The Pseudomonas aeruginosa phosphodiesterase gene nbdA is transcriptionally regulated by RpoS and AmrZ

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen causing serious infections in immune compromised persons. These infections are difficult to erase with antibiotics, due to the formation of biofilms. The biofilm lifecycle is regulated by the second messenger molecule c-di-GMP (bis-3,5-cyclic di-guanosine monophosphate). P. aeruginosa encodes 40 genes for enzymes presumably involved in the biosynthesis and degradation of c-di-GMP. A tight regulation of expression, subcellular localized function and protein interactions control the activity of these enzymes. In this work we elucidated the transcriptional regulation of the gene encoding the membrane-bound phosphodiesterase NbdA. We previously reported a transcriptional and posttranslational role of nitric oxide (NO) on nbdA and its involvement in biofilm dispersal. NO is released from macrophages during infections but can also be produced by P. aeruginosa itself during anaerobic denitrification. Recently however, contradictory results about the role of NbdA within NO-induced biofilm dispersal were published. Therefore, the transcriptional regulation of nbdA was reevaluated to obtain insights into this discrepancy. Determination of the transcriptional start site of nbdA by 5’-RACE and subsequent identification of the promoter region revealed a shortened open reading frame (ORF) in contrast to the annotated one. In addition, putative binding sites for RpoS and AmrZ were discovered in the newly defined promoter region. Employing chromosomally integrated transcriptional lacZ reporter gene fusions demonstrated a RpoS-dependent activation and AmrZ repression of nbdA transcription. In order to investigate the impact of NO on nbdA transcription, conditions mimicking exogenous and endogenous NO were applied. While neither exogenous nor endogenous NO had an influence on nbdA promoter activity, deletion of the nitrite reductase gene nirS strongly increased nbdA transcription independently of its enzymatic activity during denitrification. The latter supports a role of NirS in P. aeruginosa apart from its enzymatic function. IMPORTANCE The opportunistic pathogen Pseudomonas aeruginosa possesses a network of genes encoding proteins for the turnover of the second messenger c-di-GMP involved in regulating-among others-the lifestyle switch between planktonic, motile cells and sessile biofilms. Insight into the transcriptional regulation of these genes is important for the understanding of the protein function within the cell. Determination of the transcriptional start site of the phosphodiesterase gene nbdA revealed a new promoter region and consequently a shortened open reading frame for the corresponding protein. Binding sites for RpoS and AmrZ were identified in silico and confirmed experimentally. Previously reported regulation by nitric oxide was reevaluated and a strong influence of the moonlighting protein NirS identified.