scholarly journals Pseudomonas aeruginosa lasR mutant fitness in microoxia is supported by an Anr-regulated oxygen-binding hemerythrin

2019 ◽  
Author(s):  
Michelle E. Clay ◽  
John H. Hammond ◽  
Fangfang Zhong ◽  
Xiaolei Chen ◽  
Caitlin H. Kowalski ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcription Factor ◽  
Quorum Sensing ◽  
Opportunistic Infections ◽  
Oxygen Binding ◽  
Loss Of Function ◽  
Low Oxygen ◽  
Metabolomics Data ◽  
High Affinity ◽  
Oxygen Conditions

AbstractPseudomonas aeruginosa strains with loss-of-function mutations in the transcription factor are frequently encountered in the clinic and the environment. Among the characteristics common to LasR-defective (LasR-) strains is increased activity of the transcription factor Anr, relative to their LasR+ counterparts, in low oxygen conditions. One of the Anr-regulated genes that was highly induced in the LasR-strains encoded a putative oxygen-binding hemerythrin encoded by PA14_42860 (PA1673) which we named mhr for microoxic hemerythrin. Purified P. aeruginosa Mhr protein contained the predicted di-iron center and binds oxygen with a Kd of 1 µM. Both Anr and Mhr were necessary for fitness in lasR+ and lasR mutant strains in colony biofilms grown in microoxic conditions, and the effects were more striking in the lasR mutant. Among genes in the Anr regulon, mhr was most closely co-regulated with the Anr-controlled high affinity cytochrome c oxidase genes and in the absence of high affinity cytochrome c oxidase activity, deletion of mhr no longer caused a fitness disadvantage suggesting that Mhr works in concert with microoxic respiration. We demonstrate that Anr and Mhr contribute to LasR-strain fitness even in the normoxic biofilm conditions, and metabolomics data indicate that in a lasR mutant, expression of Anr-regulated mhr leads to differences in metabolism in cells grown on LB and artificial sputum medium. Together these data indicate that increased Anr activity in microoxically-grown lasR mutants confers an advantage in part for its regulation of the O2 binding protein Mhr.SignificancePseudomonas aeruginosa, a versatile bacterium that both lives in environmental habitats and causes life-threatening opportunistic infections, uses quorum sensing to coordinate gene expression with cell density. The lasR gene, which encodes a quorum sensing regulator, is often deleteriously mutated in clinical isolates. Interestingly, LasR-strains have high activity of the oxygen-sensitive transcription factor Anr in microoxic conditions. This report identifies and characterizes an Anr-regulated microoxic hemerythrin that reversibly binds oxygen. We showed both anr and mhr are critical to fitness in microoxia, and these genes uniquely benefit LasR-strains in normoxia. Our findings enrich our understanding of the success of P. aeruginosa as a pulmonary resident through its propensity to lose LasR functionality in the context of low-oxygen infection environments.

scholarly journals Pseudomonas aeruginosa lasR mutant fitness in microoxia is supported by an Anr-regulated oxygen-binding hemerythrin

2020 ◽  
Vol 117 (6) ◽  
pp. 3167-3173 ◽  
Author(s):  
Michelle E. Clay ◽  
John H. Hammond ◽  
Fangfang Zhong ◽  
Xiaolei Chen ◽  
Caitlin H. Kowalski ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcription Factor ◽  
Oxygen Binding ◽  
Loss Of Function ◽  
Low Oxygen ◽  
Metabolomics Data ◽  
High Affinity ◽  
Oxygen Conditions ◽  
Increased Activity ◽  
Iron Center

Pseudomonas aeruginosa strains with loss-of-function mutations in the transcription factor LasR are frequently encountered in the clinic and the environment. Among the characteristics common to LasR-defective (LasR−) strains is increased activity of the transcription factor Anr, relative to their LasR+ counterparts, in low-oxygen conditions. One of the Anr-regulated genes found to be highly induced in LasR− strains was PA14_42860 (PA1673), which we named mhr for microoxic hemerythrin. Purified P. aeruginosa Mhr protein contained the predicted di-iron center and bound molecular oxygen with an apparent Kd of ∼1 µM. Both Anr and Mhr were necessary for fitness in lasR+ and lasR mutant strains in colony biofilms grown in microoxic conditions, and the effects were more striking in the lasR mutant. Among genes in the Anr regulon, mhr was most closely coregulated with the Anr-controlled high-affinity cytochrome c oxidase genes. In the absence of high-affinity cytochrome c oxidases, deletion of mhr no longer caused a fitness disadvantage, suggesting that Mhr works in concert with microoxic respiration. We demonstrate that Anr and Mhr contribute to LasR− strain fitness even in biofilms grown in normoxic conditions. Furthermore, metabolomics data indicate that, in a lasR mutant, expression of Anr-regulated mhr leads to differences in metabolism in cells grown on lysogeny broth or artificial sputum medium. We propose that increased Anr activity leads to higher levels of the oxygen-binding protein Mhr, which confers an advantage to lasR mutants in microoxic conditions.

