The primers for cloning as well as sequencing are shown in Additional file 3. Plasmid-borne PU-H71 datasheet deletion alleles of the sseB or sseD were generated by a PCR-based method using the QuikChange II XL Site-Directed Mutagenesis Kit according to the instruction
of the supplier (# 200521-12, Stratagene, Heidelberg, Germany). All plasmids harboring mutant alleles were prescreened VX-680 purchase for successful mutagenesis, subsequently sequenced and introduced into the corresponding mutant strain by electroporation. Primers used for deletion, control PCR and DNA sequencing are listed in Additional file 3. In order to move plasmid-borne sseD deletion alleles into the Salmonella chromosome, the λ Red system was applied in combination with positive selection for the loss of a tetracycline resistance cassette on Bochner-Maloy plates as described previously [29]. For amplification of the mutations affecting the inner region of sseD, the primer pair sseD-Del-Chrom-For and seq-rev were used. Fragments for deletions in the 5′ or 3′ region were amplified using sseD-delN1-chrom-For in combination with seq-rev or sseD-Del-Chrom-For together with sseD-del-C1 (2/3/4)-chrom-rev.
All constructs were confirmed by sequencing. Sequences of primers used www.selleckchem.com/products/Trichostatin-A.html for deletion and sequencing are described in Additional file 3. Bioinformatics For bioinformatic predictions in terms of coiled-coil domains and transmembrane regions of the SPI2 translocon proteins SseB and SseD, the freely available service of the Swiss EMBnet node server http://www.ch.embnet.org:http://www.ch.embnet.org/software/COILS_form.html, http://www.ch.embnet.org/software/TMPRED_form.html was engaged. The sequence manipulation suite of the Bioinformatic
Organisation http://www.bioinformatics.org/sms/prot_mw.html was conducted in order to calculate the molecular weight of the ADP ribosylation factor SseB and SseD wild-type proteins as well as of the mutant variants of both proteins. Analyses of in vitro protein expression, surface attachment and secretion For the in vitro analyses of the expression, surface-attachment and secretion of SseB and SseD as well as the plasmid-borne or chromosomal derived mutant variants, the secretion assay described by Nikolaus et al. [7] was modified. Salmonella strains were pre-cultured overnight in PCN+P (25 mM Pi) pH 7.4, diluted 1:50 in 400 ml PCN-P media at pH 5.8 and incubated 7 h in a shaker platform with agitation at 150 rpm at 37°C. For analyses of protein synthesis, aliquots of 1 ml bacterial culture were pelleted by centrifugation in a table top centrifuge (Sigma 1-13) for 15 min at max. speed. The pellets were resuspended in sample buffer (12.5% glycerol, 4% SDS, 50 mM Tris-HCl pH 6.8, 2% β-mercaptoethanol, 0.01% bromophenol blue) according to the optical density (OD600 of 1 ml of culture × 100 = × μl of sample buffer) and heated at 95°C for 5 min.