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 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 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.

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 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 Pseudomonas aeruginosaethanol oxidation by AdhA in low oxygen environments

2019 ◽  
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 ◽  
Anoxic Conditions ◽  
Oxygen Conditions ◽  
Acetate Accumulation ◽  
Culture Supernatants

AbstractPseudomonas aeruginosahas a broad metabolic repertoire that facilitates its co-existence with different microbes. Many microbes secrete products thatP. aeruginosacan then catabolize, including ethanol, a common fermentation product. Here, we show that under oxygen limiting conditionsP. aeruginosautilizes 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, promotesP. aeruginosasurvival in LB-grown stationary phase cultures. The transcription ofadhAis elevated by hypoxia and in anoxic conditions, and we show that it is regulated by the Anr transcription factor. We have shown thatlasRmutants have higher levels of Anr-regulated transcripts in low oxygen conditions compared to their wild type counterparts. Here, we show that alasRmutant, when grown with ethanol, has an even larger decrease in pH than WT that is dependent on bothanrandadhA. The large increase in AdhA activity similar to that of a strain expressing a hyperactive Anr-D149A variant. Ethanol catabolism inP. aeruginosaby AdhA supports growth on ethanol as a sole carbon source and electron donor in oxygen-limited settings and in cells growing by denitrification in anoxic conditions. This is the first demonstration of a physiological role for AdhA in ethanol oxidation inP. aeruginosa.ImportanceEthanol is a common product of microbial fermentation, and thePseudomonas aeruginosaresponse to and utilization of ethanol is 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 in low oxygen conditions and that it is regulated by Anr.

scholarly journals Global regulator Anr represses PlcH phospholipase activity in Pseudomonas aeruginosa when oxygen is limiting

Microbiology ◽  
2014 ◽  
Vol 160 (10) ◽  
pp. 2215-2225 ◽  
Author(s):  
Angelyca A. Jackson ◽  
Emily F. Daniels ◽  
John H. Hammond ◽  
Sven D. Willger ◽  
Deborah A. Hogan
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Phospholipase C ◽  
Consensus Sequence ◽  
Mammalian Host ◽  
Low Oxygen ◽  
Hypoxic Conditions ◽  
Colonization Factor ◽  
Culture Model ◽  
A Cell ◽  
Increased Activity

Haemolytic phospholipase C (PlcH) is a potent virulence and colonization factor that is expressed at high levels by Pseudomonas aeruginosa within the mammalian host. The phosphorylcholine liberated from phosphatidylcholine and sphingomyelin by PlcH is further catabolized into molecules that both support growth and further induce plcH expression. We have shown previously that the catabolism of PlcH-released choline leads to increased activity of Anr, a global transcriptional regulator that promotes biofilm formation and virulence. Here, we demonstrated the presence of a negative feedback loop in which Anr repressed plcH transcription and we proposed that this regulation allowed for PlcH levels to be maintained in a way that promotes productive host–pathogen interactions. Evidence for Anr-mediated regulation of PlcH came from data showing that growth at low oxygen (1 %) repressed PlcH abundance and plcH transcription in the WT, and that plcH transcription was enhanced in an Δanr mutant. The plcH promoter featured an Anr consensus sequence that was conserved across all P. aeruginosa genomes and mutation of conserved nucleotides within the Anr consensus sequence increased plcH expression under hypoxic conditions. The Anr-regulated transcription factor Dnr was not required for this effect. The loss of Anr was not sufficient to completely derepress plcH transcription as GbdR, a positive regulator of plcH, was required for expression. Overexpression of Anr was sufficient to repress plcH transcription even at 21 % oxygen. Anr repressed plcH expression and phospholipase C activity in a cell culture model for P. aeruginosa–epithelial cell interactions.

scholarly journals Dynamic Oxygen Conditions Promote the Translocation of HIF-1α to the Nucleus in Mouse Blastocysts

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Jungwon Choi ◽  
Wontae Kim ◽  
Hyejin Yoon ◽  
Jaewang Lee ◽  
Jin Hyun Jun
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Protein Translocation ◽  
Cell Number ◽  
Preimplantation Embryos ◽  
Low Oxygen ◽  
Mammalian Embryo ◽  
Oxygen Conditions

Oxygen tension is one of the most critical factors for mammalian embryo development and its survival. The HIF protein is an essential transcription factor that activated under hypoxic conditions. In this study, we evaluated the effect of dynamic oxygen conditions on the expression of embryonic genes and translocation of hypoxia-inducible factor-1α (HIF-1α) in cultured mouse blastocysts. Two-pronuclear (2PN) zygotes harvested from ICR mice were subjected to either high oxygen (HO; 20%), low oxygen (LO; 5%), or dynamic oxygen (DO; 5% to 2%) conditions. In the DO group, PN zygotes were cultured in 5% O2 from days 1 to 3 and then in 2% O2 till day 5 after hCG injection. On day 5, the percentage of blastocysts in the cultured embryos from each group was estimated, and the embryos were also subjected to immunocytochemical and gene expression analysis. We found that the percentage of blastocysts was similar among the experimental groups; however, the percentage of hatching blastocysts in the DO and LO groups was significantly higher than that in the HO group. The total cell number of blastocysts in the DO group was significantly higher than that of both the HO and LO groups. Further, gene expression analysis revealed that the expression of genes related to the embryonic development was significantly higher in the DO group than that in the HO and LO groups. Interestingly, HIF-1α mRNA expression did not significantly differ; however, HIF-1α protein translocation from the cytoplasm to the nucleus was significantly higher in the DO group than in the HO and LO groups. Our study suggests that dynamic oxygen concentrations increase the developmental capacity in mouse preimplantation embryos through activation of the potent transcription factor HIF-1α.

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.

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