Gene expression in Pseudomonas aeruginosa: Evidence of iron override effects on quorum sensing and biofilm-specific gene regulation

Prior studies established that the Pseudomonas aeruginosa oxidative stress response is influenced by iron availability, whereas more recent evidence d emonstrated that it was also controlled by quorum sensing (QS) regulatory c ircuitry. In the present study, sodA (encoding manganese-cofactored superox ide dismutase [Mn-SOD]) and Mn-SOD were used as a reporter gene and endogen ous reporter enzyme, respectively, to reexamine control mechanisms that gov ern the oxidative stress response and to better understand how QS and a nut rient stress response interact or overlap in this bacterium. In cells grown in Trypticase soy broth (TSB), Mn-SOD was found in wild-type stationary-ph ase planktonic cells but not in a lasI or lasR mutant. However, Mn-SOD acti vity was completely suppressed in the wild-type strain when TSB was supplem ented with iron. Reporter gene studies indicated that sodA transcription co uld be variably induced in iron-starved cells of all three strains, dependi ng on growth stage, Iron starvation induction of sodA was greatest in the w ild-type strain and Least in the lasR mutant and was maximal in stationary- phase cells. Reporter experiments in the wild-type strain showed increased lasI::lacZ transcription in response to iron limitation, whereas the expres sion level in the las mutants was minimal and iron starvation induction of lasI::lacZ did not occur. Studies comparing Mn-SOD activity in P. aeruginos a biofilms and planktonic cultures were also initiated, In wild-type biofil ms, Mn-SOD was not detected until after 6 days, although in iron-limited wi ld-type biofilms Mn-SOD was detected within the initial 24 h of biofilm est ablishment and formation. Unlike planktonic bacteria, Mn-SOD was constituti ve in the lasI and lasR mutant biofilms but could be suppressed if the grow th medium was amended with 25 muM ferric chloride. This study demonstrated that (i) the nutritional status of the cell must be taken into account when one is evaluating QS-based gene expression; (ii) in the biofilm mode of gr owth, QS may also have negative regulatory functions; (iii) QS-based gene r egulation models based on studies with planktonic cells must be modified in order to explain biofilm gene expression behavior; and (iv) gene expressio n in biofilms is dynamic.