scholarly journals The Pseudomonas aeruginosa Exotoxin A Regulatory Gene, ptxS: Evidence for Negative Autoregulation

1999 ◽  
Vol 181 (16) ◽  
pp. 4890-4895 ◽  
Author(s):  
Britta L. Swanson ◽  
Jane A. Colmer ◽  
Abdul N. Hamood
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Upstream Region ◽  
Transcriptional Level ◽  
Exotoxin A ◽  
Specific Sequence ◽  
T7 Promoter ◽  
Gel Shift

ABSTRACT We have previously described a Pseudomonas aeruginosagene, ptxR, which enhances exotoxin A production at the transcriptional level. We have also described another gene,ptxS, which is transcribed divergently fromptxR and interferes with the enhancement of exotoxin A synthesis by ptxR. However, the mechanisms through whichptxR and/or ptxS are regulated is not known. In this study, we attempted (by using the DNA gel shift assay) to determine if P. aeruginosa contains a potential regulatory protein that binds specifically to the ptxR orptxS upstream region. In the initial analysis, different-sized gel shift bands were detected when a probe containing the ptxR-ptxS intergenic region was incubated with the lysate of P. aeruginosa PAO1. The strongest binding activity was detected with a smaller fragment that represents theptxS upstream region. Additional deletion analysis localized the binding to a 52-bp fragment immediately upstream ofptxS. The gel shift band was not detected when the 52-bp fragment was incubated with the lysate of the ptxS isogenic mutant PAO1::ptxS. However, the binding band was regenerated when a plasmid carrying ptxS intact was introduced into PAO1::ptxS. In addition, the gel shift band was detected when the 52-bp fragment was incubated with a lysate of Escherichia coli in which ptxS was overexpressed from the T7 promoter. The effect of PtxS onptxS expression was examined by using aptxS-lacZ fusion plasmid. The level of β-galactosidase activity produced by PAO1::ptxS carrying the fusion plasmid was four- to fivefold higher than that produced by PAO1 carrying the same plasmid. Using DNase I footprinting analysis, the binding region was specified to a 20-bp fragment. Within the fragment, a 14-bp palindromic sequence exists that may function as a PtxS binding site. These results suggest that PtxS autoregulates its synthesis by binding to a specific sequence within the ptxSupstream region.

Transcriptional regulation of Pseudomonas aeruginosa rhlR, encoding a quorum-sensing regulatory protein

Microbiology ◽  
2003 ◽  
Vol 149 (11) ◽  
pp. 3073-3081 ◽  
Author(s):  
Gerardo Medina ◽  
Katy Juárez ◽  
Rafael Díaz ◽  
Gloria Soberón-Chávez
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcriptional Regulation ◽  
Quorum Sensing ◽  
Regulatory Protein ◽  
Culture Conditions ◽  
Transcriptional Level ◽  
Coding Region ◽  
Upstream Gene ◽  
Transcription Start Sites ◽  
Response Systems

The Pseudomonas aeruginosa rhlR gene encodes the transcriptional regulator RhlR which has a central role in the quorum-sensing response. Different gene products involved in bacterial pathogenesis are regulated at the transcriptional level by two quorum-sensing response systems, Las and Rhl. The expression of rhlR has been reported to be under the control of the Las system, but its transcriptional regulation has not been studied in detail. Here, the rhlR promoter region has been characterized and shown to present four different transcription start sites, two of which are included in the upstream gene (rhlB) coding region. It was found that rhlR expression is not only dependent on LasR but also on different regulatory proteins such as Vfr and RhlR itself, and also on the alternative sigma factor σ 54. It is reported that rhlR expression is partially LasR-independent under certain culture conditions and is strongly influenced by environmental factors.

scholarly journals Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa.

1991 ◽  
Vol 11 (9) ◽  
pp. 4356-4362 ◽  
Author(s):  
M N Kanaan ◽  
G A Marzluf
Keyword(s):  
Dna Binding ◽  
Neurospora Crassa ◽  
Mutational Analysis ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Dna Binding Domain ◽  
Dna Binding Activity

cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.

scholarly journals The Pyrimidine Operon pyrRPB-carA fromLactococcus lactis

2001 ◽  
Vol 183 (9) ◽  
pp. 2785-2794 ◽  
Author(s):  
Jan Martinussen ◽  
Jette Schallert ◽  
Birgit Andersen ◽  
Karin Hammer
Keyword(s):  
Mutational Analysis ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Small Subunit ◽  
Transcriptional Level ◽  
Carbamoyl Phosphate Synthetase ◽  
Arginine Biosynthesis ◽  
Carbamoyl Phosphate ◽  
Biosynthetic Genes ◽  
Membrane Bound

