Molecular and Cellular Biology ◽

10.1128/mcb.14.4.2503-2515.1994 ◽

1994 ◽

Vol 14 (4) ◽

pp. 2503-2515

Author(s):

A Andrianopoulos ◽

W E Timberlake

Keyword(s):

Aspergillus Nidulans ◽

Transcriptional Activation ◽

Expression System ◽

Regulatory Gene ◽

Binding Motif ◽

Mobility Shift ◽

Genetic Switch ◽

Regulatory Regions ◽

Cis Acting ◽

Gel Mobility Shift

The Aspergillus nidulans abaA gene encodes a protein containing an ATTS DNA-binding motif and is required for the terminal stages of conidiophore development. Results from gel mobility shift and protection, missing-contact, and interference footprint assays showed that AbaA binds to the sequence 5'-CATTCY-3', where Y is a pyrimidine, making both major- and minor-groove contacts. Multiple AbaA binding sites are present in the cis-acting regulatory regions of several developmentally controlled structural genes as well as those of the upstream regulatory gene brlA, the downstream regulatory gene wetA, and abaA itself. These cis-acting regulatory regions confer AbaA-dependent transcriptional activation in a heterologous Saccharomyces cerevisiae gene expression system. From these observations, we propose that the AbaA transcription factor establishes a novel set of feedback regulatory loops responsible for determination of conidiophore development.

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The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development.

Molecular and Cellular Biology ◽

10.1128/mcb.14.4.2503 ◽

1994 ◽

Vol 14 (4) ◽

pp. 2503-2515 ◽

Cited By ~ 188

Author(s):

A Andrianopoulos ◽

W E Timberlake

Keyword(s):

Aspergillus Nidulans ◽

Transcriptional Activation ◽

Expression System ◽

Regulatory Gene ◽

Binding Motif ◽

Mobility Shift ◽

Genetic Switch ◽

Regulatory Regions ◽

Cis Acting ◽

Gel Mobility Shift

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PTS-Mediated Regulation of the Transcription Activator MtlR from Different Species: Surprising Differences despite Strong Sequence Conservation

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000369619 ◽

2015 ◽

Vol 25 (2-3) ◽

pp. 94-105 ◽

Cited By ~ 8

Author(s):

Philippe Joyet ◽

Meriem Derkaoui ◽

Houda Bouraoui ◽

Josef Deutscher

Keyword(s):

Bacillus Subtilis ◽

Lactobacillus Casei ◽

Sequence Conservation ◽

Geobacillus Stearothermophilus ◽

Transcription Activator ◽

Phosphotransferase System ◽

Transcription Regulator ◽

Regulatory Domains ◽

Genes Encoding ◽

Enzyme I

The hexitol <smlcap>D</smlcap>-mannitol is transported by many bacteria via a phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). In most Firmicutes, the transcription activator MtlR controls the expression of the genes encoding the <smlcap>D</smlcap>-mannitol-specific PTS components and <smlcap>D</smlcap>-mannitol-1-P dehydrogenase. MtlR contains an N-terminal helix-turn-helix motif followed by an Mga-like domain, two PTS regulation domains (PRDs), an EIIBGat- and an EIIAMtl-like domain. The four regulatory domains are the target of phosphorylation by PTS components. Despite strong sequence conservation, the mechanisms controlling the activity of MtlR from Lactobacillus casei, Bacillus subtilis and Geobacillus stearothermophilus are quite different. Owing to the presence of a tyrosine in place of the second conserved histidine (His) in PRD2, L. casei MtlR is not phosphorylated by Enzyme I (EI) and HPr. When the corresponding His in PRD2 of MtlR from B. subtilis and G. stearothermophilus was replaced with alanine, the transcription regulator was no longer phosphorylated and remained inactive. Surprisingly, L. casei MtlR functions without phosphorylation in PRD2 because in a ptsI (EI) mutant MtlR is constitutively active. EI inactivation prevents not only phosphorylation of HPr, but also of the PTSMtl components, which inactivate MtlR by phosphorylating its EIIBGat- or EIIAMtl-like domain. This explains the constitutive phenotype of the ptsI mutant. The absence of EIIBMtl-mediated phosphorylation leads to induction of the L. caseimtl operon. This mechanism resembles mtlARFD induction in G. stearothermophilus, but differs from EIIAMtl-mediated induction in B. subtilis. In contrast to B. subtilis MtlR, L. casei MtlR activation does not require sequestration to the membrane via the unphosphorylated EIIBMtl domain.

