Control of Transposon-mediated Activation of theglpFKOperon ofEscherichia coliby two DNA binding Proteins

Wild Type ◽

Transposon Insertion ◽

Glycerol Utilization ◽

AbstractEscherichia colicells deleted for the cyclic AMP (cAMP) receptor protein (Crp) gene (Δcrp) cannot utilize glycerol because cAMP-Crp is a required positive activator of glycerol utilization operonglpFK. We have previously shown that a transposon, Insertion Sequence 5 (IS5), can reversibly insert into the upstream regulatory region of the operon so as to activateglpFKand enable glycerol utilization. GlpR, which repressesglpFKtranscription, binds to theglpFKupstream region near the site of IS5insertion, and prevents insertion. We here show that the cAMP-Crp complex, which also binds to theglpFKupstream regulatory region, also inhibits IS5hopping into the activating site. This finding allowed us to identify conditions under which wild type cells can acquireglpFK-activating IS5insertions. Maximal rates of IS5insertion into the activating site require the presence of glycerol as well as a non-metabolizable sugar analogue that lowers cytoplasmic cAMP concentrations. Under these conditions, IS5insertional mutants accumulate and outcompete the wild type cells. Because of the widespread distribution of glucose analogues in nature, this mechanism of gene activation could have evolved by natural selection.

OmpR and LeuO Positively Regulate the Salmonella enterica Serovar Typhi ompS2 Porin Gene

10.1128/jb.186.10.2909-2920.2004 ◽

2004 ◽

Vol 186 (10) ◽

pp. 2909-2920 ◽

Cited By ~ 39

Author(s):

Marcos Fernández-Mora ◽

José Luis Puente ◽

Edmundo Calva

Keyword(s):

Salmonella Enterica ◽

Type Strain ◽

Wild Type Strain ◽

Phosphorylation Site ◽

Regulatory Region ◽

Random Mutagenesis ◽

Fold Increase ◽

Wild Type ◽

Transposon Insertion

ABSTRACT The Salmonella enterica serovar Typhi ompS2 gene codes for a 362-amino-acid outer membrane protein that contains motifs common to the porin superfamily. It is expressed at very low levels compared to the major OmpC and OmpF porins, as observed for S. enterica serovar Typhi OmpS1, Escherichia coli OmpN, and Klebsiella pneumoniae OmpK37 quiescent porins. A region of 316 bp, between nucleotides −413 and −97 upstream of the transcriptional start point, is involved in negative regulation, as its removal resulted in a 10-fold increase in ompS2 expression in an S. enterica serovar Typhi wild-type strain. This enhancement in expression was not observed in isogenic mutant strains, which had specific deletions of the regulatory ompB (ompR envZ) operon. Furthermore, ompS2 expression was substantially reduced in the presence of the OmpR D55A mutant, altered in the major phosphorylation site. Upon random mutagenesis, a mutant where the transposon had inserted into the upstream regulatory region of the gene coding for the LeuO regulator, showed an increased level of ompS2 expression. Augmented expression of ompS2 was also obtained upon addition of cloned leuO to the wild-type strain, but not in an ompR isogenic derivative, consistent with the notion that the transposon insertion had increased the cellular levels of LeuO and with the observed dependence on OmpR. Moreover, LeuO and OmpR bound in close proximity, but independently, to the 5′ upstream regulatory region. Thus, the OmpR and LeuO regulators positively regulate ompS2.

Control of Transposon-Mediated Directed Mutation by the Escherichia coli Phosphoenolpyruvate:Sugar Phosphotransferase System

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000375375 ◽

2015 ◽

Vol 25 (2-3) ◽

pp. 226-233 ◽

Cited By ~ 3

Author(s):

Milton H. Saier Jr. ◽

Zhongge Zhang

Keyword(s):

Escherichia Coli ◽

Insertion Sequence ◽

Cell Metabolism ◽

Point Mutations ◽

Receptor Protein ◽

The Novel ◽

Phosphotransferase System ◽

Directed Mutation ◽

Glycerol Utilization ◽

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) has been shown to control transport, cell metabolism and gene expression. We here present results supporting the novel suggestion that in certain instances it also regulates the mutation rate. Directed mutations are defined as mutations that occur at higher frequencies when beneficial than when neutral or detrimental. To date, the occurrence of directed point mutations has not been documented and confirmed, but a few examples of transposon-mediated directed mutations have been reported. Here we focus on the first and best-studied example of directed mutation, which involves the regulation of insertion sequence-5 hopping into a specific site upstream of the glpFK glycerol utilization operon in Escherichia coli. This insertional event specifically activates expression of the glpFK operon, allowing the growth of wild-type cells with glycerol as a carbon source in the presence of nonmetabolizable glucose analogues which normally block glycerol utilization. The sugar-transporting PTS controls this process by regulating levels of cytoplasmic glycerol-3-phosphate and cyclic (c)AMP as established in previous publications. Direct involvement of the glycerol repressor, GlpR, and the cAMP receptor protein, Crp, in the regulation of transposon-mediated directed mutation has been demonstrated.