ABSTRACT The four genes pyrR, pyrP, pyrB, and carAwere found to constitute an operon in Lactococcus lactissubsp. lactis MG1363. The functions of the different genes were established by mutational analysis. The first gene in the operon is the pyrimidine regulatory gene, pyrR, which is responsible for the regulation of the expression of the pyrimidine biosynthetic genes leading to UMP formation. The second gene encodes a membrane-bound high-affinity uracil permease, required for utilization of exogenous uracil. The last two genes in the operon, pyrBand carA, encode pyrimidine biosynthetic enzymes; aspartate transcarbamoylase (pyrB) is the second enzyme in the pathway, whereas carbamoyl-phosphate synthetase subunit A (carA) is the small subunit of a heterodimeric enzyme, catalyzing the formation of carbamoyl phosphate. The carAgene product is shown to be required for both pyrimidine and arginine biosynthesis. The expression of the pyrimidine biosynthetic genes including the pyrRPB-carA operon is subject to control at the transcriptional level, most probably by an attenuator mechanism in which PyrR acts as the regulatory protein.

scholarly journals cpc-1, the general regulatory gene for genes of amino acid biosynthesis in Neurospora crassa, is differentially expressed during the asexual life cycle

1991 ◽  
Vol 11 (2) ◽  
pp. 928-934 ◽  
Author(s):  
D J Ebbole ◽  
J L Paluh ◽  
M Plamann ◽  
M S Sachs ◽  
C Yanofsky
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Neurospora Crassa ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Recognition Sequence ◽  
Dna Binding Activity ◽  
Cell Extracts

CPCI, the principal regulatory protein required for cross-pathway control of amino acid biosynthetic genes in Neurospora crassa, contains a domain similar to the DNA-binding domain of GCN4, the corresponding general regulator in Saccharomyces cerevisiae. We examined binding by CPC1 synthesized in vitro and by CPC1 present in N. crassa whole-cell extracts. CPCI from both sources was shown to bind to the DNA sequence 5'-ATGACTCAT-3', which is also the preferred recognition sequence of GCN4, CPC1 was confirmed as the source of DNA-binding activity in extracts by immunoblotting. Slightly mobility differences between DNA complexes containing CPCI synthesized in vitro and CPC1 in mycelial extracts were observed. Analyses of N. crassa extracts from different stages of asexual development revealed that CPC1 was abundant immediately following spore germination and through early mycelial growth but was scarce subsequently. CPC1 levels could be increased at any time by imposing amino acid starvation. Copies of the CPC1 response element are located upstream of several genes regulated by cross-pathway control, including cpc-1 itself.

scholarly journals Autoregulation of the Pseudomonas aeruginosa Protein PtxS Occurs through a Specific Operator Site within the ptxS Upstream Region

2000 ◽  
Vol 182 (15) ◽  
pp. 4366-4371 ◽  
Author(s):  
Britta L. Swanson ◽  
Abdul N. Hamood
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Regulatory Protein ◽  
Site Directed Mutagenesis ◽  
Upstream Region ◽  
Palindromic Sequence ◽  
Promoter Fusion ◽  
Translation Start ◽  
Specific Operator ◽  
Operator Site ◽  
Fusion Experiments

ABSTRACT We have previously shown that the Pseudomonas aeruginosa toxA regulatory protein PtxS autoregulates its own synthesis by binding to a 52-bp fragment. The 3′ end of the 52-bp fragment is located 58 bp 5′ of the ptxS translation start site. We have identified a 14-bp palindromic sequence (TGAAACCGGTTTCA) within the 52-bp fragment. In this study, we used site-directed mutagenesis and promoter fusion experiments to determine if PtxS binds specifically to this palindromic sequence and regulatesptxS expression. We have also tried to determine the roles of specific nucleotides within the palindromic sequence in PtxS binding and ptxS expression. Initial promoter fusion experiments confirmed that the 52-bp fragment does not overlap with the region that carries the ptxS promoter activity. PtxS binding was eliminated upon the deletion of the 14-bp palindromic sequence from the 52-bp fragment. In addition, the deletion of the 14-bp sequence caused a significant enhancement in ptxS expression in theP. aeruginosa strain PAO1 and the ptxS isogenic mutant PAO::ptxS. Mutation of specific nucleotides within the 14-bp sequence eliminated, reduced, or had no effect on PtxS binding. However, mutations of several of these nucleotides produced a significant increase in ptxSexpression in both PAO1 and PAO::ptxS. These results suggest that (i) the 14-bp palindromic sequence and specific nucleotides within it play a role in PtxS binding and (ii) deletion of the palindromic sequence or changing of certain nucleotides within it interferes with another mechanism that may regulate ptxSexpression.

