2; see also Additional file 1). However, Western blot analyses indicated that the amounts of cell-associated SseB differed for the various deletion constructs. We also determined the proportion of SseB in the detached fraction, Selleckchem mTOR inhibitor corresponding to secreted protein bound to surface appendages of Salmonella,

and in the supernatant fraction corresponding to secreted proteins without association to the cell surface (Fig. 2). For Salmonella WT, a large proportion of secreted SseB was found in the detached fraction. No signal for SseB was observed for the sseB strain. An sseB strain complemented with psseB showed an equal distribution of secreted SseB in the detached and supernatant fraction and the distribution observed for this strain would be relevant for comparison to sseB strains harboring plasmids for the expression of various deletion constructs. We observed that SseBΔ4, SseBΔ5 and SseBΔ6 were not secreted or only present in secreted fractions in minute amounts. In addition, the amounts of SseBΔ5 and SseBΔ6 were highly decreased in comparison to the WT or the complemented strains and signals in Western blots were only detected this website after extended exposure times. SseBΔ1 was only detected in the detached

fraction but not the secreted fraction. The situation was Chk inhibitor opposite for SseBΔ2, which was only present in the supernatant fraction but not in the detached fraction. Control Western blots for DnaK indicated that low amounts of this cytoplasmic protein were present in the detached fraction, thus the amounts of SseB in the detached fraction of the SseBΔ1 are likely to be the result of cell lysis. The secretion and partitioning of SseBΔ3 and SseBΔC1 was similar to that of WT SseB. For SseBΔ7 and SseBΔN1, highly reduced secretion was observed and the larger proportion of the secreted protein was present in the supernatant fraction. These analyses indicate that synthesis and secretion

was affected to a different extend by deletions and that secretion similar to WT is possible Orotidine 5′-phosphate decarboxylase even with deletions of larger portions of the protein. The deletion of the coiled-coil domain had little effect on secretion and partitioning (SseBΔ3), while mutations affecting the putative transmembrane region abolished the secretion of the mutant variants of SseB (SseBΔ2 and SseBΔ4). An SseB variant that lacked the postulated chaperone binding site in the C-terminal region of SseB was still synthesized, but the amounts of this protein in the secreted fractions were highly reduced (SseBΔ7). Figure 2 Effect of various deletions in sseB on synthesis and secretion of SseB in vitro. S. Typhimurium WT or ΔsseB without plasmid, harboring plasmid psseB for complementation of the sseB deletion, or plasmids for the expression of various sseB mutant alleles (psseBΔx) were grown in 400 ml minimal medium PCN-P (0.