Faculty Opinions recommendation of Anaerobic survival of Pseudomonas aeruginosa by pyruvate fermentation requires an Usp-type stress protein.

ABSTRACT Recently, we identified a pyruvate fermentation pathway in Pseudomonas aeruginosa sustaining anaerobic survival in the absence of alternative anaerobic respiratory and fermentative energy generation systems (M. Eschbach, K. Schreiber, K. Trunk, J. Buer, D. Jahn, and M. Schobert, J. Bacteriol. 186:4596-4604, 2004). Anaerobic long-term survival of P. aeruginosa might be essential for survival in deeper layers of a biofilm and the persistent infection of anaerobic mucus plaques in the cystic fibrosis lung. Proteome analysis of P. aeruginosa cells during a 7-day period of pyruvate fermentation revealed the induced synthesis of three enzymes involved in arginine fermentation, ArcA, ArcB, and ArcC, and the outer membrane protein OprL. Moreover, formation of two proteins of unknown function, PA3309 and PA4352, increased by factors of 72- and 22-fold, respectively. Both belong to the group of universal stress proteins (Usp). Long-term survival of a PA3309 knockout mutant by pyruvate fermentation was found drastically reduced. The oxygen-sensing regulator Anr controls expression of the P PA3309-lacZ reporter gene fusion after a shift to anaerobic conditions and further pyruvate fermentation. PA3309 expression was also found induced during the anaerobic and aerobic stationary phases. This aerobic stationary-phase induction is independent of the regulatory proteins Anr, RpoS, RelA, GacA, RhlR, and LasR, indicating a currently unknown mechanism of stationary-phase-dependent gene activation. PA3309 promoter activity was detected in the deeper layers of a P. aeruginosa biofilm using a P PA3309-gfp (green fluorescent protein gene) fusion and confocal laser-scanning microscopy. This is the first description of an Anr-dependent, anaerobically induced, and functional Usp-like protein in bacteria.

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The Pseudomonas aeruginosa Universal Stress Protein PA4352 Is Essential for Surviving Anaerobic Energy Stress

Journal of Bacteriology ◽

10.1128/jb.00308-06 ◽

2006 ◽

Vol 188 (18) ◽

pp. 6529-6538 ◽

Cited By ~ 36

Author(s):

Nelli Boes ◽

Kerstin Schreiber ◽

Elisabeth Härtig ◽

Lothar Jaensch ◽

Max Schobert

Keyword(s):

Pseudomonas Aeruginosa ◽

Persistent Infection ◽

Regulatory Protein ◽

Stress Protein ◽

Stress Conditions ◽

Anaerobic Conditions ◽

Knockout Mutant ◽

Universal Stress Protein ◽

Energy Stress ◽

Anaerobic Energy

ABSTRACT During infection of the cystic fibrosis (CF) lung, Pseudomonas aeruginosa microcolonies are embedded in the anaerobic CF mucus. This anaerobic environment seems to contribute to the formation of more robust P. aeruginosa biofilms and to an increased antibiotic tolerance and therefore promotes persistent infection. This study characterizes the P. aeruginosa protein PA4352, which is important for survival under anaerobic energy stress conditions. PA4352 belongs to the universal stress protein (Usp) superfamily and harbors two Usp domains in tandem. In Escherichia coli, Usp-type stress proteins are involved in survival during aerobic growth arrest and under various other stresses. A P. aeruginosa PA4352 knockout mutant was tested for survival under several stress conditions. We found a decrease in viability of this mutant compared to the P. aeruginosa wild type during anaerobic energy starvation caused by the missing electron acceptors oxygen and nitrate. Consistent with this phenotype under anaerobic conditions, the PA4352 knockout mutant was also highly sensitive to carbonyl cyanide m-chlorophenylhydrazone, the chemical uncoupler of the electron transport chain. Primer extension experiments identified two promoters upstream of the PA4352 gene. One promoter is activated in response to oxygen limitation by the oxygen-sensing regulatory protein Anr. The center of a putative Anr binding site was identified 41.5 bp upstream of the transcriptional start site. The second promoter is active only in the stationary phase, however, independently of RpoS, RelA, or quorum sensing. This is the second P. aeruginosa Usp-type stress protein that we have identified as important for survival under anaerobic conditions, which resembles the environment during persistent infection.

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The Pseudomonas aeruginosa complement of lactate dehydrogenases enables use of D- and L-lactate and metabolic crossfeeding

10.1101/313593 ◽

2018 ◽

Author(s):

Yu-Cheng Lin ◽

William-Cole Cornell ◽

Alexa Price-Whelan ◽

Lars E.P. Dietrich

Keyword(s):

