The secretion defect was reversed by mutant complementation with a gene copy into a plasmid of moderate copy number (Figure ?(Figure3).3). (gene favors the symbiotic competitive ability on cv. Esmeralda and affects negatively the secretion of proteins through T3SS. Here we localize Y4yS in the bacterial membrane using a translational reporter peptide fusion. analysis indicated that this protein presents a tetratricopeptide repeat (TPR) domain, a signal peptide Neochlorogenic acid and a canonical lipobox LGCC in the N-terminal sequence. These features that are shared with proteins required for the formation of the secretin complex in type IV secretion systems and in the Tad system, together with its localization, suggest that the T3SS secretin (RhcC2) complex. Remarkably, analysis of RhcC2 in the wild-type and mutant strains indicated that the absence Rabbit Polyclonal to CCDC45 of Y4yS affects negatively the accumulation of normal levels of RhcC2 in the membrane. box consensus sequence in its promoter region (Krause et al., 2002; Marie et al., 2004). TtsI binds to boxes (TB motifs) in the promoter regions of genes encoding T3SS components, inducing their transcription (Wassem et al., 2008). MAFF303099 has a functional T3SS (Snchez et al., 2009; Okazaki et al., 2010). The T3SS gene cluster is part of the symbiotic island (Kaneko et al., 2000a,b). Regulation of the MAFF303099 T3SS is similar to that of other rhizobia; a box precedes its gene homolog (Figure ?(Figure1)1) (Snchez et al., 2009). The cluster of T3SS genes of MAFF303099 also contains conserved Neochlorogenic acid TB motifs upstream of the orthologs of (((USDA257 NopP and NopE1/NopE2 (Schechter et al., 2010; Wenzel et al., 2010). Depending on the nodulated legume, a mutation affecting T3SS functionality can alter its nodulation competitiveness (Snchez et al., 2012). Genes that code for proteins secreted by this system in and with functionality in nodulation competitiveness (gene are shown. The lipobox and the region containing the TPR domain are underlined by a thin and a wide line respectively. The MAFF303099 T3SS cluster, which contains all the conserved genes required for the formation of the T3SS apparatus, also harbors an additional three genes, codes for a putative lipoprotein with homology to a protein of involved in the assembly of the extracellular filament (CpaD) (Skerker and Shapiro, 2000; Tampakaki, 2014; Rhizobase data bank). codes for a protein similar to members of the T3SS SctO protein family with unknown function. is a homolog to the gene of Rhizobium sp. NGR234, USDA110, and (Marie et al., 2001; Gazi et al., 2012). The (box upstream the gene (Figure ?(Figure1).1). The gene codes for a small unknown protein (165 aa) with a tetratricopeptide repeat (TPR) domain. TPR domains are imperfect 34-amino acid repeats often arranged in tandem arrays (Edqvist et al., 2006) that are involved in protein-protein interactions and the assembly of multiprotein complexes (D’Andrea and Regan, 2003). TPR domains were described in several T3SS proteins such as chaperones, regulators and exceptionally in one T3SS effector. TPR domains are found in class II Neochlorogenic acid and class V T3SS chaperones. Class II T3SS chaperones are translocator-chaperones and class V T3SS chaperones are required for T3SS needle formation in pathogens (Sun et al., 2008; Francis, 2010). T3SS of rhizobia have pili instead of a needle (Saad et al., 2008; Abby and Rocha, 2012). NopX, NopA, and NopB have been described as components of rhizobial T3SS pili where NopX has been suggested to be the translocator protein in the system (Marie et al., 2001; Saad et al., 2008). No chaperone for T3SS effectors (named class I chaperones) or for pili components has been described for T3SS until now. The existence of tetratricopeptide-like repeats has also been reported in transcriptional regulators of T3SS such as HilA from and HrpB from (Pallen et al., 2003). Also a T3SS effector of Xanthomonas (PthA) was found to have a TPR domain (Murakami et al., 2010). It has also been reported that TPR proteins are involved in the functionality of other secretion systems, including pilotins and some accessory proteins of type IV secretion systems (T4SS) (Korotkov et al., 2011; Koo et al., 2012). Pilotins are small membrane lipoproteins required for the localization and/or stability of the secretin complex formed at the outer membrane (OM) in T2SS, T3SS, and T4SS (Koo et al., 2012). The secretin complex is a homo-multimeric complex that.