Mutations That StimulateflhDCExpression in Escherichia coli K-12

10.1128/jb.00455-15 ◽

2015 ◽

Vol 197 (19) ◽

pp. 3087-3096 ◽

Cited By ~ 20

Author(s):

Karen A. Fahrner ◽

Howard C. Berg

Keyword(s):

Escherichia Coli ◽

Transcription Factors ◽

Insertion Sequence ◽

Receptor Protein ◽

Upstream Region ◽

Single Nucleotide ◽

Content Type ◽

Sequence Elements ◽

Camp Receptor ◽

K 12

ABSTRACTMotility is a beneficial attribute that enables cells to access and explore new environments and to escape detrimental ones. The organelle of motility inEscherichia coliis the flagellum, and its production is initiated by the activating transcription factors FlhD and FlhC. The expression of these factors by theflhDCoperon is highly regulated and influenced by environmental conditions. TheflhDCpromoter is recognized by σ70and is dependent on the transcriptional activator cyclic AMP (cAMP)-cAMP receptor protein complex (cAMP-CRP). A number of K-12 strains exhibit limited motility due to low expression levels offlhDC. We report here a large number of mutations that stimulateflhDCexpression in such strains. They include single nucleotide changes in the −10 element of the promoter, in the promoter spacer, and in the cAMP-CRP binding region. In addition, we show that insertion sequence (IS) elements or a kanamycin gene located hundreds of base pairs upstream of the promoter can effectively enhance transcription, suggesting that the topology of a large upstream region plays a significant role in the regulation offlhDCexpression. None of the mutations eliminated the requirement for cAMP-CRP for activation. However, several mutations allowed expression in the absence of the nucleoid organizing protein, H-NS, which is normally required forflhDCexpression.IMPORTANCETheflhDCoperon ofEscherichia coliencodes transcription factors that initiate flagellar synthesis, an energetically costly process that is highly regulated. Few deregulating mutations have been reported thus far. This paper describes new single nucleotide mutations that stimulateflhDCexpression, including a number that map to the promoter spacer region. In addition, this work shows that insertion sequence elements or a kanamycin gene located far upstream from the promoter or repressor binding sites also stimulate transcription, indicating a role of regional topology in the regulation offlhDCexpression.

Expression of the cpdA Gene, Encoding a 3′,5′-Cyclic AMP (cAMP) Phosphodiesterase, Is Positively Regulated by the cAMP-cAMP Receptor Protein Complex

10.1128/jb.01350-08 ◽

2008 ◽

Vol 191 (3) ◽

pp. 922-930 ◽

Cited By ~ 18

Author(s):

Han-Suk Kim ◽

Sung-Min Kim ◽

Hyun-Jung Lee ◽

Soon-Jung Park ◽

Kyu-Ho Lee

Keyword(s):

Regulatory Region ◽

Site Directed Mutagenesis ◽

Receptor Protein ◽

Upstream Region ◽

Intracellular Camp ◽

Dependent Manner ◽

Multicopy Plasmid ◽

Camp Receptor Protein ◽

Camp Phosphodiesterase ◽

ABSTRACT The intracellular level of cyclic 3′,5′-AMP (cAMP), a signaling molecule that mediates a variety of cellular processes, is finely modulated by the regulation of its synthesis, excretion, and degradation. In this study, cAMP phosphodiesterase (CpdA), an enzyme that catalyzes the conversion of cAMP to AMP, was characterized in a pathogenic bacterium, Vibrio vulnificus. The cpdA gene exists in an operon composed of mutT, yqiB, cpdA, and yqiA, the transcription of which was initiated at position −22 upstream of mutT. A cpdA-null mutant of V. vulnificus contained significantly higher levels of cAMP than the wild type but showed no detectable cAMP when a multicopy plasmid of the cpdA gene was provided in trans, suggesting that CpdA is responsible for cAMP degradation. Cellular contents of the CpdA protein decreased dramatically in both cya and crp mutants. In addition, levels of expression of the cpdA::luxAB transcription fusion decreased in cya and crp mutants. The level of expression of cpdA::luxAB in the cya mutant increased in a concentration-dependent manner upon the exogenous addition of cAMP. The cAMP-cAMP receptor protein (CRP) complex bound directly to the upstream region of mutT, which includes a putative CRP-binding sequence centered at position −95.5 relative to the transcription start site. Site-directed mutagenesis or the deletion of this sequence in the cpdA::luxAB transcription fusion resulted in the loss of regulation by cAMP and CRP. Thus, this study demonstrates that CpdA plays a crucial role in determining the intracellular cAMP level and shows for the first time that the expression of cpdA is activated by the cAMP-CRP complex via direct binding to the regulatory region.

