scholarly journals Binding of Pseudomonas aeruginosa AlgZ to Sites Upstream of the algZ Promoter Leads to Repression of Transcription

2005 ◽  
Vol 187 (13) ◽  
pp. 4430-4443 ◽  
Author(s):  
Deborah M. Ramsey ◽  
Patricia J. Baynham ◽  
Daniel J. Wozniak
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Sites ◽  
Transcriptional Control ◽  
Specific Binding ◽  
Transcriptional Start Site ◽  
Opportunistic Pathogen ◽  
Single Copy ◽  
Amino Acid Identity ◽  
Mobility Shift ◽  
Base Pairs

ABSTRACT Mucoid variants of the opportunistic pathogen Pseudomonas aeruginosa produce the exopolysaccharide alginate and colonize the respiratory tracts of cystic fibrosis patients. The genes encoding the alginate biosynthetic enzymes are clustered in a single operon, which is under tight transcriptional control. One essential activator of the alginate operon is AlgZ, a proposed ribbon-helix-helix DNA binding protein that shares 30% amino acid identity with the Mnt repressor of Salmonella enterica serovar Typhimurium bacteriophage P22. In the current study, we examined the role of AlgZ as an autoregulator. Using single-copy algZ-lacZ transcription fusions, an increase in algZ transcription was observed in an algZ mutant compared to the isogenic wild-type strain, suggesting that AlgZ may have an additional role as a repressor. To identify the AlgZ binding site, overlapping regions upstream of algZ were incubated with AlgZ and analyzed by electrophoretic mobility shift assays. Specific binding activity was localized to a region spanning from 66 to 185 base pairs upstream of the algZ transcriptional start site. Two AlgZ binding sites were defined using copper-phenanthroline footprinting and deletion analyses, with one site centered at 93 base pairs and the other centered at 161 base pairs upstream of the algZ promoter. Deletion of both binding sites resulted in the loss of AlgZ binding. These results indicate that AlgZ represses algZ transcription, and this activity is mediated by multiple AlgZ-DNA interactions.

scholarly journals Regulation of Pseudomonas Quinolone Signal Synthesis in Pseudomonas aeruginosa

2005 ◽  
Vol 187 (13) ◽  
pp. 4372-4380 ◽  
Author(s):  
Dana S. Wade ◽  
M. Worth Calfee ◽  
Edson R. Rocha ◽  
Elizabeth A. Ling ◽  
Elana Engstrom ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Transcriptional Start Site ◽  
Opportunistic Pathogen ◽  
Homoserine Lactone ◽  
Mobility Shift ◽  
Sensing System ◽  
Regulator Protein ◽  
Quorum Sensing System

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis patients and is a major source of nosocomial infections. This bacterium controls many virulence factors by using two quorum-sensing systems, las and rhl. The las system is composed of the LasR regulator protein and its cell-to-cell signal, N-(3-oxododecanoyl) homoserine lactone, and the rhl system is composed of RhlR and the signal N-butyryl homoserine lactone. A third intercellular signal, the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4-quinolone), also regulates numerous virulence factors. PQS synthesis requires the expression of multiple operons, one of which is pqsABCDE. Previous experiments showed that the transcription of this operon, and therefore PQS production, is negatively regulated by the rhl quorum-sensing system and positively regulated by the las quorum-sensing system and PqsR (also known as MvfR), a LysR-type transcriptional regulator protein. With the use of DNA mobility shift assays and β-galactosidase reporter fusions, we have studied the regulation of pqsR and its relationship to pqsA, lasR, and rhlR. We show that PqsR binds the promoter of pqsA and that this binding increases dramatically in the presence of PQS, implying that PQS acts as a coinducer for PqsR. We have also mapped the transcriptional start site for pqsR and found that the transcription of pqsR is positively regulated by lasR and negatively regulated by rhlR. These results suggest that a regulatory chain occurs where pqsR is under the control of LasR and RhlR and where PqsR in turn controls pqsABCDE, which is required for the production of PQS.

scholarly journals Identification of the Regulon of AphB and Its Essential Roles in LuxR and Exotoxin Asp Expression in the Pathogen Vibrio alginolyticus

