Inner primers contained 18 bp of homology to the tet cassette, which was amplified together with flanking sequences in a second PCR reaction. The resulting 4000 bp fragment was used for transformation of PY79 wild type

cells, selecting for tetracycline (tet) resistance, giving rise to HW2 (dynA::tet). As an alternative strategy, 500 bp internal of dynA (starting at bp 1480) were amplified S63845 and cloned into pMutin, using HindIII and EcoRI restriction sites. PY79 cells were transformed with plasmid DNA, selecting for Mls resistance, giving rise to a DynA truncation missing the last 500 amino acids. For the generation of a dynA floT double mutant strain, strain DML1541 ΔfloT (yuaG) (in frame deletion of yuaG, kind gift from M. Hinderhofer, University of Konstanz) was transformed with chromosomal DNA from strain HW2, selecting for tet resistance. For the generation of a C-terminal YFP fusion to DynA, the last 500 bp of dynA were amplified by PCR and were closed into pSG1164YFP [43] using ApaI and EcoRI restriction sites. PY79 cells were transformed with the resulting plasmid, which integrated at the dynA locus via single crossover integration (this was verified by PCR using a pair of primers that binds within the yfp gene and upstream of the 500 bp used

for integration). Expression of full length DynA-YFP was verified by Western blotting. For simultaneous visualization of DynA and of FtsZ, strain HW1 (DynA-YFP) Dorsomorphin clinical trial was transformed with chromosomal DNA from strain BS1059 [39], in which FtsZ-CFP is expressed from a xylose inducible fusion at the amylase

locus. The resulting colonies were obtained through selection on spectinomycin containing plates. For the localization of FtsZ-CFP or of YFP-MreB in dynA mutant cells, strain BS1059 or JS12 was transformed with chromosomal DNA from strain HW2, respectively. To visualize FloT-YFP in the absence of DynA, strain HW2 (Δ dynA) was transformed with chromosomal DNA of strain FD295 (floT yfp). To create a dynA mreB double deletion, Phosphatidylinositol diacylglycerol-lyase strain 3725 [36] (ΔmreB) was transformed with chromosomal DNA of strain HW2 (Δ dynA) and incubated at 25°C using PAB/SMM agar [44]. The ezrA dynA double deletion was created by transformation of strain HW2 (ΔdynA) with chromosomal DNA of strain FG375 (kind gift from F. Gueiros-Filho, University of São Paulo, Brasil). The plasmids used for S2 cell transfection were created by cloning the complete coding sequence of DynA or of FloT into the vector pFD1 [45], using KpnI and XhoI or ApaI and ClaI, respectively. Schneider cell culture and transient transfection D. melanogaster S2 Schneider cells were grown in Schneider’s www.selleckchem.com/products/PLX-4720.html Drosophila medium (Lonza Group Ltd.) supplemented with 5-10% (v/v) fetal calf serum (FCS) at 25°C without addition of CO2. Cells were passaged every 2 to 3 days to maintain optimal growth.