Short repeated elements in the upstream regulatory region of the SUC2 gene of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2324-2333.1986 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2324-2333

Author(s):

L Sarokin ◽

M Carlson

Keyword(s):

Carbon Catabolite Repression ◽

Consensus Sequence ◽

Regulatory Region ◽

Lacz Fusion ◽

Upstream Region ◽

Heterologous Promoter ◽

Suc2 Gene ◽

High Level ◽

Repeated Motif

Expression of secreted invertase from the SUC2 gene is regulated by carbon catabolite repression. Previously, an upstream regulatory region that is required for derepression of secreted invertase was identified and shown to confer glucose-repressible expression to the heterologous promoter of a LEU2-lacZ fusion. In this paper we show that tandem copies of a 32-base pair (bp) sequence from the upstream regulatory region activate expression of the same LEU2-lacZ fusion. The level of expression increased with the number of copies of the element, but was independent of their orientation; the expression from constructions containing four copies of the sequence was only twofold lower than that when the entire SUC2 upstream regulatory region was present. This activation was not significantly glucose repressible. The 32-bp sequence includes a 7-bp motif with the consensus sequence (A/C)(A/G)GAAAT that is repeated at five sites within the upstream regulatory region. Genetic evidence supporting the functional significance of this repeated motif was obtained by pseudoreversion of a SUC2 deletion mutant lacking part of the upstream region, including two copies of the 7-bp element. In three of five pseudorevertants, the mutations that restored high-level SUC2 expression altered one of the remaining copies of the 7-bp element.

Expression System for High Levels of GAG Lyase Gene Expression and Study of the hepA Upstream Region in Flavobacterium heparinum

10.1128/jb.184.12.3242-3252.2002 ◽

2002 ◽

Vol 184 (12) ◽

pp. 3242-3252 ◽

Cited By ~ 11

Author(s):

Françoise Blain ◽

A. Lydia Tkalec ◽

Zhongqi Shao ◽

Catherine Poulin ◽

Marc Pedneault ◽

...

Keyword(s):

Transcriptional Start Site ◽

Expression System ◽

Regulatory Region ◽

Start Codon ◽

Upstream Region ◽

Wild Type ◽

Heparinase I ◽

High Level ◽

Flavobacterium Heparinum ◽

Transcriptional Start Sites

ABSTRACT A system for high-level expression of heparinase I, heparinase II, heparinase III, chondroitinase AC, and chondroitinase B in Flavobacterium heparinum is described. hepA, along with its regulatory region, as well as hepB, hepC, cslA, and cslB, cloned downstream of the hepA regulatory region, was integrated in the chromosome to yield stable transconjugant strains. The level of heparinase I and II expression from the transconjugant strains was approximately fivefold higher, while heparinase III expression was 10-fold higher than in wild-type F. heparinum grown in heparin-only medium. The chondroitinase AC and B transconjugant strains, grown in heparin-only medium, yielded 20- and 13-fold increases, respectively, in chondroitinase AC and B expression, compared to wild-type F. heparinum grown in chondroitin sulfate A-only medium. The hepA upstream region was also studied using cslA as a reporter gene, and the transcriptional start site was determined to be 26 bp upstream of the start codon in the chondroitinase AC transconjugant strain. The transcriptional start sites were determined for hepA in both the wild-type F. heparinum and heparinase I transconjugant strains and were shown to be the same as in the chondroitinase AC transconjugant strain. The five GAG lyases were purified from these transconjugant strains and shown to be identical to their wild-type counterparts.

RUNX1 Directly Regulates Band 3 Transcription.