scholarly journals Links between Anr and Quorum Sensing in Pseudomonas aeruginosa Biofilms

2015 ◽  
Vol 197 (17) ◽  
pp. 2810-2820 ◽  
Author(s):  
John H. Hammond ◽  
Emily F. Dolben ◽  
T. Jarrod Smith ◽  
Sabin Bhuju ◽  
Deborah A. Hogan
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcription Factor ◽  
Quorum Sensing ◽  
Cellular Response ◽  
Iron Acquisition ◽  
Laboratory Strain ◽  
Interspecies Interactions ◽  
Low Oxygen ◽  
Chronic Infections ◽  
Content Type

ABSTRACTInPseudomonas aeruginosa, the transcription factor Anr controls the cellular response to low oxygen or anoxia. Anr activity is high in oxygen-limited environments, including biofilms and populations associated with chronic infections, and Anr is necessary for persistence in a model of pulmonary infection. In this study, we characterized the Anr regulon in biofilm-grown cells at 1% oxygen in the laboratory strain PAO1 and in a quorum sensing (QS)-deficient clinical isolate, J215. As expected, transcripts related to denitrification, arginine fermentation, high-affinity cytochrome oxidases, and CupA fimbriae were lower in the Δanrderivatives. In addition, we observed that transcripts associated with quorum sensing regulation, iron acquisition and storage, type VI secretion, and the catabolism of aromatic compounds were also differentially expressed in the Δanrstrains. Prior reports have shown that quorum sensing-defective mutants have higher levels of denitrification, and we found that multiple Anr-regulated processes, including denitrification, were strongly inversely proportional to quorum sensing in both transcriptional and protein-based assays. We also found that in LasR-defective strains but not their LasR-intact counterparts, Anr regulated the production of the 4-hydroxy-2-alkylquinolines, which play roles in quorum sensing and interspecies interactions. These data show that Anr was required for the expression of important metabolic pathways in low-oxygen biofilms, and they reveal an expanded and compensatory role for Anr in the regulation of virulence-related genes in quorum sensing mutants, such as those commonly isolated from infections.IMPORTANCEPseudomonas aeruginosacauses acute ocular, soft tissue, and pulmonary infections, as well as chronic infections in the airways of cystic fibrosis patients.P. aeruginosauses quorum sensing (QS) to regulate virulence, but mutations in the gene encoding the master regulator of QS,lasR, are frequently observed in clinical isolates. We demonstrated that the regulon attributed to Anr, an oxygen-sensitive transcription factor, was more highly expressed inlasRmutants. Furthermore, we show that Anr regulates the production of several different secreted factors inlasRmutants. These data demonstrate the importance of Anr in naturally occurring quorum sensing mutants in the context of chronic infections.

scholarly journals Intra-species signaling between Pseudomonas aeruginosa genotypes increases production of quorum sensing controlled virulence factors

2020 ◽  
Author(s):  
Dallas L. Mould ◽  
Nico J. Botelho ◽  
Deborah A. Hogan
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcription Factor ◽  
Quorum Sensing ◽  
Opportunistic Pathogen ◽  
Diverse Population ◽  
Loss Of Function ◽  
Wild Type ◽  
Natural Settings ◽  
Citrate Release ◽  
Virulence Factor Production

AbstractThe opportunistic pathogen Pseudomonas aeruginosa damages hosts through the production of diverse secreted products, many of which are regulated by quorum sensing. The lasR gene, which encodes a central quorum-sensing regulator, is frequently mutated, and loss of LasR function impairs the activity of downstream regulators RhlR and PqsR. We found that in diverse models, the presence of P. aeruginosa wild type causes LasR loss-of-function strains to hyperproduce RhlR/I-regulated antagonistic factors, and autoinducer production by the wild type is not required for this effect. We uncovered a reciprocal interaction between isogenic wild type and lasR mutant pairs wherein the iron-scavenging siderophore pyochelin, specifically produced by the lasR mutant, induces citrate release and cross-feeding from wild type. Citrate stimulates RhlR signaling and RhlI levels in LasR-but not in LasR+ strains, and the interactions occur in diverse media. Co-culture interactions between strains that differ by the function of a single transcription factor may explain worse outcomes associated with mixtures of LasR+ and LasR loss-of-function strains. More broadly, this report illustrates how interactions within a genotypically diverse population, similar to those that frequently develop in natural settings, can promote net virulence factor production.

