Transcriptome Analysis ofPseudomonas aeruginosaGrowth: Comparison of Gene Expression in Planktonic Cultures and Developing and Mature Biofilms

Abstract

The transcriptomes of logarithmic-and stationary-phase Pseudomonas aeruginosa planktonic cultures and static biofilms of different stages of development were compared. Developing and confluent biofilm transcriptomes were found to be related to those of logarithmic-and stationary-phase planktonic cultures, respectively. In addition, a number of novel genes were up-regulated in developing and confluent biofilms, including genes encoding putative solute transport proteins and transcriptional regulators, respectively.Pseudomonas aeruginosa is a versatile organism that can survive in soil, marshes, and marine habitats, on plant and animal tissue, and on nonliving surfaces (24). At 6.3 million base pairs (5,570 open reading frames [ORFs]), the genome of P. aeruginosa is one of the largest bacterial genomes that have been sequenced (24), and its size and complexity are thought to enable survival in diverse environments. Central to this survival is the ability to adopt and switch between free-living (planktonic) and biofilm (surface-attached) lifestyles. Biofilms are populations of microorganisms adhered to a surface or interface, and P. aeruginosa biofilms arise in a variety of clinical settings, including the cystic fibrosis lung, urinary catheters, and contact lenses (22,23,29). Biofilm infections are notoriously difficult to eradicate, even after prolonged antimicrobial therapy, and it is well documented that biofilms are less susceptible to antimicrobial agents than free-living (planktonic) bacteria and provide protection from the host immune response (5, 9, 11).Control of gene expression in P. aeruginosa is the key determinant of its flexibility, and a variety of highly integrated regulatory mechanisms have been described previously. These include the LasR-LasI and RhlR-RhlI cell density-dependent quorum-sensing (QS) systems and a large number of twocomponent regulatory systems (6,13,16,24,27). Knowledge of the specific genes required by P. aeruginosa for survival under different environmental conditions will improve our understanding of the biology of this organism and form the basis for the rational design of novel therapeutic approaches. The recent development of an Affymetrix GeneChip microarray representing 5,549 ORFs on the P. aeruginosa chromosome now enables the analysis of global gene expression of this organism under defined growth conditions and the comparison of different growth states. Here we provide the first study in which the transcriptional profiles of two bacterial planktonic phases (logarithmic phase [LP] and stationary phase [SP]) and multiple biofilm time points (8,14,24, and 48 h) are compared.Biofilm characterization. Previous studies using continuous culture biofilm systems (10, 17) have shown that biofilms form in a sequential process: (i) attachment, (ii) microcolony formation, and (iii) biofilm maturation. Using a Zeiss LSM510 confocal laser scanning microscope (40ϫ magnification), we characterized biofilm development of wild-type P. aeruginosa strain PAO1 (7) tagged with green fluores...

Transcriptional profiling of Legionella pneumophila biofilm cells and the influence of iron on biofilm formation

“…Interestingly, proteomic studies on a P. aeruginosa biofilm revealed the induction of AhpC, together with L-ornithine 5-monooxygenase, whose gene is located within the pvd locus involved in pyoverdine synthesis (Visca et al, 1994;Sauer et al, 2002). In contrast, transcriptome analyses of P. aeruginosa biofilms (Whiteley et al, 2001;Waite et al, 2005) revealed no induction of the pyoverdine genes, except when the gene expression profile of biofilm cells was compared to that of cells released from the biofilm (Sauer et al, 2004). However, only pvcAB appeared to be ironregulated during the planktonic lifestyle, suggesting that iron does not constitute the only signal inducing expression of the biofilm-regulated genes.…”

“…Although these models do not reflect the complexity found in natural environmental biofilms, monospecies biofilms were the first step towards understanding adaptation to the sessile lifestyle for many bacteria, including two recent studies on L. pneumophila (Mampel et al, 2006;Piao et al, 2006). These models pave the way for the study of complicated microbial interactions and have proven to be useful for assessing the gene expression profile of sessile cells even if the different cell types present within a biofilm cannot be analysed independently by this approach (Schembri et al, 2003;Beloin et al, 2004;Waite et al, 2005;Whiteley et al, 2001).…”

Subinhibitory concentrations of the cationic antimicrobial peptide colistin induce the pseudomonas quinolone signal in Pseudomonas aeruginosa

“…While our genetic and phenotypic data would suggest that subinhibitory concentrations of colistin influence biofilm formation in P. aeruginosa, no significant difference was observed in biomass using flow cells (data not shown). It is important to note that gene expression varies in P. aeruginosa planktonic cultures relative to the biofilm mode of growth (Waite et al, 2005(Waite et al, , 2006Whiteley et al, 2001). Furthermore, culture conditions may influence the patterns of gene expression in response to subinhibitory concentrations of colistin.…”

“…Flow-cell analysis also revealed no difference in biomass or ability to form mushroom structures in the presence of the antimicrobial (data not shown). This may be a reflection of the distinct environments and expression profiles that exist in planktonic cultures and mature biofilms (Waite et al, 2005(Waite et al, , 2006Whiteley et al, 2001). It must also be considered that other genes, which are not responsive to subinhibitory concentrations of colistin under the conditions tested, are required for induction of the biofilm mode of growth.…”

The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus

“…Several S. aureus genes have been reported to contribute to its biofilm-forming ability, including agr (3)(4)(5), sarA (5-7), sigB (8), ica (9), rbf (10), tcaR (11), arlRS (12,13), and alsSD (14). Microarray analyses of S. aureus (15,16), Pseudomonas aeruginosa (17,18), Bacillus subtilis (19,20), and Escherichia coli (21) have also illustrated that there are major differences in gene expression between biofilm and planktonic cultures of these organisms. These results underscore the need to study biofilm physiology as a way to better understand how the bacteria exist in vivo.…”