Blood ◽

10.1182/blood.v106.11.1735.1735 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1735-1735

Author(s):

Jingping Xie ◽

Scott W. Hiebert ◽

Mark J. Koury ◽

Stephen J. Brandt

Keyword(s):

Histone Acetylation ◽

Gene Knockout ◽

Regulatory Region ◽

Erythroid Differentiation ◽

Band 3 ◽

Upstream Region ◽

Pcr Analysis ◽

Terminal Erythroid Differentiation ◽

Chip Analysis

Abstract RUNX1 (AML1 or CBFA2) regulates the expression of a number of genes important to hematopoiesis. Gene knockout studies demonstrated that a heterodimeric complex of RUNX1 and its DNA binding partner, core binding factor-beta (CBFbeta), is essential for definitive hematopoiesis. Here, we report that RUNX1 directly represses expression of the Band 3 gene prior to terminal erythroid differentiation. Band 3 is one of four major components of the erythrocyte membrane skeleton and is important for maintenance of cytoskeletal architecture and electroneutral Cl-/HCO3− exchange across the red cell membrane. Band 3 expression, like that of beta-globin, increases dramatically with terminal erythroid differentiation. In a previous study, we identified an upstream region in the mouse Band 3 gene designated as B3URE (for Band 3 upstream regulatory region) bound by multiple transcription factors, including TAL1 (also known as SCL), RUNX1, Ldb1, and GATA1, that acts as an orientation- and position-independent and tissue-specific repressive element. Chromatin immunoprecipitation (ChIP) analysis showed that RUNX1 was associated with the B3URE in intact MEL cells and electrophoretic mobility shift analysis confirmed specific RUNX1 interaction with RUNX1 binding sites in the B3URE. Together with CBFbeta, RUNX1 inhibited reporter activity from a construct linking the B3URE with 1 kb of Band 3 promoter in transiently transfected MEL but not COS cells. DNA affinity precipitation analysis with wild-type and mutant oligos established that RUNX1 and CBFbeta in MEL cell nuclear extracts could interact with the B3URE in vitro and suggested that RUNX1 recruits TAL1 and Ldb1 to DNA. Northern blot and quantitative real-time PCR analysis demonstrated that enforced expression of RUNX1 dramatically inhibited dimethylsulfoxide (DMSO)-induced Band 3 gene expression. Quantitative ChIP analysis showed that histone acetylation in the B3URE increased more than 4-fold, while histone methylation decreased ~50% after 3 days of DMSO-induced differentiation. Over the same time frame, the promoter region underwent significantly less acetylation but more extensive demethylation. Finally, changes in B3URE acetylation and methylation were attenuated and inhibited, respectively, in RUNX1-transfected MEL cells relative to vector controls. In sum, these results demonstrate that the Band 3 gene is a direct target of RUNX1 in erythroid cells and indicate that the B3URE contributes to the tightly regulated expression of this gene in differentiating erythroid progenitors. One mechanism by which RUNX1 regulates Band 3 transcription may be by influencing histone acetylation/methylation in this upstream regulatory region.

Cyclic AMP (cAMP) and cAMP Receptor Protein Influence both Synthesis and Uptake of Extracellular Autoinducer 2 in Escherichia coli

10.1128/jb.187.6.2066-2076.2005 ◽

2005 ◽

Vol 187 (6) ◽

pp. 2066-2076 ◽

Cited By ~ 130

Author(s):

Liang Wang ◽

Yoshifumi Hashimoto ◽

Chen-Yu Tsao ◽

James J. Valdes ◽

William E. Bentley

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

Carbon Sources ◽

Cell Communication ◽

Receptor Protein ◽

Upstream Region ◽

E Coli ◽

Autoinducer 2 ◽

Serovar Typhimurium ◽

ABSTRACT Bacterial autoinducer 2 (AI-2) is proposed to be an interspecies mediator of cell-cell communication that enables cells to operate at the multicellular level. Many environmental stimuli have been shown to affect the extracellular AI-2 levels, carbon sources being among the most important. In this report, we show that both AI-2 synthesis and uptake in Escherichia coli are subject to catabolite repression through the cyclic AMP (cAMP)-CRP complex, which directly stimulates transcription of the lsr (for “luxS regulated”) operon and indirectly represses luxS expression. Specifically, cAMP-CRP is shown to bind to a CRP binding site located in the upstream region of the lsr promoter and works with the LsrR repressor to regulate AI-2 uptake. The functions of the lsr operon and its regulators, LsrR and LsrK, previously reported in Salmonella enterica serovar Typhimurium, are confirmed here for E. coli. The elucidation of cAMP-CRP involvement in E. coli autoinduction impacts many areas, including the growth of E. coli in fermentation processes.