scholarly journals Molecular Insights into Function and Competitive Inhibition of Pseudomonas aeruginosa Multiple Virulence Factor Regulator

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Tomoe Kitao ◽  
Francois Lepine ◽  
Seda Babloudi ◽  
Frederick Walte ◽  
Stefan Steinbacher ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcription Factor ◽  
Drug Discovery ◽  
Dna Binding ◽  
Quorum Sensing ◽  
Binding Domain ◽  
Antimicrobial Drug ◽  
Single Amino Acid ◽  
Persistent Infections ◽  
New Approaches

ABSTRACT New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by multidrug-resistant (MDR) bacteria. Multiple virulence factor regulator (MvfR or PqsR), a Pseudomonas aeruginosa quorum sensing transcription factor, regulates functions important in both acute and persistent infections. Recently identified non-ligand-based benzamine-benzimidazole (BB) inhibitors of MvfR suppress both acute and persistent P. aeruginosa infections in mice without perturbing bacterial growth. Here, we elucidate the crystal structure of the MvfR ligand binding domain (LBD) in complex with one potent BB inhibitor, M64. Structural analysis indicated that M64 binds, like native ligands, to the MvfR hydrophobic cavity. A hydrogen bond and pi interaction were found to be important for MvfR-M64 affinity. Surface plasmon resonance analysis demonstrated that M64 is a competitive inhibitor of MvfR. Moreover, a protein engineering approach revealed that Gln194 and Tyr258 are critical for the interaction between MvfR and M64. Random mutagenesis of the full-length MvfR protein identified a single-amino-acid substitution, I68F, at a DNA binding linker domain that confers M64 insensitivity. In the presence of M64, I68F but not the wild-type (WT) MvfR protein retained DNA binding ability. Our findings strongly suggest that M64 promotes conformational change at the DNA binding domain of MvfR and that the I68F mutation may compensate for this change, indicating allosteric inhibition. This work provides critical new insights into the molecular mechanism of MvfR function and inhibition that could aid in the optimization of anti-MvfR compounds and improve our understanding of MvfR regulation. IMPORTANCE Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes serious acute, persistent, and relapsing infections. New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by this pathogen. The Pseudomonas aeruginosa quorum sensing transcription factor MvfR regulates functions important in both acute and persistent infections. We used recently identified inhibitors of MvfR to perform structural studies and reveal important insights that would benefit the optimization of anti-MvfR compounds. Altogether, the results reported here provide critical detailed mechanistic insights into the function of MvfR domains that may benefit the optimization of the chemical, pharmacological, and safety properties of MvfR antagonist series.

scholarly journals Modulation of Pseudomonas aeruginosa Quorum Sensing by Glutathione

2019 ◽  
Vol 201 (9) ◽  
Author(s):  
Hui Zhou ◽  
Meizhen Wang ◽  
Nicole E. Smalley ◽  
Maxim Kostylev ◽  
Amy L. Schaefer ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Quorum Sensing ◽  
Cell Density ◽  
Redox State ◽  
Gene Activation ◽  
Cellular Redox ◽  
Content Type ◽  
Cellular Redox State

ABSTRACT Pseudomonas aeruginosa uses quorum sensing (QS) to regulate the production of a battery of secreted products. At least some of these products are shared among the population and serve as public goods. When P. aeruginosa is grown on casein as the sole carbon and energy source, the QS-induced extracellular protease elastase is required for growth. We isolated a P. aeruginosa variant, which showed increased production of QS-induced factors after repeated transfers in casein broth. This variant, P. aeruginosa QS*, had a mutation in the glutathione synthesis gene gshA. We describe several experiments that show a gshA coding variant and glutathione affect the QS response. The P. aeruginosa QS transcription factor LasR has a redox-sensitive cysteine (C79). We report that GshA variant cells with a LasR C79S substitution show a similar QS response to that of wild-type P. aeruginosa. Surprisingly, it is not LasR but the QS transcription factor RhlR that is more active in bacteria containing the variant gshA. Our results demonstrate that QS integrates information about cell density and the cellular redox state via glutathione levels. IMPORTANCE Pseudomonas aeruginosa and other bacteria coordinate group behaviors using a chemical communication system called quorum sensing (QS). The QS system of P. aeruginosa is complex, with several regulators and signals. We show that decreased levels of glutathione lead to increased gene activation in P. aeruginosa, which did not occur in a strain carrying the redox-insensitive variant of a transcription factor. The ability of P. aeruginosa QS transcription factors to integrate information about cell density and cellular redox state shows these transcription factors can fine-tune levels of the gene products they control in response to at least two types of signals or cues.

