8fold), the enzyme uridylate kinase (6.4fold), and the downregulated sellectchem proteins polyketide synthase (?4.3fold) and luminal binding protein (?3.4fold). Gene expression differences between acute and chronic infection AES-1 isolates Against expectations that chronic infection leads to a downregulation of virulence-related genes, a number of genes with virulence functions were upregulated in chronic AES-1M compared to its acute infection counterpart (Table 2). These included alcohol dehydrogenase adhA, (5.5fold), the T3SS regulator pscD (3.2fold), uroporphyrinogen decarboxylase hemE (16-fold), peptidyl-prolyl cis-trans isomerase ppiA (2.5-fold), alginate-associated genes algD,E,F,8 and amrZ (1.9 to 10.7fold), dihydroorotase pyrC (12.1fold), uridylate kinase pyrH (6.4fold), the extracellular polysaccharide genes pelC (5.

6fold) and pelE (3.7fold) part of the pelABCDEF operon, phospholipase N (plcN) (3.2fold) and cardiolipin synthase cls (7.0fold). Genes with putative or probable virulence roles upregulated in chronic AES-1M included a probable haloacid dehalogenase (21.6fold), threonine dehydratase ilvA1 (9.7fold) xanthine phosphoribosyltransferase xpt (16.7fold) and polyhydroxyalkanoate synthesis protein phaF (6.0fold) (Table S2). The downregulation of homogentisate 1,2-dioxygenase hmgA (-7.5fold) is also virulence related since its downregulation de-represses pyomelanin production, which enhances persistence. Table 2 Known virulence-related genes differentially expressed in P. aeruginosa AES-1M compared to AES-1R (p<0.05).

Gene expression by quantitative PCR The average quantitative PCR ratios of the selected virulence-related (Table 2) and other genes correlated well with their microarray expression ratios (correlation coefficient: R2=0.8053 ? Fig. 4). All genes showed either up- or downregulation consistent with the microarray results, despite eight of these being quantified using different RNA samples for array and qPCR. Figure 4 Differential expression of virulence-related genes by microarray and qPCR. Discussion The sequencing of the genome of the Australian epidemic strain AES-1 (isolate R) has provided the first opportunity to examine the similarities and differences between this frequent clone widespread in eastern Australia and other frequent clones such as PaLES and c3719. In terms of overall size, AES-1R (6.254 Mbp) falls between c3719 (6.

146 Mbp) and PaLES (6.601 Mbp), and close to that of PAO1 (6.264 Mbp). As the c3719 and PaLES genomes are closed and the AES-1R genome is not, a direct comparison of CDS is not possible, however in terms of genes of known function there are significant differences between AES-1R and c3719. In subcellular Entinostat localisation (Fig. 1) AES-1R has a significantly smaller proportion of cytoplasmic genes and a significantly greater proportion (3.4% against 2.7%) of outer membrane genes compared to c3719 (Pearson’s ��2 test: p=0.047 and p=0.019, respectively,).