2017 ◽  
Vol 199 (20) ◽  
Author(s):  
Xiating Gao ◽  
Yang Liu ◽  
Huan Liu ◽  
Zhen Yang ◽  
Qin Liu ◽  
...  
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Vibrio Alginolyticus ◽  
Physiological Adaptation ◽  
Pcr Analysis ◽  
Growth Stages ◽  
Mobility Shift ◽  
Marine Animals ◽  
Content Type ◽  
Virulence Gene Expression

ABSTRACT In Vibrio species, AphB is essential to activate virulence cascades by sensing low-pH and anaerobiosis signals; however, its regulon remains largely unknown. Here, AphB is found to be a key virulence regulator in Vibrio alginolyticus, a pathogen for marine animals and humans. Chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) enabled the detection of 20 loci in the V. alginolyticus genome that contained AphB-binding peaks. An AphB-specific binding consensus was confirmed by electrophoretic mobility shift assays (EMSAs), and the regulation of genes flanking such binding sites was demonstrated using quantitative real-time PCR analysis. AphB binds directly to its own promoter and positively controls its own expression in later growth stages. AphB also activates the expression of the exotoxin Asp by binding directly to the promoter regions of asp and the master quorum-sensing (QS) regulator luxR. DNase I footprinting analysis uncovered distinct AphB-binding sites (BBS) in these promoters. Furthermore, a BBS in the luxR promoter region overlaps that of LuxR-binding site I, which mediates the positive control of luxR promoter activity by AphB. This study provides new insights into the AphB regulon and reveals the mechanisms underlying AphB regulation of physiological adaptation and QS-controlled virulence in V. alginolyticus. IMPORTANCE In this work, AphB is determined to play essential roles in the expression of genes associated with QS, physiology, and virulence in V. alginolyticus, a pathogen for marine animals and humans. AphB was found to bind directly to 20 genes and control their expression by a 17-bp consensus binding sequence. Among the 20 genes, the aphB gene itself was identified to be positively autoregulated, and AphB also positively controlled asp and luxR expression. Taken together, these findings improve our understanding of the roles of AphB in controlling physiological adaptation and QS-controlled virulence gene expression.

scholarly journals Structure and promoter characterization of the gene encoding the large subunit (R1 protein) of mouse ribonucleotide reductase

1993 ◽  
Vol 90 (23) ◽  
pp. 11322-11326 ◽  
Author(s):  
S Björklund ◽  
K Hjortsberg ◽  
E Johansson ◽  
L Thelander
Keyword(s):  
Ribonucleotide Reductase ◽  
Genomic Library ◽  
Protein Complexes ◽  
Specific Binding ◽  
Transcriptional Start Site ◽  
Large Subunit ◽  
Mobility Shift ◽  
Direct Role ◽  
Encoding Gene ◽  
R1 Gene

Mammalian ribonucleotide reductase (EC 1.17.4.1) is composed of two nonidentical subunits, proteins R1 and R2, both required for enzyme activity. The structure of the genomic mouse ribonucleotide reductase R1 gene was compiled from a number of overlapping lambda clones isolated from a Charon 4A mouse sperm genomic library. The R1-encoding gene covers 26 kb and consists of 19 exons. All exon-intron boundaries were located by dideoxynucleotide sequencing, showing that intron 7 starts with the variant GC instead of GT. About 3.5 kb of DNA from the 5'-flanking region of the R1-encoding gene were cloned and sequenced, and the transcriptional start site was determined by nuclease S1 mapping of RNA. DNase I footprinting assays on the R1 promoter identified two nearly identical 23-bp-long protein-binding regions. Three protein complexes binding to one of the 23-mer regions were resolved and partially identified by using gel-retardation mobility-shift assays and UV crosslinking. One complex most likely contained Sp1, and another complex showed S-phase-specific binding, suggesting a direct role in the cell-cycle-dependent R1 gene expression.

scholarly journals Independent Regulation of MucD, an HtrA-Like Protease in Pseudomonas aeruginosa, and the Role of Its Proteolytic Motif in Alginate Gene Regulation