scholarly journals Pseudomonas aeruginosa Ethanol Oxidation by AdhA in Low-Oxygen Environments

2019 ◽  
Vol 201 (23) ◽  
Author(s):  
Alex W. Crocker ◽  
Colleen E. Harty ◽  
John H. Hammond ◽  
Sven D. Willger ◽  
Pedro Salazar ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alcohol Dehydrogenase ◽  
Ethanol Oxidation ◽  
Physiological Role ◽  
Wild Type ◽  
Low Oxygen ◽  
Content Type ◽  
Anoxic Conditions ◽  
Oxygen Conditions ◽  
Acetate Accumulation

ABSTRACT Pseudomonas aeruginosa has a broad metabolic repertoire that facilitates its coexistence with different microbes. Many microbes secrete products that P. aeruginosa can then catabolize, including ethanol, a common fermentation product. Here, we show that under oxygen-limiting conditions P. aeruginosa utilizes AdhA, an NAD-linked alcohol dehydrogenase, as a previously undescribed means for ethanol catabolism. In a rich medium containing ethanol, AdhA, but not the previously described PQQ-linked alcohol dehydrogenase, ExaA, oxidizes ethanol and leads to the accumulation of acetate in culture supernatants. AdhA-dependent acetate accumulation and the accompanying decrease in pH promote P. aeruginosa survival in LB-grown stationary-phase cultures. The transcription of adhA is elevated by hypoxia and under anoxic conditions, and we show that it is regulated by the Anr transcription factor. We have shown that lasR mutants, which lack an important quorum sensing regulator, have higher levels of Anr-regulated transcripts under low-oxygen conditions than their wild-type counterparts. Here, we show that a lasR mutant, when grown with ethanol, has an even larger decrease in pH than the wild type (WT) that is dependent on both anr and adhA. The large increase in AdhA activity is similar to that of a strain expressing a hyperactive Anr-D149A variant. Ethanol catabolism in P. aeruginosa by AdhA supports growth on ethanol as a sole carbon source and electron donor in oxygen-limited settings and in cells growing by denitrification under anoxic conditions. This is the first demonstration of a physiological role for AdhA in ethanol oxidation in P. aeruginosa. IMPORTANCE Ethanol is a common product of microbial fermentation, and the Pseudomonas aeruginosa response to and utilization of ethanol are relevant to our understanding of its role in microbial communities. Here, we report that the putative alcohol dehydrogenase AdhA is responsible for ethanol catabolism and acetate accumulation under low-oxygen conditions and that it is regulated by Anr.

scholarly journals GidA Posttranscriptionally Regulates rhl Quorum Sensing in Pseudomonas aeruginosa

2009 ◽  
Vol 191 (18) ◽  
pp. 5785-5792 ◽  
Author(s):  
Rashmi Gupta ◽  
Timothy R. Gobble ◽  
Martin Schuster
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Flavin Adenine Dinucleotide ◽  
Skim Milk ◽  
Opportunistic Pathogen ◽  
Homoserine Lactone ◽  
Trna Modification ◽  
Loss Of Function ◽  
Novel Genes ◽  
Lasa Protease

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa utilizes two interconnected acyl-homoserine lactone quorum-sensing (acyl-HSL QS) systems, LasRI and RhlRI, to regulate the expression of hundreds of genes. The QS circuitry itself is integrated into a complex network of regulation by other factors. However, our understanding of this network is still unlikely to be complete, as a comprehensive, saturating approach to identifying regulatory components has never been attempted. Here, we utilized a nonredundant P. aeruginosa PA14 transposon library to identify additional genes that regulate QS at the level of LasRI/RhlRI. We initially screened all 5,459 mutants for loss of function in one QS-controlled trait (skim milk proteolysis) and then rescreened attenuated candidates for defects in other QS phenotypes (LasA protease, rhamnolipid, and pyocyanin production) to exclude mutants defective in functions other than QS. We identified several known and novel genes, but only two novel genes, gidA and pcnB, affected all of the traits assayed. We characterized gidA, which exhibited the most striking QS phenotypes, further. This gene is predicted to encode a conserved flavin adenine dinucleotide-binding protein involved in tRNA modification. Inactivation of the gene primarily affected rhlR-dependent QS phenotypes such as LasA, pyocyanin, and rhamnolipid production. GidA affected RhlR protein but not transcript levels and also had no impact on LasR and acyl-HSL production. Overexpression of rhlR in a gidA mutant partially restored QS-dependent phenotypes. Taken together, these results indicate that GidA selectively controls QS gene expression posttranscriptionally via RhlR-dependent and -independent pathways.

