Unexpected role for a serine/threonine-rich domain in the Candida albicans Iff protein family.

Glycosylphosphatidylinositol (GPI)-anchored proteins are an important class of cell wall proteins in Candida albicans because of their localization and their function, even if more than half of them have no characterized homolog in the databases. In this study, we focused on the IFF protein family, investigating their exposure on the cell surface and the sequences that determine their subcellular localization. Protein localization and surface exposure were monitored by the addition of a V5 tag on all members of the family. The data obtained using the complete proteins showed for Iff3 (or -9), Iff5, Iff6, and Iff8 a covalent linkage to the ß-1,6-glucan network but, remarkably, showed that Iff2/Hyr3 was linked through disulfide bridges or NaOH-labile bonds. However, since some proteins of the Iff family were undetectable, we designed chimeric constructions using the last 60 amino acids of these proteins to test the localization signal. These constructions showed a ß-1,6-glucan linkage for Iff1/Rbr3, Iff2/Hyr3, Iff4 and Iff7/Hyr4 C-terminal-Iff5 fusion proteins, and a membrane localization for the Iff10/Flo9 C terminus-Iff5 fusion protein. Immunofluorescence analyses coupled to these cell fraction data confirmed the importance of the length of the central serine/threonine-rich region for cell surface exposure. Further analysis of the Iff2/Hyr3 linkage to the cell surface showed for the first time that a serine/threonine central region of a GPI-anchored protein may be responsible for the disulfide and the NaOH bonds to the glucan and glycoproteins network and may also override the signal of the proximal ω site region.

Analysis of the LacI family regulators of Erwinia chrysanthemi 3937, involvement in the bacterial phytopathogenicity.

Analysis of the regulators of the LacI family was performed in order to identify those potentially involved in pathogenicity of Erwinia chrysanthemi (Dickeya dadantii). Among the 18 members of the LacI family, the function of 11 members is either known or predicted and only 7 members have, as yet, no proposed function. Inactivation of these seven genes, called lfaR, lfbR, lfcR, lfdR, lfeR, lffR, and lfgR, demonstrated that four of them are important for plant infection. The lfaR and lfcR mutants showed a reduced virulence on chicory, Saintpaulia sp., and Arabidopsis. The lfeR mutant showed a reduced virulence on Arabidopsis. The lfdR mutant was more efficient than the wild-type strain in initiating maceration on Saintpaulia sp. The genetic environment of each regulator was examined to detect adjacent genes potentially involved in a common function. Construction of transcriptional fusions in these neighboring genes demonstrated that five regulators, LfaR, LfcR, LfeR, LffR, and LfgR, act as repressors of adjacent genes. Analysis of these fusions also indicated that the genes controlled by LfaR, LfcR, LfgR, and LffR are expressed during plant infection. Moreover, addition of crude plant extracts to culture medium demonstrated that the expression of the LfaR- and LfgR-controlled genes is specifically induced by plant components.