Expression ofptxRand its effect ontoxAandregAexpression during the growth cycle ofPseudomonas aeruginosastrain PAO1

1999 ◽  
Vol 45 (12) ◽  
pp. 1008-1016 ◽  
Author(s):  
Jane A Colmer ◽  
Abdul N Hamood
Keyword(s):  
Regulatory Gene ◽  
Blot Hybridization ◽  
Sufficient Conditions ◽  
Growth Cycle ◽  
Transcriptional Level ◽  
Northern Blot Hybridization ◽  
Exotoxin A ◽  
Strain Pao1 ◽  
Iron Deficient ◽  
Multiple Copies

The expression of the toxA and regA genes in Pseudomonas aeruginosa is negatively regulated by iron at the transcriptional level. We have previously described ptxR, an exotoxin A regulatory gene which appears to enhance toxA expression through regA. In this study, we have tried to determine if ptxR expression correlates with its effect on toxA and regA expression throughout the growth cycle of P. aeruginosa strain PAO1. This was done using Northern blot hybridization experiments (with toxA, regA, and ptxR probes), and ptxR transcriptional fusion studies. To avoid problems related to the presence of multiple copies of ptxR in PAO1, we have constructed a PAO1 strain (PAO1-XR) that carries only two ptxR genes in its chromosome. Our results showed that when PAO1-XR was grown in iron-limited conditions, the increase in exotoxin A activity and the accumulation of toxA mRNA appeared at about mid- to late-exponential phase. A similar increase in the accumulation of regA mRNA was detected. Both regA transcripts, T1 and T2, were enhanced in PAO1-XR. In iron-sufficient medium, neither toxA nor regA mRNA was detected at any time point in the growth cycle of PAO1-XR. In contrast, the accumulation of ptxR mRNA was detected throughout the growth cycle of PAO1-XR under both iron-deficient and iron-sufficient conditions. The presence of iron in the growth medium also had no effect on the level of β-galactosidase activity produced by a ptxR-lacZ fusion in PAO1. These results suggest that (i) the enhancement in toxA expression by ptxR correlates with the enhancement in regA expression; (ii) ptxR affects the expression of the regA P1 and P2 promoters; (iii) ptxR expression precedes its effect on toxA and regA expression; and (iv) unlike toxA and regA, the overall expression of ptxR throughout the growth cycle of PAO1 is not negatively regulated by iron.Key words: ptxR, differential expression, transcriptional regulation, regA, toxA.

scholarly journals Negative Control of Quorum Sensing by RpoN (σ54) in Pseudomonas aeruginosa PAO1

2003 ◽  
Vol 185 (7) ◽  
pp. 2227-2235 ◽  
Author(s):  
Karin Heurlier ◽  
Valerie Dénervaud ◽  
Gabriella Pessi ◽  
Cornelia Reimmann ◽  
Dieter Haas
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Hydrogen Cyanide ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Homoserine Lactone ◽  
Negative Control ◽  
Signal Molecules ◽  
Cell Densities ◽  
Global Regulatory Gene

ABSTRACT In Pseudomonas aeruginosa PAO1, the expression of several virulence factors such as elastase, rhamnolipids, and hydrogen cyanide depends on quorum-sensing regulation, which involves the lasRI and rhlRI systems controlled by N-(3-oxododecanoyl)-l-homoserine lactone and N-butyryl-l-homoserine lactone, respectively, as signal molecules. In rpoN mutants lacking the transcription factor σ54, the expression of the lasR and lasI genes was elevated at low cell densities, whereas expression of the rhlR and rhlI genes was markedly enhanced throughout growth by comparison with the wild type and the complemented mutant strains. As a consequence, the rpoN mutants had elevated levels of both signal molecules and overexpressed the biosynthetic genes for elastase, rhamnolipids, and hydrogen cyanide. The quorum-sensing regulatory protein QscR was not involved in the negative control exerted by RpoN. By contrast, in an rpoN mutant, the expression of the gacA global regulatory gene was significantly increased during the entire growth cycle, whereas another global regulatory gene, vfr, was downregulated at high cell densities. In conclusion, it appears that GacA levels play an important role, probably indirectly, in the RpoN-dependent modulation of the quorum-sensing machinery of P. aeruginosa.

Mutational analysis of the DNA-binding domain of the CYS3 regulatory protein of Neurospora crassa

1991 ◽  
Vol 11 (9) ◽  
pp. 4356-4362
Author(s):  
M N Kanaan ◽  
G A Marzluf
Keyword(s):  
Dna Binding ◽  
Neurospora Crassa ◽  
Mutational Analysis ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Dna Binding Domain ◽  
Dna Binding Activity

cys-3, the major sulfur regulatory gene of Neurospora crassa, activates the expression of a set of unlinked structural genes which encode sulfur catabolic-related enzymes during conditions of sulfur limitation. The cys-3 gene encodes a regulatory protein of 236 amino acid residues with a leucine zipper and an upstream basic region (the b-zip region) which together may constitute a DNA-binding domain. The b-zip region was expressed in Escherichia coli to examine its DNA-binding activity. The b-zip domain protein binds to the promoter region of the cys-3 gene itself and of cys-14, the sulfate permease II structural gene. A series of CYS3 mutant proteins obtained by site-directed mutagenesis were expressed and tested for function, dimer formation, and DNA-binding activity. The results demonstrate that the b-zip region of cys-3 is critical for both its function in vivo and specific DNA-binding in vitro.

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