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Regulation of the Bacillus subtilis mannitol utilization genes: promoter structure and transcriptional activation by the wild-type regulator (MtlR) and its mutants

Microbiology ◽

10.1099/mic.0.071233-0 ◽

2014 ◽

Vol 160 (1) ◽

pp. 91-101 ◽

Cited By ~ 10

Author(s):

Kambiz Morabbi Heravi ◽

Josef Altenbuchner

Keyword(s):

Bacillus Subtilis ◽

Transcriptional Activation ◽

Transcription Initiation ◽

Phosphotransferase System ◽

Mobility Shift ◽

Dnase I Footprinting ◽

Proposed Model ◽

Rna Polymerase Holoenzyme ◽

Electrophoretic Mobility Shift Assays

Expression of mannitol utilization genes in Bacillus subtilis is directed by P mtlA , the promoter of the mtlAFD operon, and P mtlR , the promoter of the MtlR activator. MtlR contains phosphoenolpyruvate-dependent phosphotransferase system (PTS) regulation domains, called PRDs. The activity of PRD-containing MtlR is mainly regulated by the phosphorylation/dephosphorylation of its PRDII and EIIBGat-like domains. Replacing histidine 342 and cysteine 419 residues, which are the targets of phosphorylation in these two domains, by aspartate and alanine provided MtlR-H342D C419A, which permanently activates P mtlA in vivo. In the mtlR-H342D C419A mutant, P mtlA was active, even when the mtlAFD operon was deleted from the genome. The mtlR-H342D C419A allele was expressed in an Escherichia coli strain lacking enzyme I of the PTS. Electrophoretic mobility shift assays using purified MtlR-H342D C419A showed an interaction between the MtlR double-mutant and the Cy5-labelled P mtlA and P mtlR DNA fragments. These investigations indicate that the activated MtlR functions regardless of the presence of the mannitol-specific transporter (MtlA). This is in contrast to the proposed model in which the sequestration of MtlR by the MtlA transporter is necessary for the activity of MtlR. Additionally, DNase I footprinting, construction of P mtlA -P licB hybrid promoters, as well as increasing the distance between the MtlR operator and the −35 box of P mtlA revealed that the activated MtlR molecules and RNA polymerase holoenzyme likely form a class II type activation complex at P mtlA and P mtlR during transcription initiation.

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Mutations in the Regulatory Gene hrpG ofXanthomonas campestris pv. vesicatoria Result in Constitutive Expression of All hrp Genes

Journal of Bacteriology ◽

10.1128/jb.181.21.6828-6831.1999 ◽

1999 ◽

Vol 181 (21) ◽

pp. 6828-6831 ◽

Cited By ~ 110

Author(s):

Kai Wengelnik ◽

Ombeline Rossier ◽

Ulla Bonas

Keyword(s):

Gene Expression ◽

Amino Acid ◽

Transcriptional Activation ◽

Xanthomonas Campestris ◽

Regulatory Gene ◽

Constitutive Expression ◽

Amino Acid Substitutions ◽

Pathogenicity Genes ◽

Hrp Genes ◽

Related Proteins

ABSTRACT hrpG is a key regulatory gene for transcriptional activation of pathogenicity genes (hrp) ofXanthomonas campestris pv. vesicatoria. We identified three mutations in hrpG which render hrp gene expression constitutive in normally suppressing medium. The mutations in hrpG result in novel amino acid substitutions compared to mutations in related proteins, such as OmpR. In addition, mutatedhrpG enhances the timing and intensity of plant reactions in infection assays.

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Functional Analysis of 14 Genes That Constitute the Purine Catabolic Pathway in Bacillus subtilis and Evidence for a Novel Regulon Controlled by the PucR Transcription Activator

Journal of Bacteriology ◽

10.1128/jb.183.11.3293-3302.2001 ◽

2001 ◽

Vol 183 (11) ◽

pp. 3293-3302 ◽

Cited By ~ 87

Author(s):

Anna C. Schultz ◽

Per Nygaard ◽

Hans H. Saxild

Keyword(s):

