Transcriptome analyses of continuous cultures of Pseudomonas aeruginosa growing slowly under oxygen and nitrate limitation. Pseudomonas aeruginosa PAO1

Chemostats have been used for decades in studying cell growth under controlled environments. Whole transcriptome sequencing by RNA-seq is a relatively new method for gene expression analysis. We utilized both tools for expression analysis of Pseudomonas aeruginosa growing slowly in synthetic cystic fibrosis medium under growth-limiting nitrate and oxygen levels. We established steady-state cultures under two divergent growth rates and found that during slower growth at a doubling time of 9.8 h, 76 known quorum genes were up-expressed while just 11 were down-expressed. Quorum-controlled genes were also more expressed in response to oxygen limitation. 21% of up-expressed genes under slow, oxygen-limited growth are quorum controlled while just 6% are up-expressed under slow, nitrate-limited growth. We found that the autoinducer for regulator RhlR, C4-HSL, is ~3.4 times higher when cells are growing slowly under oxygen limitation while the concentration of the autoinducer for LasR remained unchanged. Experiments with deletion mutants show the importance of rhlR for expression of quorum-controlled genes under slow, oxygen-limited conditions. This is an intriguing observation since P. aeruginosa is likely growing in similar environments in the cystic fibrosis lung, and lasR mutations are known to arise during chronic infection. Overall design: RNA-seq analysis of continuously grown cultures grown in duplicate per growth rate tested (2.4 h and 9.8 h doubling times) per conditions tested (anaerobic and microaerobic)