Iron-binding compounds impair Pseudomonas aeruginosa biofilm formation, especially under anaerobic conditions

2009 ◽  
Vol 58 (6) ◽  
pp. 765-773 ◽  
Author(s):  
Che Y. O'May ◽  
Kevin Sanderson ◽  
Louise F. Roddam ◽  
Sylvia M. Kirov ◽  
David W. Reid
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Low Oxygen ◽  
Anaerobic Conditions ◽  
Chronic Infections ◽  
Strain Pao1 ◽  
Biofilm Inhibition ◽  
Oxygen Conditions ◽  
Biofilm Development ◽  
Chelating Compounds

The success of Pseudomonas aeruginosa in cystic fibrosis (CF) and other chronic infections is largely attributed to its ability to grow in antibiotic-resistant biofilm communities. This study investigated the effects of limiting iron levels as a strategy for preventing/disrupting P. aeruginosa biofilms. A range of synthetic and naturally occurring iron-chelating agents were examined. Biofilm development by P. aeruginosa strain PAO1 and CF sputum isolates from chronically infected individuals was significantly decreased by iron removal under aerobic atmospheres. CF strains formed poor biofilms under anaerobic conditions. Strain PAO1 was also tested under anaerobic conditions. Biofilm formation by this model strain was almost totally prevented by several of the chelators tested. The ability of synthetic chelators to impair biofilm formation could be reversed by iron addition to cultures, providing evidence that these effective chelating compounds functioned by directly reducing availability of iron to P. aeruginosa. In contrast, the biological chelator lactoferrin demonstrated enhanced anti-biofilm effects as iron supplementation increased. Hence biofilm inhibition by lactoferrin appeared to occur through more complex mechanisms to those of the synthetic chelators. Overall, our results demonstrate the importance of iron availability to biofilms and that iron chelators have potential as adjunct therapies for preventing biofilm development, especially under low oxygen conditions such as encountered in the chronically infected CF lung.

scholarly journals ThePseudomonas aeruginosaOrphan Quorum Sensing Signal Receptor QscR Regulates Global Quorum Sensing Gene Expression by Activating a Single Linked Operon

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Fengming Ding ◽  
Ken-Ichi Oinuma ◽  
Nicole E. Smalley ◽  
Amy L. Schaefer ◽  
Omar Hamwy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Transcription Factor ◽  
Quorum Sensing ◽  
Cell Communication ◽  
Homoserine Lactone ◽  
Acyl Homoserine Lactone ◽  
Null Mutant ◽  
Content Type ◽  
Single Operon

ABSTRACTPseudomonas aeruginosauses two acyl-homoserine lactone signals and two quorum sensing (QS) transcription factors, LasR and RhlR, to activate dozens of genes. LasR responds toN-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and RhlR toN-butanoyl-homoserine lactone (C4-HSL). There is a thirdP. aeruginosaacyl-homoserine-lactone-responsive transcription factor, QscR, which acts to dampen or delay activation of genes by LasR and RhlR by an unknown mechanism. To better understand the role of QscR inP. aeruginosaQS, we performed a chromatin immunoprecipitation analysis, which showed this transcription factor bound the promoter of only a single operon of three genes linked toqscR, PA1895 to PA1897. Other genes that appear to be regulated by QscR in transcriptome studies were not direct targets of QscR. Deletion of PA1897 recapitulates the early QS activation phenotype of a QscR-null mutant, and the phenotype of a QscR-null mutant was complemented by PA1895-1897 but not by PA1897 alone. We conclude that QscR acts to modulate quorum sensing through regulation of a single operon, apparently raising the QS threshold of the population and providing a “brake” on QS autoinduction.IMPORTANCEQuorum sensing, a cell-cell communication system, is broadly distributed among bacteria and is commonly used to regulate the production of shared products. An important consequence of quorum sensing is a delay in production of certain products until the population density is high. The bacteriumPseudomonas aeruginosahas a particularly complicated quorum sensing system involving multiple signals and receptors. One of these receptors, QscR, downregulates gene expression, unlike the other receptors inP. aeruginosa. QscR does so by inducing the expression of a single operon whose function provides an element of resistance to a population reaching a quorum. This finding has importance for design of quorum sensing inhibitory strategies and can also inform design of synthetic biological circuits that use quorum sensing receptors to regulate gene expression.

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