The Pseudomonas aeruginosa transcriptome in planktonic cultures and static biofilms using RNA sequencing.

In this study, we evaluated how gene expression differs in mature Pseudomonas aeruginosa biofilms as opposed to planktonic cells by the use of RNA sequencing technology that gives rise to both quantitative and qualitative information on the transcriptome. Although a large proportion of genes were consistently regulated in both the stationary phase and biofilm cultures as opposed to the late exponential growth phase cultures, the global biofilm gene expression pattern was clearly distinct indicating that biofilms are not just surface attached cells in stationary phase. A large amount of the genes found to be biofilm specific were involved in adaptation to microaerophilic growth conditions, repression of type three secretion and production of extracellular matrix components. Additionally, we found many small RNAs to be differentially regulated most of them similarly in stationary phase cultures and biofilms. A qualitative analysis of the RNA-seq data revealed more than 3000 putative transcriptional start sites (TSS). By the use of rapid amplification of cDNA ends (5'-RACE) we confirmed the presence of three different TSS associated with the pqsABCDE operon, two in the promoter of pqsA and one upstream of the second gene, pqsB. Taken together, this study reports the first transcriptome study on P. aeruginosa that employs RNA sequencing technology and provides insights into the quantitative and qualitative transcriptome including the expression of small RNAs in P. aeruginosa biofilms.

An orphan sensor kinase controls quinolone signal production via MexT in Pseudomonas aeruginosa.

Pseudomonas aeruginosa employs both N-acylhomoserine lactone and 2-alkyl-4(1H)-quinolone (AQ)-mediated interbacterial signalling for the orchestration of a genome-wide gene regulatory network. Despite the many advances that have been made in understanding the target genes of quorum sensing regulation, little is known on how quorum sensing systems are influenced by environmental cues. In this study, we show that AQ production is modulated by an orphan P. aeruginosa sensor kinase. Transcriptional studies of the sensor kinase (MxtR) mutant demonstrated that an induced expression of MexT, a LysR-type transcriptional regulator, largely determined the global transcriptional profile. Thereby, overexpression of the MexT-regulated MexEF-OprN efflux pump led to a delayed expression of the AQ biosynthetic genes and of AQ-dependent virulence factors. Furthermore, we demonstrated that autophosphorylation of MxtR was inhibited by ubiquinone, the central electron carrier of respiration in in vitro experiments. Our results elucidate on a mechanism by which P. aeruginosa senses environmental conditions and adapts by controlling the production of interbacterial AQ signal molecules. A regulatory function of a sensor kinase may indicate that there is a pre-emptive role of adaptation mechanisms that are turned on under distinct environmental conditions and that are important for efficient colonization and pathogenesis.

Intrinsic thermal sensing controls proteolysis of Yersinia virulence regulator RovA.

Pathogens, which alternate between environmental reservoirs and a mammalian host, frequently use thermal sensing devices to adjust virulence gene expression. Here, we identify the Yersinia virulence regulator RovA as a protein thermometer. Thermal shifts encountered upon host entry lead to a reversible conformational change of the autoactivator, which reduces its DNA-binding functions and renders it more susceptible for proteolysis. Cooperative binding of RovA to its target promoters is significantly reduced at 37 degrees C, indicating that temperature control of rovA transcription is primarily based on the autoregulatory loop. Thermally induced reduction of DNA-binding is accompanied by an enhanced degradation of RovA, primarily by the Lon protease. This process is also subject to growth phase control. Studies with modified/chimeric RovA proteins indicate that amino acid residues in the vicinity of the central DNA-binding domain are important for proteolytic susceptibility. Our results establish RovA as an intrinsic temperature-sensing protein in which thermally induced conformational changes interfere with DNA-binding capacity, and secondarily render RovA susceptible to proteolytic degradation.