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.

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 oxygensensing 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.

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 ⌬anr ⌬relA ⌬spoT. 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 ⌬relA 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.