Pseudomonas Aeruginosa ◽

Metabolic Pathways ◽

Bacterial Colonization ◽

Expression Patterns ◽

Chronic Infections ◽

Lactate Oxidation ◽

Growth And Survival ◽

Liquid Cultures ◽

Pyruvate Fermentation ◽

Lactate Dehydrogenases

ABSTRACTPseudomonas aeruginosa is the most common cause of chronic, biofilm-based lung infections in patients with cystic fibrosis (CF). Sputum from patients with CF has been shown to contain oxic and hypoxic subzones as well as millimolar concentrations of lactate. Here, we describe the physiological roles and expression patterns of P. aeruginosa lactate dehydrogenases in the contexts of different growth regimes. P. aeruginosa produces four enzymes annotated as lactate dehydrogenases, three of which are known to contribute to anaerobic or aerobic metabolism in liquid cultures. These three are LdhA, which reduces pyruvate to D-lactate during anaerobic survival, and LldE and LldD, which oxidize D-lactate and L-lactate, respectively, during aerobic growth. We demonstrate that the fourth enzyme, LldA, performs redundant L-lactate oxidation during growth in aerobic cultures in both a defined MOPS-based medium and synthetic CF sputum medium. However, LldA differs from LldD in that its expression is induced specifically by the L-enantiomer of lactate. We also show that all four enzymes perform functions in colony biofilms that are similar to their functions in liquid cultures. Finally, we provide evidence that the enzymes LdhA and LldE have the potential to support metabolic cross-feeding in biofilms, where LdhA can catalyze the production of D-lactate in the anaerobic zone that is then used as a substrate in the aerobic zone. Together, these observations further our understanding of the metabolic pathways that can contribute to P. aeruginosa growth and survival during CF lung infection.IMPORTANCELactate is thought to serve as a carbon and energy source during chronic infections. Sites of bacterial colonization can contain two enantiomers of lactate: the L-form, generally produced by the host, and the D-form, which is usually produced by bacteria including the pulmonary pathogen Pseudomonas aeruginosa. Here, we characterize P. aeruginosa’s set of four enzymes that it can use to interconvert pyruvate and lactate, the functions of which depend on the availability of oxygen and specific enantiomers of lactate. We also show that anaerobic pyruvate fermentation triggers production of the aerobic D-lactate dehydrogenase in both liquid cultures and biofilms, thereby enabling metabolic cross-feeding of lactate over time and space between subpopulations of cells. These metabolic pathways could contribute to P. aeruginosa growth and survival in the lung.

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SpoT-Triggered Stringent Response Controls usp Gene Expression in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00600-08 ◽

2008 ◽

Vol 190 (21) ◽

pp. 7189-7199 ◽

Cited By ~ 29

Author(s):

Nelli Boes ◽

Kerstin Schreiber ◽

Max Schobert

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Stationary Phase ◽

Stress Proteins ◽

Stringent Response ◽

Triple Mutant ◽

Pyruvate Fermentation ◽

Anaerobic Energy ◽

In The Wild ◽

Layer Chromatography

ABSTRACT The universal stress proteins (Usps) UspK (PA3309) and UspN (PA4352) of Pseudomonas aeruginosa are essential for surviving specific anaerobic energy stress conditions such as pyruvate fermentation and anaerobic stationary phase. Expression of the respective genes is under the control of the oxygen-sensing regulator Anr. In this study we investigated the regulation of uspN and three additional P. aeruginosa usp genes: uspL (PA1789), uspM (PA4328), and uspO (PA5027). Anr induces expression of these genes in response to anaerobic conditions. Using promoter-lacZ fusions, we showed that P uspL -lacZ, P uspM -lacZ, and P uspO -lacZ were also induced in stationary phase as described for P uspN -lacZ. However, stationary phase gene expression was abolished in the P. aeruginosa triple mutant Δanr ΔrelA ΔspoT. The relA and spoT genes encode the regulatory components of the stringent response. We determined pppGpp and ppGpp levels using a thin-layer chromatography approach and detected the accumulation of ppGpp in the wild type and the ΔrelA mutant in stationary phase, indicating a SpoT-derived control of ppGpp accumulation. Additional investigation of stationary phase in LB medium revealed that alkaline pH values are involved in the regulatory process of ppGpp accumulation.

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75 Anaerobic survival of Pseudomonas aeruginosa requires the Usp-like stress protein PA3309

Journal of Cystic Fibrosis ◽

10.1016/s1569-1993(06)80066-4 ◽

2006 ◽

Vol 5 ◽

pp. S17

Author(s):

K. Schreiber ◽

N. Bös ◽

T. Bjarnsholt ◽

M. Hentzer ◽

M. Givskov ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Stress Protein

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Long-Term Anaerobic Survival of the Opportunistic Pathogen Pseudomonas aeruginosa via Pyruvate Fermentation

Journal of Bacteriology ◽

10.1128/jb.186.14.4596-4604.2004 ◽

2004 ◽

Vol 186 (14) ◽

pp. 4596-4604 ◽

Cited By ~ 149

Author(s):

Martin Eschbach ◽

Kerstin Schreiber ◽

Katharina Trunk ◽

Jan Buer ◽

Dieter Jahn ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Opportunistic Pathogen ◽

Integration Host Factor ◽

Anaerobic Growth ◽

Nitrate Respiration ◽

Term Survival ◽

Long Term Survival ◽

Pyruvate Fermentation ◽

Activity Measurements

ABSTRACT Denitrification and arginine fermentation are central metabolic processes performed by the opportunistic pathogen Pseudomonas aeruginosa during biofilm formation and infection of lungs of patients with cystic fibrosis. Genome-wide searches for additional components of the anaerobic metabolism identified potential genes for pyruvate-metabolizing NADH-dependent lactate dehydrogenase (ldhA), phosphotransacetylase (pta), and acetate kinase (ackA). While pyruvate fermentation alone does not sustain significant anaerobic growth of P. aeruginosa, it provides the bacterium with the metabolic capacity for long-term survival of up to 18 days. Detected conversion of pyruvate to lactate and acetate is dependent on the presence of intact ldhA and ackA-pta loci, respectively. DNA microarray studies in combination with reporter gene fusion analysis and enzyme activity measurements demonstrated the anr- and ihfA-dependent anaerobic induction of the ackA-pta promoter. Potential Anr and integration host factor binding sites were localized. Pyruvate-dependent anaerobic long-term survival was found to be significantly reduced in anr and ihfA mutants. No obvious ldhA regulation by oxygen tension was observed. Pyruvate fermentation is pH dependent. Nitrate respiration abolished pyruvate fermentation, while arginine fermentation occurs independently of pyruvate utilization.

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