Gene Expression ◽

Bacillus Subtilis ◽

Uric Acid ◽

Nitrogen Source ◽

Nitrogen Sources ◽

Fold Increase ◽

Degradation Pathway ◽

Enzyme Analysis ◽

Transcription Activator ◽

Purine Degradation

ABSTRACT The soil bacterium Bacillus subtilis has developed a highly controlled system for the utilization of a diverse array of low-molecular-weight compounds as a nitrogen source when the preferred nitrogen sources, e.g., glutamate plus ammonia, are exhausted. We have identified such a system for the utilization of purines as nitrogen source in B. subtilis. Based on growth studies of strains with knockout mutations in genes, complemented with enzyme analysis, we could ascribe functions to 14 genes encoding enzymes or proteins of the purine degradation pathway. A functional xanthine dehydrogenase requires expression of five genes (pucA, pucB, pucC, pucD, and pucE). Uricase activity is encoded by thepucL and pucM genes, and a uric acid transport system is encoded by pucJ and pucK. Allantoinase is encoded by the pucH gene, and allantoin permease is encoded by the pucI gene. Allantoate amidohydrolase is encoded by pucF. In a pucRmutant, the level of expression was low for all genes tested, indicating that PucR is a positive regulator of puc gene expression. All 14 genes except pucI are located in a gene cluster at 284 to 285° on the chromosome and are contained in six transcription units, which are expressed when cells are grown with glutamate as the nitrogen source (limiting conditions), but not when grown on glutamate plus ammonia (excess conditions). Our data suggest that the 14 genes and the gde gene, encoding guanine deaminase, constitute a regulon controlled by the pucR gene product. Allantoic acid, allantoin, and uric acid were all found to function as effector molecules for PucR-dependent regulation ofpuc gene expression. When cells were grown in the presence of glutamate plus allantoin, a 3- to 10-fold increase in expression was seen for most of the genes. However, expression of thepucABCDE unit was decreased 16-fold, while expression ofpucR was decreased 4-fold in the presence of allantoin. We have identified genes of the purine degradation pathway in B. subtilis and showed that their expression is subject to both general nitrogen catabolite control and pathway-specific control.

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The Lactobacillus casei ptsHI47T Mutation Causes Overexpression of a LevR-Regulated but RpoN-Independent Operon Encoding a Mannose Class Phosphotransferase System

Journal of Bacteriology ◽

10.1128/jb.186.14.4543-4555.2004 ◽

2004 ◽

Vol 186 (14) ◽

pp. 4543-4555 ◽

Cited By ~ 19

Author(s):

Alain Mazé ◽

Grégory Boël ◽

Sandrine Poncet ◽

Ivan Mijakovic ◽

Yoann Le Breton ◽

...

Keyword(s):

Catabolite Repression ◽

Lactobacillus Casei ◽

Constitutive Expression ◽

Proteome Analysis ◽

Phosphoryl Group ◽

Transcription Activator ◽

Phosphotransferase System ◽

Reading Frame ◽

Elevated Expression ◽

Dna Fragment

ABSTRACT A proteome analysis of Lactobacillus casei mutants that are affected in carbon catabolite repression revealed that a 15-kDa protein was strongly overproduced in a ptsHI47T mutant. This protein was identified as EIIA of a mannose class phosphotransferase system (PTS). A 7.1-kb DNA fragment containing the EIIA-encoding open reading frame and five other genes was sequenced. The first gene encodes a protein resembling the RpoN (σ54)-dependent Bacillus subtilis transcription activator LevR. The following pentacistronic operon is oriented in the opposite direction and encodes four proteins with strong similarity to the proteins of the B. subtilis Lev-PTS and one protein of unknown function. The genes present on the 7.1-kb DNA fragment were therefore called levR and levABCDX. The levABCDX operon was induced by fructose and mannose. No “−12, −24” promoter typical of RpoN-dependent genes precedes the L. casei lev operon, and its expression was therefore RpoN independent but required LevR. Phosphorylation of LevR by P∼His-HPr stimulates its activity, while phosphorylation by P∼EIIBLev inhibits it. Disruption of the EIIBLev-encoding levB gene therefore led to strong constitutive expression of the lev operon, which was weaker in a strain carrying a ptsI mutation preventing phosphorylation by both P∼EIIBLev and P∼His-HPr. Expression of the L. casei lev operon is also subject to P-Ser-HPr-mediated catabolite repression. The observed slow phosphoenolpyruvate- and ATP-dependent phosphorylation of HPrI47T as well as the slow phosphoryl group transfer from the mutant P∼His-HPr to EIIALev are assumed to be responsible for the elevated expression of the lev operon in the ptsHI47T mutant.

