scholarly journals Functional Analysis and Regulation of the Divergent spuABCDEFGH-spuI Operons for Polyamine Uptake and Utilization in Pseudomonas aeruginosa PAO1

2002 ◽  
Vol 184 (14) ◽  
pp. 3765-3773 ◽  
Author(s):  
Chung-Dar Lu ◽  
Yoshifumi Itoh ◽  
Yuji Nakada ◽  
Ying Jiang
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcription Initiation ◽  
Regulatory Protein ◽  
Induction Effect ◽  
Two Component System ◽  
S1 Nuclease ◽  
Inducible Promoters ◽  
Divergent Promoters ◽  
Knockout Mutants ◽  
Polyamine Uptake

ABSTRACT A multiple-gene locus for polyamine uptake and utilization was discovered in Pseudomonas aeruginosa PAO1. This locus contained nine genes designated spuABCDEFGHI (spu for spermidine and putrescine utilization). The physiological functions of the spu genes in utilization of two polyamines (putrescine and spermidine) were analyzed by using Tn5 transposon-mediated spu knockout mutants. Growth and uptake experiments support that the spuDEFGH genes specify components of a major ABC-type transport system for spermidine uptake, and enzymatic measurements indicated that spuC encodes putrescine aminotransferase with pyruvate as the amino group receptor. Although spuA and spuB mutants showed an apparent defect in spermidine utilization, the biochemical functions of the gene products have yet to be elucidated. Assays of lacZ fusions demonstrated the presence of agmatine-, putrescine-, and spermidine-inducible promoters for the spuABCDEFGH operon and the divergently transcribed spuI gene of unknown function. Since the observed induction effect of agmatine was abolished in an aguA mutant where conversion of agmatine into putrescine was blocked, putrescine or spermidine, but not agmatine, serves as the inducer molecule of the spuA-spuI divergent promoters. S1 nuclease mappings confirmed further the induction effects of the polyamines on transcription of the divergent promoters and localized the transcription initiation sites. Gel retardation assays with extracts from the cells grown on putrescine or spermidine demonstrated the presence of a polyamine-responsive regulatory protein interacting with the divergent promoter region. Finally, the absence of the putrescine-inducible spuA expression and putrescine aminotransferase (spuC) formation in the cbrB mutant indicated that the spu operons are regulated by the global CbrAB two-component system perhaps via the putative polyamine-responsive transcriptional activator.

scholarly journals Involvement of AlgQ in Transcriptional Regulation of Pyoverdine Genes in Pseudomonas aeruginosa PAO1

2005 ◽  
Vol 187 (15) ◽  
pp. 5097-5107 ◽  
Author(s):  
Cecilia Ambrosi ◽  
Federica Tiburzi ◽  
Francesco Imperi ◽  
Lorenza Putignani ◽  
Paolo Visca
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sigma Factor ◽  
Transcription Initiation ◽  
Regulatory Networks ◽  
Regulatory Protein ◽  
Single Copy ◽  
Virulence Determinants ◽  
Iron Starvation ◽  
Inorganic Polyphosphate ◽  
E Coli