2006 ◽  
Vol 188 (8) ◽  
pp. 3134-3137 ◽  
Author(s):  
Lynn F. Wood ◽  
Dennis E. Ohman
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Gene Regulation ◽  
Transcriptional Start Site ◽  
Single Copy ◽  
Start Site ◽  
Temperature Resistance

ABSTRACT Expression of mucD, encoding a homologue of the HtrA(DegP) family of endoserine proteases, was investigated in Pseudomonas aeruginosa. Expressed from the algT-mucABCD operon, MucD was detected in mucoid (FRD1) and nonmucoid (PAO1) parental strains and also when polar insertions were placed upstream in algT or mucB. A transcriptional start site for a mucD promoter (PmucD) was mapped within mucC. Expression of single-copy mucD217, encoding MucD altered in the protease motif (S217A), was defective in temperature resistance and alginate gene regulation.

scholarly journals PrtR Homeostasis Contributes to Pseudomonas aeruginosa Pathogenesis and Resistance against Ciprofloxacin

2014 ◽  
Vol 82 (4) ◽  
pp. 1638-1647 ◽  
Author(s):  
Ziyu Sun ◽  
Jing Shi ◽  
Chang Liu ◽  
Yongxin Jin ◽  
Kewei Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Bacterial Colonization ◽  
Mrna Level ◽  
Sos Response ◽  
Opportunistic Pathogen ◽  
Host Cells ◽  
Chronic Infections ◽  
Mobility Shift ◽  
Content Type

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that causes acute and chronic infections in humans. Pyocins are bacteriocins produced byP. aeruginosathat are usually released through lysis of the producer strains. Expression of pyocin genes is negatively regulated by PrtR, which gets cleaved under SOS response, leading to upregulation of pyocin synthetic genes. Previously, we demonstrated that PrtR is required for the expression of type III secretion system (T3SS), which is an important virulence component ofP. aeruginosa. In this study, we demonstrate that mutation inprtRresults in reduced bacterial colonization in a mouse acute pneumonia model. Examination of bacterial and host cells in the bronchoalveolar lavage fluids from infected mice revealed that expression of PrtR is induced by reactive oxygen species (ROS) released by neutrophils. We further demonstrate that treatment with hydrogen peroxide or ciprofloxacin, known to induce the SOS response and pyocin production, resulted in an elevated PrtR mRNA level. Overexpression of PrtR by atacpromoter repressed the endogenousprtRpromoter activity, and electrophoretic mobility shift assay revealed that PrtR binds to its own promoter, suggesting an autorepressive mechanism of regulation. A high level of PrtR expressed from a plasmid resulted in increased T3SS gene expression during infection and higher resistance against ciprofloxacin. Overall, our results suggest that the autorepression of PrtR contributes to the maintenance of a relatively stable level of PrtR, which is permissive to T3SS gene expression in the presence of ROS while increasing bacterial tolerance to stresses, such as ciprofloxacin, by limiting pyocin production.

scholarly journals Hepatic transcription of the acute-phase alpha 1-inhibitor III gene is controlled by a novel combination of cis-acting regulatory elements.

1990 ◽  
Vol 10 (7) ◽  
pp. 3483-3491 ◽  
Author(s):  
L J Abraham ◽  
A D Bradshaw ◽  
B R Shiels ◽  
W Northemann ◽  
G Hudson ◽  
...  
Keyword(s):  
Binding Sites ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Nuclear Factor ◽  
Base Pairs ◽  
Cis Acting ◽  
Expression Studies ◽  
Positive Regulators ◽  
Flanking Region ◽  
Related Proteins

mRNA coding for the abundant broad-range plasma proteinase inhibitor alpha 1-inhibitor III (alpha 1I3) was detected only in rat liver, while mRNA for the related proteins alpha 1-macroglobulin and alpha 2-macroglobulin was also found in a variety of nonhepatic tissues. cis-Acting control elements necessary for the hepatic transcription of alpha 1I3 were mapped by transfection and expression studies of control-region constructs in cultured hepatic and nonhepatic cells. The promoter-proximal 5'-flanking region contained four control elements, I to IV, located between -109 and -196 base pairs upstream of the transcriptional start site relevant for the hepatic transcription of this gene. Elements II and III were essential, and I and IV exerted strong modulatory effects. Elements I to III acted as positive regulators, and IV acted as a negative element. Element II contained the sequence TGGCA and is probably a binding site for a nuclear factor related to the known transcription factor NF1. The other three elements did not resemble consensus binding sites for known transcription factors that are involved in the hepatocyte-specific transcription of other well-characterized plasma protein genes, such as the prototype factor HNF-1. Thus, the alpha 1I3 gene achieves its highly hepatocyte-specific transcription through a novel combination of cis-acting control elements and trans-acting factors.