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Transcriptional Organization and Dynamic Expression of thehbpCAD Genes, Which Encode the First Three Enzymes for 2-Hydroxybiphenyl Degradation in Pseudomonas azelaica HBP1

Journal of Bacteriology ◽

10.1128/jb.183-1.270-279.2001 ◽

2001 ◽

Vol 183 (1) ◽

pp. 270-279 ◽

Cited By ~ 28

Author(s):

Marco C. M. Jaspers ◽

Andreas Schmid ◽

Mark H. J. Sturme ◽

David A. M. Goslings ◽

Hans-Peter E. Kohler ◽

...

Keyword(s):

Transcriptional Activation ◽

Regulatory Gene ◽

Toxic Substance ◽

Active Role ◽

Integration Host Factor ◽

Host Factor ◽

Northern Analysis ◽

Structural Genes ◽

Transcription Activator ◽

Intergenic Regions

ABSTRACT Pseudomonas azelaica HBP1 degrades the toxic substance 2-hydroxybiphenyl (2-HBP) by means of three enzymes that are encoded by structural genes hbpC, hbpA, andhbpD. These three genes form a small noncontiguous cluster. Their expression is activated by the product of regulatory genehbpR, which is located directly upstream of thehbpCAD genes. The HbpR protein is a transcription activator and belongs to the so-called XylR/DmpR subclass within the NtrC family of transcriptional activators. Transcriptional fusions between the different hbp intergenic regions and the luxABgenes of Vibrio harveyi in P. azelaica and inEscherichia coli revealed the existence of two HbpR-regulated promoters; one is located in front of hbpC, and the other one is located in front of hbpD. Northern analysis confirmed that the hbpC and hbpA genes are cotranscribed, whereas the hbpD gene is transcribed separately. No transcripts comprising the entire hbpCADcluster were detected, indicating that transcription from P hbpC is terminated after the hbpAgene. E. coli mutant strains lacking the structural genes for the RNA polymerase ς54 subunit or for the integration host factor failed to express bioluminescence from P hbpC - and P hbpD -luxABfusions when a functional hbpR gene was provided intrans. This pointed to the active role of ς54and integration host factor in transcriptional activation from these promoters. Primer extension analysis revealed that both P hbpC and P hbpD contain the typical motifs at position −24 (GG) and −12 (GC) found in ς54-dependent promoters. Analysis of changes in the synthesis of the hbp mRNAs, in activities of the 2-HBP pathway enzymes, and in concentrations of 2-HBP intermediates during the first 4 h after induction of continuously grown P. azelaica cells with 2-HBP demonstrated that the specific transcriptional organization of the hbp genes ensured smooth pathway expression.

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209 Overexpression of WAVE1 activates pluripotency-related genes in porcine somatic cells

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab209 ◽

2019 ◽

Vol 31 (1) ◽

pp. 230

Author(s):

K. Carey ◽

K. Uh ◽

J. Ryu ◽

K. Lee

Keyword(s):