ABSTRACT In response to iron limitation, Pseudomonas aeruginosa produces the fluorescent siderophore pyoverdine. Transcription of pyoverdine biosynthetic (pvd) genes is driven by the iron starvation sigma factor PvdS, which is negatively regulated by the Fur-Fe(II) holorepressor. We studied the effect of AlgQ, the Escherichia coli Rsd orthologue, on pyoverdine production by P. aeruginosa PAO1. AlgQ is a global regulatory protein which activates alginate, ppGpp, and inorganic polyphosphate synthesis through a cascade involving nucleoside diphosphate kinase (Ndk). AlgQ is also capable of interacting with region 4 of RpoD. In a reconstituted E. coli system, PvdS-dependent transcription from the pvdA promoter was doubled by the multicopy algQ gene. The P. aeruginosa ΔalgQ mutant exhibited a moderate but reproducible reduction in pyoverdine production compared with wild-type PAO1, as a result of a decline in transcription of pvd genes. PvdS expression was not affected by the algQ mutation. Single-copy algQ fully restored pyoverdine production and expression of pvd genes in the ΔalgQ mutant, while ndk did not. An increased intracellular concentration of RpoD mimicked the ΔalgQ phenotype, whereas PvdS overexpression suppressed the algQ mutation. E. coli rsd could partially substitute for algQ in transcriptional modulation of pvd genes. We propose that AlgQ acts as an anti-sigma factor for RpoD, eliciting core RNA polymerase recruitment by PvdS and transcription initiation at pvd promoters. AlgQ provides a link between the pyoverdine and alginate regulatory networks. These systems have similarities in responsiveness and physiological function: both depend on alternative sigma factors, respond to nutrient starvation, and act as virulence determinants for P. aeruginosa.

scholarly journals Molecular Characterization and Regulation of an Operon Encoding a System for Transport of Arginine and Ornithine and the ArgR Regulatory Protein in Pseudomonas aeruginosa

1998 ◽  
Vol 180 (21) ◽  
pp. 5559-5566 ◽  
Author(s):  
Takayuki Nishijyo ◽  
Seung-Moon Park ◽  
Chung-Dar Lu ◽  
Yoshifumi Itoh ◽  
Ahmed T. Abdelal
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Consensus Sequence ◽  
Regulatory Protein ◽  
Enteric Bacteria ◽  
Open Reading Frames ◽  
Significant Similarity ◽  
S1 Nuclease ◽  
Bacteria Transport ◽  
Transport Studies ◽  
Upstream Promoter

ABSTRACT The complete nucleotide sequence for the aot operon ofPseudomonas aeruginosa PAO1 was determined. This operon contains six open reading frames. The derived sequences for four of these, aotJ, aotQ, aotM, andaotP, show high similarity to those of components of the periplasmic binding protein-dependent ABC (ATP binding cassette) transporters of enteric bacteria. Transport studies with deletion derivatives established that these four genes function in arginine-inducible uptake of arginine and ornithine but not lysine. TheaotO gene, which encodes a polypeptide with no significant similarity to any known proteins, is not essential for arginine and ornithine uptake. The sixth and terminal gene in the operon encodes ArgR, which has been recently shown to function in regulation of arginine metabolism. Studies with anaotJ::lacZ translational fusion showed that expression of the aot operon is strongly induced by arginine and that this effect is mediated by ArgR. S1 nuclease and primer extension experiments showed the presence of two promoters, P1 and P2. The downstream promoter, P2, is induced by arginine and appears to be subject to carbon catabolite repression. The upstream promoter, P1, is induced by glutamate. Footprinting experiments established the presence of a 44-bp ArgR binding site that overlaps the −35 region for P2, as was shown to be the case for the arginine-inducible aru promoter, and the −10 region for P1, as was shown to be the case for arginine-repressible operons in P. aeruginosa. Sequence alignment confirms the architecture and the consensus sequence of the ArgR binding sites, as was previously reported.

scholarly journals Molecular Characterization and Regulation of theaguBA Operon, Responsible for Agmatine Utilization inPseudomonas aeruginosa PAO1

2001 ◽  
Vol 183 (22) ◽  
pp. 6517-6524 ◽  
Author(s):  
Yuji Nakada ◽  
Ying Jiang ◽  
Takayuki Nishijyo ◽  
Yoshifumi Itoh ◽  
Chung-Dar Lu
Keyword(s):  
Amino Acids ◽  
Sequence Similarity ◽  
Regulatory Protein ◽  
Regulatory Region ◽  
Negative Regulator ◽  
Induction Effect ◽  
Significant Similarity ◽  
S1 Nuclease ◽  
Agmatine Deiminase ◽  
Nuclease Mapping

