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...

Background: Pseudomonas aeruginosa is a genetically complex bacterium which can adopt and switch between a free-living or biofilm lifestyle, a versatility that enables it to thrive in many different environments and contributes to its success as a human pathogen.

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The Pseudomonas aeruginosa type III secretion system (TTSS), enabling direct injection of toxins into host cells, has been shown to be crucial to virulence in several models of P. aeruginosa pathogenesis. Using the strain PA14 and its isogenic mutant, PA14exsA, we investigated the role of the TTSS during infection of the nematode Caenorhabditis elegans. Although C. elegans N2 was killed by PA14 in an infection like process over 48 to 72 h the same effect was observed following infection with PA14exsA, implying that a functional TTSS was not essential for virulence. This was despite the TTSS being actively expressed during C. elegans infection as demonstrated by the use of green fluorescent reporter constructs and RT-PCR. However, compared to the wild type PA14, PA14exsA did display a reduced rate of killing of C. elegans strain AU1 which harbours a mutation in the sek-1 gene encoding a MAP kinase involved in nematode innate immunity. A fuller understanding of the mechanism of resistance to type III attack in C. elegans may lead to the identification and development of novel therapeutic targets affording protection to TTSS products in man.

Tetanus toxin is a potent neurotoxin synthesized by Clostridium tetani. Immunization with fragment C protein, the nontoxic C-terminal domain of tetanus toxin, will protect mice against lethal challenge with tetanus toxin. A synthetic gene encoding fragment C (tetC) had previously been shown to express high levels of fragment C in Saccharomyces cerevisiae. A plasmid, pcDNA3/tetC, which encodes the synthetic tetC gene expressed under the control of the human cytomegalovirus major intermediate-early promoter/enhancer region, was constructed. Expression of fragment C was observed in eukaryotic cells growing in vitro following transfection with pcDNA3/tetC. The immune response induced by intramuscular immunization with pure pcDNA3/tetC DNA was evaluated in a murine model. Anti-fragment C serum immunoglobulin and proliferative responses in splenocytes were observed in BALB/c mice following two immunizations with pcDNA3/tetC. The major immunoglobulin G subclass that recognized fragment C was immunoglobulin G2a, and the stimulated splenocytes secreted high levels of gamma interferon. Immunity to tetanus is dependent on the presence of neutralizing serum antibodies against tetanus toxin. Sufficient anti-fragment C serum immunoglobulins were induced by DNA-mediated immunization to protect mice against lethal challenge with tetanus toxin.