Another interesting difference observed was the maximum population density achieved. The PA23 wild type consistently reached a higher OD600 in stationary phase compared to PA23-443 (Figure 4). A similar altered pattern of growth has been observed ARRY-162 in vivo for gacS mutants of PA23 and 30–84 which exhibit a shorter lag phase and earlier entry into logarithmic growth phase [4, 29]. LTTRs have previously been implicated in the regulation of cellular growth factors. For example, the well-studied LTTR OxyR is involved in regulating the expression of various metabolic genes such as tRNA nucleotidyl transferases and synthetases, ribosomal proteins and QS-regulated targets [30]. Figure 4 Growth rate analysis
of wild-type PA23 and mutant PA23-443. Cells were grown in M9 minimal media supplemented with 1 mM MgSO4 and 0.2% glucose. Spectrophotometric optical densities were taken at 600 nm. Evofosfamide manufacturer Diamonds; PA23wt, circles; PA23-443. PtrA negatively affects motility Our iTRAQ proteomic data indicated upregulation of the flagellin and related hook-associated protein (MOK_01499) in PA23-443. Further inspection
of the locus tags upstream of MOK_01499 also indicated upregulation of proteins FliG (MOK_01489; Vdiff = +0.72) and FliS (MOK_01496; Vdiff = +0.66), although this upregulation was not considered significant. The upregulated flagellin and related hook-associated protein, therefore, is likely part of the Fli operon based on its proximity to upstream genes. To verify the results of the proteomic analysis, motility assays were conducted. As outlined in Table 4, swimming (flagellar) motility was almost 3-fold greater in PA23-443 compared to the wild type, indicating that PtrA is having a repressive effect on this phenotype. In a similar fashion, proteomic analysis
of a P. aeruginosa gacA mutant revealed a 7.5-fold and 8.8-fold increase in expression of a flagellin (FliC) and flagellar-capping protein (FliD), respectively [27]. Introduction of ptrA in trans caused a modest reduction in motility, but did not Methocarbamol fully restore the wild-type phenotype. It is important to bear in mind that for our complementation studies, multiple copies of the ptrA gene were provided rather than a single chromosomal copy. Because LTTRs bind both activation SC79 research buy binding sites and regulatory binding sites upstream of target genes [14], the number of copies of the regulator may be of critical importance for proper binding and subsequent regulation of target genes. This observation was noted with complementation studies involving the LTTR OxyR in restoration of rhamnolipid and pyocyanin production in P. aeruginosa[31]. When multiple copies of oxyR were present in the cell, the wild-type phenotype was not restored; whereas insertion of single chromosomal copy of the LTTR gene resulted in full complementation [31]. Table 4 Motility analysis of P.