. plantarum Septation OatA. To assess its ability at rescuing the cell-cycle defect of the oatA mutant, complementation and time-lapse experiments were performed with the truncated OatATM110-YFP protein as reported above for the wild type and the OatAD510A/S511A catalytic mutant. Intriguingly, the OatATM110-YFP fusion was able to complement the cell cycle defect of the mutant, showing that the first 10 TM segments of OatA are sufficient to restore the wild-type phenotype. Fluorescent microscopy was then used to reveal the sublocalization of OatATM110-YFP. A similarly constructed fusion protein between OatB and YFP as well as a cytoplasmic-localized free YFP, were used as a control. The OatB protein displays a predicted topological organization that is similar to OatA with 11 TM segments and a surface-exposed O-acetyltransferase domain. While OatA is involved in MurNAc Oacetylation, OatB is responsible for GlcNAc O-acetylation in L. plantarum. Fluorescent microscopy revealed different localization patterns for these fluorescent proteins. OatBTM110-YFP harbored a global membrane-associated localization pattern compared to the cytoplasmic-localized YFP, whereas OatATM110-YFP was even more restricted to a septal location . Fluorescence ratios between the septum and the pole were determined. In the case of the OatB fusion, the measured mean fluorescence ratio was,2 as expected for a global membrane localization, while the mean ratio was largely higher than 2 for the OatA fusion, showing that the increased fluorescence observed at the septum was not due to the presence of the septal double membrane, but well to a specific location of OatATM110-YFP. Due to low fluorescence level and quick bleaching of the YFP fusion, we were unable to perform time lapse experiments with the OatA/BTM110-YFP fusions. However, the localization of the OatA fusion was followed 
during the cell cycle by selecting of a set of cell images at different stages of growth. The OatA fusion could be detected at mid-cell before the invagination of the septal membrane as revealed by FM464 fluorescent labeling. After membrane invagination and septum formation, OatATM110-YFP fluorescence formed a marked transversal band that shrunk as constriction proceeded. At a late septation stage, cells retained a polar localization of the OatA fusion that persisted until the next division cycle of daughter cells under fast growth conditions. These data suggest that Role of OatA in L. plantarum Septation OatA is recruited at a septal position in the early stage of the division process and that it remains at the septum until the ultimate stage of division before cell separation. Overexpression of oatA Leads to Curved Cells and Aberrant Septation As OatA is localized at a septal position during all steps of the division process and as its depletion affects the uncoupling of the elongation-septation process, we next investigated the impact of its overproduction on cell morphology. The three versions of the oatA gene were overexpressed by a high concentration of the nisin inducer in wild type and oatA mutant strains. The empty vector and the oatBTM110::yfp fusion were used as a control. Surprisingly, although oatA deletion did not lead to any obvious cell morphology defect, except for the lack of cell length control, overexpression of the three OatA variants led to similar morphological 300817-68-9 site aberrations in a significant part of the population in both wild type and OatA2 backgrounds and 50% abnormal c
