2, but SseD was detected using a polyclonal antiserum raised against recombinant SseD. For the cytosolic portion of SseD, we observed a lower molecular weight form in addition to the protein found in the secreted fraction. The quantification of the signal intensities is shown in Additional file 1. Similar effects were observed for the secretion and partitioning of SseC in strains expressing various alleles of sseB (data not shown). Effect of deletion of SseB domains on formation of translocon structures on Salmonella We have previously observed that secreted translocon proteins

SseB, SseC and SseD were #Ispinesib order randurls[1|1|,|CHEM1|]# predominantly located in surface structures that occurred in single or low copy number resulting in a punctuated staining in immune-fluorescence analyses [7, 8]. To test the effect of deletions in SseB on the formation of such surface structures, we used immunofluorescence to analyze various strains grown under secretion-inducing culture conditions (Fig. 4). The treatment of cells with lysozyme prior to immuno-labeling allowed the estimation of the cytoplasmic pool of SseB variants SGC-CBP30 mw (Fig. 4A). The staining intensities for SseB observed correlated well with the data shown in Fig. 2. The investigation of surface-located SseB (Fig. 4B) indicated that WT SseB,

SseBΔN1, SseBΔ1 and SseBΔC1 showed punctuate staining of single or low ADAMTS5 numbers of complexes per cell. A more intense and evenly distributed staining was observed for the psseB complemented sseB strain and a strains expressing sseBΔ2. No or only very rare staining for SseB was found for the other mutant forms of SseB. Figure 4 Surface location of SseB variants under

in vitro culture conditions. S. Typhimurium wild type (WT), the sseB strain, or the sseB strain harboring psseB for expression of WT sseB, or plasmids for the expression of various mutant alleles of sseB (psseBΔx) were grown in vitro under conditions that induced the synthesis and secretion of SseB. At 8 h of culture, the bacteria were fixed on chitosan-pretreated cover slips. The bacterial cells were stained with rabbit anti-Salmonella O-1,4,5,12,27 antiserum conjugated with DyLight 547 NHS ester (red). SseB was immuno-stained using rabbit polyclonal antibody against recombinant SseB as primary antibody and anti rabbit Alexa488 was used as secondary antibody (green). A) Presence of SseB within the bacterial cytoplasm was investigated by immunofluorescence labeling of SseB after lysozyme permeabilization of the bacteria. B) For analyses of SseB secretion and surface location, the lysozyme treatment was omitted. We next investigated the function of the various mutant forms of SseB in intracellular Salmonella (Fig. 5).