Hepatic transcription of the acute-phase alpha 1-inhibitor III gene is controlled by a novel combination of cis-acting regulatory elements

1990 ◽  
Vol 10 (7) ◽  
pp. 3483-3491
Author(s):  
L J Abraham ◽  
A D Bradshaw ◽  
B R Shiels ◽  
W Northemann ◽  
G Hudson ◽  
...  
Keyword(s):  
Binding Sites ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Nuclear Factor ◽  
Base Pairs ◽  
Cis Acting ◽  
Expression Studies ◽  
Positive Regulators ◽  
Flanking Region ◽  
Related Proteins

mRNA coding for the abundant broad-range plasma proteinase inhibitor alpha 1-inhibitor III (alpha 1I3) was detected only in rat liver, while mRNA for the related proteins alpha 1-macroglobulin and alpha 2-macroglobulin was also found in a variety of nonhepatic tissues. cis-Acting control elements necessary for the hepatic transcription of alpha 1I3 were mapped by transfection and expression studies of control-region constructs in cultured hepatic and nonhepatic cells. The promoter-proximal 5'-flanking region contained four control elements, I to IV, located between -109 and -196 base pairs upstream of the transcriptional start site relevant for the hepatic transcription of this gene. Elements II and III were essential, and I and IV exerted strong modulatory effects. Elements I to III acted as positive regulators, and IV acted as a negative element. Element II contained the sequence TGGCA and is probably a binding site for a nuclear factor related to the known transcription factor NF1. The other three elements did not resemble consensus binding sites for known transcription factors that are involved in the hepatocyte-specific transcription of other well-characterized plasma protein genes, such as the prototype factor HNF-1. Thus, the alpha 1I3 gene achieves its highly hepatocyte-specific transcription through a novel combination of cis-acting control elements and trans-acting factors.

scholarly journals The sarcomeric actin CArG-binding factor is indistinguishable from the c-fos serum response factor.

1989 ◽  
Vol 9 (2) ◽  
pp. 515-522 ◽  
Author(s):  
L M Boxer ◽  
R Prywes ◽  
R G Roeder ◽  
L Kedes
Keyword(s):  
Transcriptional Control ◽  
Serum Response Factor ◽  
Specific Binding ◽  
Immune Serum ◽  
Response Factor ◽  
Mobility Shift ◽  
Binding Factor ◽  
Carg Box ◽  
Zinc Addition ◽  
Serum Response

The c-fos serum response element (SRE) and a sarcomeric actin promoter element (CArG box) are similar in sequence and are recognized, respectively, by the serum response factor (SRF) and the CArG-binding factor (CBF). Although the transcriptional controls for the c-fos and sarcomeric actin genes are rather different, SRF and CBF have been found to be indistinguishable by all criteria tested. They exhibited similar chromatographic properties, sedimentation rates, and temperature stabilities. In mobility shift assays, the SRE competed more strongly than the actin CArG box for formation of either the SRF-SRE or the CBF-CArG complex. The symmetric inverted repeat of the left side of the Xenopus cytoskeletal actin SRE also competed, even more strongly, for each complex. The site-specific binding of each protein was inhibited both by orthophenanthroline, whose effects were reversed by zinc addition, and by treatment with potato acid phosphatase. Furthermore, immune serum raised against the c-fos SRF also recognized the actin CBF. We discuss how transcriptional control of these diverse genes might be obtained with a single similar factor.

scholarly journals Regulation of the Pseudomonas aeruginosa Quorum-Sensing Regulator VqsR