Stem Cells ◽

Transcriptional Activation ◽

Pluripotent Stem Cells ◽

Target Genes ◽

Somatic Cells ◽

Constitutive Expression ◽

Fold Increase ◽

Cellular Reprogramming ◽

Transcript Levels ◽

Fetal Fibroblasts

Despite extensive efforts, cellular reprogramming in livestock species has had limited success. Induced pluripotent stem cells (iPSC) have been established; however, these cells often show incomplete reprogramming status, and constitutive expression of exogenous reprogramming factors is required due to inactivation of endogenous pluripotency-related genes. A previous study reported that overexpression of the Xenopus egg-derived WAVE1 gene assists reprogramming of murine somatic cells into the pluripotent state. The WAVE1 gene is also required for oocyte-mediated reprogramming by transcriptional activation of embryonic genes. In this study, we investigated the role of porcine WAVE1 in cellular reprogramming by inducing overexpression of WAVE1 in porcine fetal fibroblasts (PFF). Previously, we cloned the coding sequences (CDS) of porcine WAVE1 using porcine expressed sequence tags (EST) and predicted porcine WAVE1 sequences. The WAVE1 CDS, derived from porcine mature oocytes, was overexpressed in PFF by transfection using the Neon system. Then, G418-based antibiotic selection was performed to enrich cells constitutively overexpressing WAVE1. After cell culture for 4 weeks, RNA was extracted from the WAVE1 transfected and control PFF, and cDNA was synthesised from the RNA using random hexamers. The cDNAs were used for quantitative reverse transcription PCR to analyse the expression pattern of pluripotency- and reprogramming-related genes: POU5F1, NANOG, KLF2, SOX2, DPPA3, ZFP42, ESRRB, TET1, TET2, and TET3. The expression of target genes were normalized to GAPDH level and the ΔΔCt algorithm was used for analysis. Three technical replications and 4 biological replications were performed. Student’s t-test was used for the comparison and P-values<0.05 were considered significant. On average, a 20-fold increase of WAVE1 was observed in the transfected cells compared with control cells. Interestingly, overexpression of WAVE1 activated some of the pluripotency-related genes in porcine PFF. Specifically, transcript levels of NANOG, KLF2, and SOX2 were increased compared with those in the control cells (P<0.05). In addition, levels of POU5F1 and DPPA3 tended to be higher in WAVE1-overexpressing cells compared with those in the control cells (P<0.1). However, transcript levels of other pluripotency-related genes (ZFP42, DPPA3, and ESRRB) did not change in WAVE1-overexpressing cells. The expression level of TET family (TET1, TET2, and TET3), which is enriched in pluripotent stem cells and a key regulator of DNA methylation, was not changed in WAVE1-overexpressing cells. These results indicate that WAVE1 can be a novel factor in porcine cellular reprogramming. Considering that a key defect of current porcine iPSC generation is insufficient expression of endogenous pluripotency genes, application of WAVE1 may enhance quality of porcine iPSC. We intend to evaluate expression of pluripotency markers at the protein level in WAVE1-overexpressing cells and investigate mechanisms underpinning WAVE1-mediated reprogramming process in future studies.

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Mlc of Thermus thermophilus: a Glucose-Specific Regulator for a Glucose/Mannose ABC Transporter in the Absence of the Phosphotransferase System

Journal of Bacteriology ◽

10.1128/jb.00715-06 ◽

2006 ◽

Vol 188 (18) ◽

pp. 6561-6571 ◽

Cited By ~ 8

Author(s):

Fabienne F. V. Chevance ◽

Marc Erhardt ◽

Christina Lengsfeld ◽

Sung-Jae Lee ◽

Winfried Boos

Keyword(s):

Abc Transporter ◽

Thermophilic Bacteria ◽

Thermus Thermophilus ◽

Sugar Metabolism ◽

Enteric Bacteria ◽

Thermophilic Bacterium ◽

Binding Motif ◽

Phosphotransferase System ◽

Sequence Alignments ◽

Glucose Binding

ABSTRACT We report the presence of Mlc in a thermophilic bacterium. Mlc is known as a global regulator of sugar metabolism in gram-negative enteric bacteria that is controlled by sequestration to a glucose-transporting EIIGlc of the phosphotransferase system (PTS). Since thermophilic bacteria do not possess PTS, Mlc in Thermus thermophilus must be differently controlled. DNA sequence alignments between Mlc from T. thermophilus (MlcTth) and Mlc from E. coli (MlcEco) revealed that MlcTth conserved five residues of the glucose-binding motif of glucokinases. Here we show that MlcTth is not a glucokinase but is indeed able to bind glucose (KD = 20 μM), unlike MlcEco. We found that mlc of T. thermophilus is the first gene within an operon encoding an ABC transporter for glucose and mannose, including a glucose/mannose-binding protein and two permeases. malK1, encoding the cognate ATP-hydrolyzing subunit, is located elsewhere on the chromosome. The system transports glucose at 70°C with a Km of 0.15 μM and a V max of 4.22 nmol per min per ml at an optical density (OD) of 1. MlcTth negatively regulates itself and the entire glucose/mannose ABC transport system operon but not malK1, with glucose acting as an inducer. MalK1 is shared with the ABC transporter for trehalose, maltose, sucrose, and palatinose (TMSP). Mutants lacking malK1 do not transport either glucose or maltose. The TMSP transporter is also able to transport glucose with a Km of 1.4 μM and a V max of 7.6 nmol per min per ml at an OD of 1, but it does not transport mannose.

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