Half a decade has
gone by since the publication of the genome sequence of Xac (da Silva et al., 2002), and apparently, Selleck Midostaurin its large-scale proteome analyses are confined to a few reports using techniques such as two-dimensional protein gels, yeast two-hybrid, and nuclear magnetic resonance scans for folded proteins (Mehta & Rosato, 2001, 2003; Galvao-Botton et al., 2003; Alegria et al., 2004, 2005; Khater et al., 2007). Moreover, the two last methods used heterologous expression of proteins in organisms different from the one under investigation. Most of the limitation to explore the biology of Xac lies in a complete lack of protein expression systems adapted to this bacterium. Here, we showed
the construction and test of new protein expression vectors dedicated to Xac, and the subsequent utilization of our system to characterize the hypothetical protein XAC3408. XAC3408 is 30% identical (at the amino acid level) to the B. subtilis cell division protein ZapA, and our subcellular localization experiments using GFP-XAC3408 support the hypothesis that XAC3408 is the Xac orthologue of ZapABsu. MS-275 cost ZapA-like proteins are conserved among bacteria, in which they function by promoting the FtsZ bundling and stabilization of FtsZ polymers (Gueiros-Filho & Losick, 2002; Low et al., 2004; Scheffers, 2008). ZapAXac exhibited a localization pattern similar to that observed for ZapABsu (Gueiros-Filho & Losick, 2002), and the availability of Xac mutants labeled at the septum
provides a new perspective for antimicrobial drug development trials with Xac aimed to disrupt cell division. The vectors described here are Phosphatidylinositol diacylglycerol-lyase integrative and allow the ectopic expression of proteins from the amy locus of Xac. Such a strategy has been used extensively in the Gram-positive rod B. subtilis (Lewis & Marston, 1999; Gueiros-Filho & Losick, 2002), and it is believed to avoid disturbances to genes/chromosomal regions that might produce undesirable effects in cell growth and altered phenotypes. Besides, integration into amy has the advantage of allowing the characterization of essential genes, which may not accommodate changes in their coding sequences. Finally, the disruption of amy produces a bacterial phenotype easily detectable on a plate and allows the distinction of insertions that had occurred in amy from those in the gene being under investigation. In the present work, we showed that the α-amylase gene was not essential for Xac to grow on a plate, neither was it found to play any key role during infection, an outcome somewhat expected since it has been demonstrated that in bacteria α-amylases may be essential for growth on starch, but dispensable for growth on other carbon sources (Worthington et al., 2003).