ABSTRACT Pseudomonas aeruginosa PAO1 utilizes agmatine as the sole carbon and nitrogen source via two reactions catalyzed successively by agmatine deiminase (encoded by aguA; also called agmatine iminohydrolase) andN-carbamoylputrescine amidohydrolase (encoded byaguB). The aguBA and adjacentaguR genes were cloned and characterized. The predicted AguB protein (M r 32,759; 292 amino acids) displayed sequence similarity (≤60% identity) to enzymes of the β-alanine synthase/nitrilase family. While the deduced AguA protein (M r 41,190; 368 amino acids) showed no significant similarity to any protein of known function, assignment of agmatine deiminase to AguA in this report discovered a new family of carbon-nitrogen hydrolases widely distributed in organisms ranging from bacteria to Arabidopsis. The aguR gene encoded a putative regulatory protein (M r24,424; 221 amino acids) of the TetR protein family. Measurements of agmatine deiminase and N-carbamoylputrescine amidohydrolase activities indicated the induction effect of agmatine and N-carbamoylputrescine on expression of theaguBA operon. The presence of an inducible promoter for the aguBA operon in theaguR-aguB intergenic region was demonstrated by lacZ fusion experiments, and the transcription start of this promoter was localized 99 bp upstream from the initiation codon of aguB by S1 nuclease mapping. Experiments with knockout mutants of aguR established that expression of the aguBA operon became constitutive in the aguR background. Interaction of AguR overproduced in Escherichia coli with the aguBAregulatory region was demonstrated by gel retardation assays, supporting the hypothesis that AguR serves as the negative regulator of the aguBA operon, and binding of agmatine andN-carbamoylputrescine to AguR would antagonize its repressor function.

scholarly journals The Fur-Regulated Gene Encoding the Alternative Sigma Factor PvdS Is Required for Iron-Dependent Expression of the LysR-Type Regulator PtxR in Pseudomonas aeruginosa

1998 ◽  
Vol 180 (24) ◽  
pp. 6784-6788 ◽  
Author(s):  
Michael L. Vasil ◽  
Urs A. Ochsner ◽  
Zaiga Johnson ◽  
Jane A. Colmer ◽  
Abdul N. Hamood
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sigma Factor ◽  
Transcription Initiation ◽  
Regulatory Protein ◽  
Alternative Sigma Factor ◽  
Rnase Protection ◽  
Aerobic Conditions ◽  
Regulated Expression ◽  
P2 Promoter ◽  
Microaerobic Conditions

ABSTRACT We previously identified a novel regulator of the exotoxin A gene (toxA) in Pseudomonas aeruginosa, PtxR, that belongs to the LysR family of prokaryotic regulatory proteins. Preliminary data also suggest that PtxR affects the expression of siderophores in P. aeruginosa. Because toxAexpression and siderophore production in this organism are coordinately regulated by the ferric uptake regulator (Fur) and the Fur-regulated alternative sigma factor PvdS, regulation of ptxR itself in the context of these regulators was examined. RNase protection analyses of ptxR transcription revealed that there are two independent transcription initiation sites (T1 and T2). While transcription from the promoter of T1 is constitutive throughout the growth cycle of PAO1, transcription from the second promoter (P2) is negatively affected by iron. Transcription from the P2 promoter is constitutive in a fur mutant under microaerobic conditions but still iron regulated during aerobic growth. High concentrations (>100 nM) of the ferric uptake regulatory protein (Fur) failed to bind to either of the promoter regions of ptxR in either gel mobility shift assays or DNase I footprint experiments. These results indicate that Fur indirectly regulates the iron-dependent expression ofptxR. Iron-regulated transcription of ptxR from the P2 promoter, but not constitutive expression from the P1 promoter, was dependent on the Fur-regulated alternative sigma factor genepvdS, even under aerobic conditions. Consequently, there are two levels of iron-regulated expression of ptxR. The iron-regulated expression of ptxR under microaerobic conditions from the P2 promoter of ptxR is mediated indirectly by Fur through the iron-regulated expression ofpvdS. In contrast, pvdS-mediated iron regulation of ptxR under aerobic conditions is Fur independent.