Two-State Allosteric Modeling Suggests Protein Equilibrium as an Integral Component for Cyclic AMP (cAMP) Specificity in the cAMP Receptor Protein of Escherichia coli

10.1128/jb.00074-08 ◽

2008 ◽

Vol 190 (13) ◽

pp. 4532-4540 ◽

Cited By ~ 14

Author(s):

Hwan Youn ◽

Junseock Koh ◽

Gary P. Roberts

Keyword(s):

Escherichia Coli ◽

Binding Pocket ◽

Receptor Protein ◽

Ligand Specificity ◽

Wild Type ◽

Camp Receptor Protein ◽

Active Protein ◽

Common Basis ◽

Affinity Ligand ◽

ABSTRACT Activation of the cAMP receptor protein (CRP) from Escherichia coli is highly specific to its allosteric ligand, cAMP. Ligands such as adenosine and cGMP, which are structurally similar to cAMP, fail to activate wild-type CRP. However, several cAMP-independent CRP variants (termed CRP*) exist that can be further activated by both adenosine and cGMP, as well as by cAMP. This has remained a puzzle because the substitutions in many of these CRP* variants lie far from the cAMP-binding pocket (>10 Å) and therefore should not directly affect that pocket. Here we show a surprising similarity in the altered ligand specificity of four CRP* variants with a single substitution in D53S, G141K, A144T, or L148K, and we propose a common basis for this phenomenon. The increased active protein population caused by an equilibrium shift in these variants is hypothesized to preferentially stabilize ligand binding. This explanation is completely consistent with the cAMP specificity in the activation of wild-type CRP. The model also predicts that wild-type CRP should be activated even by the lower-affinity ligand, adenosine, which we experimentally confirmed. The study demonstrates that protein equilibrium is an integral factor for ligand specificity in an allosteric protein, in addition to the direct effects of ligand pocket residues.

Sycrp2 Is Essential for Twitching Motility in the CyanobacteriumSynechocystissp. Strain PCC 6803

10.1128/jb.00436-18 ◽

2018 ◽

Vol 200 (21) ◽

Cited By ~ 3

Author(s):

Wei-Yu Song ◽

Sha-Sha Zang ◽

Zheng-Ke Li ◽

Guo-Zheng Dai ◽

Ke Liu ◽

...

Keyword(s):

Cell Motility ◽

Receptor Protein ◽

Upstream Region ◽

Twitching Motility ◽

Camp Receptor Protein ◽

Mobility Shift ◽

Content Type ◽

Receptor Proteins ◽

Camp Receptor ◽

Pcc 6803

ABSTRACTTwo cAMP receptor proteins (CRPs), Sycrp1 (encoded bysll1371) and Sycrp2 (encoded bysll1924), exist in the cyanobacteriumSynechocystissp. strain PCC 6803. Previous studies have demonstrated that Sycrp1 has binding affinity for cAMP and is involved in motility by regulating the formation of pili. However, the function of Sycrp2 remains unknown. Here, we report thatsycrp2disruption results in the loss of motility ofSynechocystissp. PCC 6803, and that the phenotype can be recovered by reintroducing thesycrp2gene into the genome ofsycrp2-disrupted mutants. Electron microscopy showed that thesycrp2-disrupted mutant lost the pilus apparatus on the cell surface, resulting in a lack of cell motility. Furthermore, the transcript level of thepilA9-pilA11operon (essential for cell motility and regulated by the cAMP receptor protein Sycrp1) was markedly decreased insycrp2-disrupted mutants compared with the wild-type strain. Moreover, yeast two-hybrid analysis and a pulldown assay demonstrated that Sycrp2 interacted with Sycrp1 to form a heterodimer and that Sycrp1 and Sycrp2 interacted with themselves to form homodimers. Gel mobility shift assays revealed that Sycrp1 specifically binds to the upstream region ofpilA9. Together, these findings indicate that inSynechocystissp. PCC 6803, Sycrp2 regulates the formation of pili and cell motility by interacting with Sycrp1.IMPORTANCEcAMP receptor proteins (CRPs) are widely distributed in cyanobacteria and play important roles in regulating gene expression. Although many cyanobacterial species have two cAMP receptor-like proteins, the functional links between them are unknown. Here, we found that Sycrp2 in the cyanobacteriumSynechocystissp. strain PCC 6803 is essential for twitching motility and that it interacts with Sycrp1, a known cAMP receptor protein involved with twitching motility. Our findings indicate that the two cAMP receptor-like proteins in cyanobacteria do not have functional redundancy but rather work together.