The location of fluorescent PI3K activation signals in single cells was then investigated in each case by fluorescence microscopy, the most informative results being shown in Fig 4. In most cases, green signals appeared to be somehow localized in specific cell sites or compartments. This was not altogether surprising for proteins known or predicted to be associated

with the membrane. CyoD::GFP was clearly bound to the cell contour (more intense at the poles), as mTOR inhibitor would be expected of a protein that forms part of the membrane-bound respiratory chain [45]. LapA::GFP originates in a large loosely surface-associated protein that is exported through an ABC transporter system [46]. That fluorescence appears in this case in regularly spaced foci along the longitudinal cell axis suggests the dots to be the sites of export to the extracellular medium. Yet, the

most unusual appearance was that of the PP1794::GFP fusion. This ORF encodes a protein predicted to have a putative outer membrane location. The hybrid product resulting from its fusion to GFP was near entirely confined to the cell poles and displayed a clear-cut boundary with the rest of the cell, an unprecedented {Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|buy Anti-infection Compound Library|Anti-infection Compound Library ic50|Anti-infection Compound Library price|Anti-infection Compound Library cost|Anti-infection Compound Library solubility dmso|Anti-infection Compound Library purchase|Anti-infection Compound Library manufacturer|Anti-infection Compound Library research buy|Anti-infection Compound Library order|Anti-infection Compound Library mouse|Anti-infection Compound Library chemical structure|Anti-infection Compound Library mw|Anti-infection Compound Library molecular weight|Anti-infection Compound Library datasheet|Anti-infection Compound Library supplier|Anti-infection Compound Library in vitro|Anti-infection Compound Library cell line|Anti-infection Compound Library concentration|Anti-infection Compound Library nmr|Anti-infection Compound Library in vivo|Anti-infection Compound Library clinical trial|Anti-infection Compound Library cell assay|Anti-infection Compound Library screening|Anti-infection Compound Library high throughput|buy Antiinfection Compound Library|Antiinfection Compound Library ic50|Antiinfection Compound Library price|Antiinfection Compound Library cost|Antiinfection Compound Library solubility dmso|Antiinfection Compound Library purchase|Antiinfection Compound Library manufacturer|Antiinfection Compound Library research buy|Antiinfection Compound Library order|Antiinfection Compound Library chemical structure|Antiinfection Compound Library datasheet|Antiinfection Compound Library supplier|Antiinfection Compound Library in vitro|Antiinfection Compound Library cell line|Antiinfection Compound Library concentration|Antiinfection Compound Library clinical trial|Antiinfection Compound Library cell assay|Antiinfection Compound Library screening|Antiinfection Compound Library high throughput|Anti-infection Compound high throughput screening| behaviour that will be the subject of future studies. Apart of such envelope-related proteins we also found a non-homogenous distribution of GFP in fusions to ribosomal proteins (Figure 4). We believe that these high-fluorescent sites can be related to the so-called translation factories that seem to gather most of the ribosomal machinery

of individual cells [47]. More unexpected was the high signal brought about by the NusA::GFP fusion. In E. coli, this protein is a transcription termination/anti-termination factor that acts either way depending on its association to other cellular proteins [48]. While its high level of expression in P. putida was unexpected, its uneven distribution in single cell probably reflected also the occurrence of transcription factories [47] in this bacterium. Finally, one FliC::GFP fusion was found HA-1077 to give an uniform GFP signal throughout individual cells. The flagellin protein FliC is the main structural component of the flagella [49]. That fliC::gfp cells lacked any swimming motility (data not shown) indicated that the function had been knocked-out. It is hence likely that the FliC::GFP cannot enter the secretion pathway and it freely diffuses in the cytoplasm as a result. However, the FlgM::GFP fusion also originated evenly fluorescent cells (Figure 4), but in this case the transposition did not affect its function since this strain was still motile (not shown). Figure 4 Subcellular localization of high-fluorescence GFP fusions generated by mutagenesis of P. putida with mini-Tn 5 GFPKm. Cultures of the cells under examination were grown until stationary phase in LB medium and prepared for epifluorescence microscopy as explained in Materials and Methods.