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.

Signals for transcription initiation and termination in the Saccharomyces cerevisiae plasmid 2 micron circle

1985 ◽  
Vol 5 (10) ◽  
pp. 2770-2780
Author(s):  
A Sutton ◽  
J R Broach
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Unknown Function ◽  
Transcription Initiation ◽  
Coding Region ◽  
S1 Nuclease ◽  
Plasmid Maintenance ◽  
Transcriptional Regulatory ◽  
Polyadenylation Sites ◽  
Plasmid Genes ◽  
Extension Analysis

By S1 nuclease protection experiments and primer extension analysis, we determined precisely the cap and polyadenylation sites of transcripts from the four genes of the yeast 2 micron circle plasmid, as well as those of other plasmid transcripts of unknown function. In addition, we used deletion analysis to identify sequences necessary for polyadenylation in plasmid transcripts. Our results indicate that plasmid genes constitute independent transcription units and that plasmid mRNAs are not derived by extensive processing of precursor transcripts. In addition, we found that the D coding region of 2 micron circle is precisely encompassed by a polyadenylated transcript, suggesting that this coding region constitutes a functional plasmid gene. Our identification of the position of plasmid polyadenylation sites and of sequences necessary for polyadenylation provides support for a tripartite signal for polyadenylation as proposed by Zaret and Sherman (K.S. Zaret and F. Sherman, Cell 28:563-573, 1982). Finally, these data highlight salient features of the transcriptional regulatory circuitry that underlies the control of plasmid maintenance in the cell.

Unusual aspects of the polyamine transport system affect the design of strategies for use of polyamine analogues in chemotherapy

2007 ◽  
Vol 35 (2) ◽  
pp. 318-321 ◽  
Author(s):  
J.L.A. Mitchell ◽  
T.K. Thane ◽  
J.M. Sequeira ◽  
R. Thokala
Keyword(s):  
Regulatory Protein ◽  
Feedback System ◽  
Uptake System ◽  
Polyamine Synthesis ◽  
Polyamine Analogues ◽  
Binding Partners ◽  
Polyamine Transport ◽  
Polyamine Uptake ◽  
Tumour Cell Growth

One strategy for inhibiting tumour cell growth is the use of polyamine mimetics to depress endogenous polyamine levels and, ideally, obstruct critical polyamine-requiring reactions. Such polyamine analogues make very unusual drugs, in that extremely high intracellular concentrations are required for growth inhibition or cytotoxicity. Cells exposed to even sub-micromolar concentrations of such analogues can achieve effective intracellular levels because these compounds are incorporated by the very aggressive polyamine uptake system. Once incorporated to these levels, many of these analogues induce the synthesis of a regulatory protein, antizyme, which inhibits both polyamine synthesis and the transporter they used to enter the cell. Thus this feedback system allows steady-state maintenance of effective cellular doses of such analogues. Accordingly, effective cellular levels of polyamine analogues are generally inversely related to their capacity to induce antizyme. Antizyme activity is down-regulated by interaction with several binding partners, most notably antizyme inhibitor, and at least a few tumour tissues exhibit deficiencies in antizyme expression. Our studies explore the role of antizyme induction by several polyamine analogues in their physiological response and the possibility that cell-to-cell differences in antizyme expression may contribute to variable sensitivities to these agents.

scholarly journals Amino Acid-Mediated Induction of the Basic Amino Acid-Specific Outer Membrane Porin OprD from Pseudomonas aeruginosa

1999 ◽  
Vol 181 (17) ◽  
pp. 5426-5432 ◽  
Author(s):  
Martina M. Ochs ◽  
Chung-Dar Lu ◽  
Robert E. W. Hancock ◽  
Ahmed T. Abdelal
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Regulatory Protein ◽  
Basic Amino Acid ◽  
Sole Source ◽  
Activation Mechanism ◽  
Beta Lactam ◽  
Mobility Shift ◽  
Carbon And Nitrogen