2007 ◽  
Vol 189 (12) ◽  
pp. 4367-4374 ◽  
Author(s):  
Luen-Luen Li ◽  
Jane E. Malone ◽  
Barbara H. Iglewski
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Mutational Analysis ◽  
Transcriptional Start Site ◽  
Transcriptional Regulators ◽  
Mobility Shift ◽  
Operator Sequence ◽  
Biofilm Development ◽  
Microarray Studies ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Bacteria communicate with each other to regulate cell density-dependent gene expression via a quorum-sensing (QS) cascade. In Pseudomonas aeruginosa, two known QS systems, las and rhl, control the expression of many factors that relate to virulence, pathogenicity, and biofilm development. Microarray studies of the las and rhl regulons led to our hypothesis that a complicated hierarchy in the QS regulon is composed of multiple transcriptional regulators. Here, we examined a QS-regulated gene, vqsR, which encodes a probable transcriptional regulator with a putative 20-bp operator sequence (las box) upstream. The transcriptional start site for vqsR was determined. The vqsR promoter was identified by examining a series of vqsR promoter-lacZ fusions. In addition, an Escherichia coli system where either LasR or RhlR protein was expressed from a plasmid indicated that the las system was the dominant regulator for vqsR. Electrophoretic mobility shift assays (EMSA) demonstrate that purified LasR protein binds directly to the vqsR promoter in the presence of 3O-C12-HSL. Point mutational analysis of the vqsR las box suggests that positions 3 and 18 in the las box are important for vqsR transcription, as assayed with a series of vqsRp-lacZ fusions. EMSA also shows that positions 3 and 18 are important for binding between the vqsR promoter and LasR. Our results demonstrate that the las system directly regulates vqsR, and certain nucleotides in the las box are crucial for LasR binding and activation of the vqsR promoter.

scholarly journals Functional Analyses of the RsmY and RsmZ Small Noncoding Regulatory RNAs inPseudomonas aeruginosa

2018 ◽  
Vol 200 (11) ◽  
Author(s):  
Kayley H. Janssen ◽  
Manisha R. Diaz ◽  
Matthew Golden ◽  
Justin W. Graham ◽  
Wes Sanders ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Site ◽  
Binding Sites ◽  
Rna Binding ◽  
Opportunistic Pathogen ◽  
Binding Properties ◽  
Content Type ◽  
Preferential Binding ◽  
Regulatory Rnas

ABSTRACTPseudomonas aeruginosais a Gram-negative opportunistic pathogen with distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA and RsmF are both members of the CsrA family of RNA-binding proteins and regulate protein synthesis at the posttranscriptional level. RsmA activity is controlled by two small noncoding regulatory RNAs (RsmY and RsmZ). Bioinformatic analyses suggest that RsmY and RsmZ each have 3 or 4 putative RsmA binding sites. Each predicted binding site contains a GGA sequence presented in the loop portion of a stem-loop structure. RsmY and RsmZ regulate RsmA, and possibly RsmF, by sequestering these proteins from target mRNAs. In this study, we used selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) chemistry to determine the secondary structures of RsmY and RsmZ and functional assays to characterize the contribution of each GGA site to RsmY/RsmZ activity. Our data indicate that RsmA has two preferential binding sites on RsmY and RsmZ, while RsmF has one preferential binding site on RsmY and two sites on RsmZ. Despite RsmF and RsmA sharing a common consensus site, RsmF binding properties are more restrictive than those of RsmA.IMPORTANCECsrA homologs are present in many bacteria. The opportunistic pathogenPseudomonas aeruginosauses RsmA and RsmF to inversely regulate factors associated with acute and chronic virulence phenotypes. RsmA has an affinity for RsmY and RsmZ higher than that of RsmF. The goal of this study was to understand the differential binding properties of RsmA and RsmF by using the RsmY and RsmZ regulatory small RNAs (sRNAs) as a model. Mutagenesis of the predicted RsmA/RsmF binding sites on RsmY and RsmZ revealed similarities in the sites required to control RsmA and RsmF activityin vivo. Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RsmF binding activityin vivoandin vitroare more stringent than those for RsmA.

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