ABSTRACT Pseudomonas aeruginosa can utilize arginine and other amino acids as both carbon and nitrogen sources. Earlier studies have shown that the specific porin OprD facilitates the diffusion of basic amino acids as well as the structurally analogous beta-lactam antibiotic imipenem. The studies reported here showed that the expression of OprD was strongly induced when arginine, histidine, glutamate, or alanine served as the sole source of carbon. The addition of succinate exerted a negative effect on induction ofoprD, likely due to catabolite repression. The arginine-mediated induction was dependent on the regulatory protein ArgR, and binding of purified ArgR to its operator upstream of theoprD gene was demonstrated by gel mobility shift and DNase assays. The expression of OprD induced by glutamate as the carbon source, however, was independent of ArgR, indicating the presence of more than a single activation mechanism. In addition, it was observed that the levels of OprD responded strongly to glutamate and alanine as the sole sources of nitrogen. Thus, that the expression ofoprD is linked to both carbon and nitrogen metabolism ofPseudomonas aeruginosa.

scholarly journals The two-component system CopRS maintains femtomolar levels of free copper in the periplasm of Pseudomonas aeruginosa using a phosphatase-based mechanism

2020 ◽  
Author(s):  
Lorena Novoa-Aponte ◽  
Fernando C. Soncini ◽  
José M. Argüello
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Phosphatase Activity ◽  
Response Regulator ◽  
Redox Enzymes ◽  
Two Component System ◽  
Component System ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems ◽  
Systems Control

ABSTRACTTwo component systems control periplasmic Cu+ homeostasis in Gram-negative bacteria. In characterized systems such as Escherichia coli CusRS, upon Cu+ binding to the periplasmic sensing domain of CusS, a cytoplasmic phosphotransfer domain phosphorylates the response regulator CusR. This drives the expression of efflux transporters, chaperones, and redox enzymes to ameliorate metal toxic effects. Here, we show that the Pseudomonas aeruginosa two component sensor histidine kinase CopS exhibits a Cu-dependent phosphatase activity that maintains a non-phosphorylated CopR when the periplasmic Cu levels are below its activation threshold. Upon Cu+ binding to the sensor, the phosphatase activity is blocked and the phosphorylated CopR activates transcription of the CopRS regulon. Supporting the model, mutagenesis experiments revealed that the ΔcopS strain showed constitutive high expression of the CopRS regulon, lower intracellular Cu+ levels, and larger Cu tolerance when compared to wild type cells. The invariant phospho-acceptor residue His235 of CopS was not required for the phosphatase activity itself, but necessary for its Cu-dependency. To sense the metal, the periplasmic domain of CopS binds two Cu+ ions at its dimeric interface. Homology modeling of CopS based on CusS structure (four Ag+ binding sites) clearly explains the different binding stoichiometries in both systems. Interestingly, CopS binds Cu+/2+ with 30 × 10−15 M affinities, pointing to the absence of free (hydrated) Cu+/2+ in the periplasm.IMPORTANCECopper is a micronutrient required as cofactor in redox enzymes. When free, copper is toxic, mismetallating proteins, and generating damaging free radicals. Consequently, copper overload is a strategy that eukaryotic cells use to combat pathogens. Bacteria have developed copper sensing transcription factors to control copper homeostasis. The cell envelope is the first compartment that has to cope with copper stress. Dedicated two component systems control the periplasmic response to metal overload. This manuscript shows that the copper sensing two component system present in Pseudomonadales exhibits a signal-dependent phosphatase activity controlling the activation of the response regulator, distinct from previously described periplasmic Cu sensors. Importantly, the data show that the sensor is activated by copper levels compatible with the absence of free copper in the cell periplasm. This emphasizes the diversity of molecular mechanisms that have evolved in various bacteria to manage the copper cellular distribution.

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