Visualizing Interactions along the Escherichia coli Twin-Arginine Translocation Pathway Using Protein Fragment Complementation

The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways

Transport of Folded Proteins by the Tat System

The twin-arginine protein translocation (Tat) system has been characterized in bacteria, archaea and the chloroplast thylakoidal membrane. This system is distinct from other protein transport systems with respect to two key features. Firstly, it accepts cargo proteins with an N-terminal signal peptide that carries the canonical twin-arginine motif, which is essential for transport. Second, the Tat system only accepts and translocates fully folded cargo proteins across the respective membrane. Here, we review the core essential features of folded protein transport via the bacterial Tat system, using the three-component TatABC system of Escherichia coli and the two-component TatAC systems of Bacillus subtilis as the main examples. In particular, we address features of twin-arginine signal peptides, the essential Tat components and how they assemble into different complexes, mechanistic features and energetics of Tat-dependent protein translocation, cytoplasmic chaperoning of Tat cargo proteins, and the remarkable proofreading capabilities of the Tat system. In doing so, we present the current state of our understanding of Tat-dependent protein translocation across biological membranes, which may serve as a lead for future investigations

Comparação de Meios de Enriquecimento e de Plaqueamento Utilizados na Pesquisa de Salmonella em Carcaças de Frango e Fezes de Aves Comparison of Different Enrichment Broth and Plating Media Used to Isolating Salmonella from Chicken Carcasses and Poultry Faeces Samples

Este trabalho foi desenvolvido para avaliar, comparativamente, os caldos de enriquecimento Rapapport-novobiocina (RVN), selenito-cistinanovobiocina (SCN) e tetrationato-novobiocina (TN) e os meios para plaqueamento ágar Hektoen (HE), ágar MacConkey (MC), ágar Salmonella-Shigella (SS), ágar verde brilhante (VB) e ágar xilose lisina desoxicolato (XLD), utilizados no isolamento de Salmonella em carcaças de frango e fezes de aves. O procedimento bacteriológico consistiu das etapas de pré-enriquecimento, enriquecimento em caldos seletivos, plaqueamento, testes bioquímicos presuntivos e confirmação sorológica com soros polivalentes anti antígenos somáticos e flagelares de Salmonella.. As fezes foram experimentalmente contaminadas com 8 sorotipos de Salmonella (Agona, Anatum, Enteritidis, Havana, Infantis, Owakam, Schwazengrund e Typhimurium) e a concentração final foi aproximadamente de 1,2 x 10² ufc/g. As fezes foram inoculadas nos caldos enriquecedores e a partir daí, seguiu-se o mesmo procedimento utilizado para as carcaças. Os resultados referentes às carcaças de frango não foram estatisticamente diferentes (p>0,01) para os caldos e as placas. Todavia, verificou-se superioridade numérica em relação aos caldos SCN e TN sobre o caldo RVN, e em relação ao ágar XLD sobre os demais. Verificou-se também que com o emprego de dois caldos de enriquecimento e de dois meios para plaqueamento pode-se obter maior positividade. Quanto ao exame das fezes, o caldo TN mostrou-se superior aos demais (p>0,01), não havendo diferença (p>0,01) de resultados para os meios de plaqueamento. Os resultados sugerem que a utilização de mais de um meio de enriquecimento e de plaqueamento poderia aumentar as chances de isolamento de Salmonella.<br>This study was undertaken to compare selenite-cystine-novobiocin (SCN) broth, tetrationate-novobiocin (TN) broth and Rappaport-Vassiliadisnovobiocin (RVN) broth as enrichment and MacConkey (MC) agar, brilliant green (BG) agar, Hektoen (HE) agar, Salmonella-Shigella (SS) agar and xylose Iysine desoxycholate (XLD) agar as plating media for isolating Salmonella from broiler carcasses and chicken feces. The carcasses were washed with 300mL of Buffer Peptone Water 0.1% (BPW). The BPW solation was incubated 6h at ambient temperature and up to 24 h/43ºC. From this, 2 mL were inoculate in 20 mL of enrichment broth, incubated at 43ºC/24h. The enrichment broth was plated on the five tested selective agars, also incubated at 43ºC/24h. Up to five colonies from each plate were inoculated in TSI agar and LIA agar, incubated at 37ºC/24h. The genus of Salmonella was confirmed by slide agglutination test using poly serum anti-somatic Salmonella antigens (O) and poly serum anti-fiagellar (H) Salmonella antigens. The feces were experimentally contaminated with eight Salmonella serotypos (Agona, Anatum, Enteritidis, Havana, Intantis, Owakam, Schwazengrundand Typhimurium). Each serotype was tested five times. Salmonella serotypes were added in the sample of feces individually so that the final concentration of the bacterium was 1,2 x iO³ ufclg. Two grams of each sample were inoculated in 20 mL of the enrichment broth. The proceduring from this point wes the same adopted to carcasses examination. To broiler carcasses there was no statistic difference (p>0, 001) among the enrichment broth and among plating media. However the use of two enrichment broth (SCN and TN) and two agars between XLD agar, SS agar and HE agar gave the bost results. The assay done with feces showed that the TN broth was much better than the others and, in this case it can be plated either HE agar, or VB agar or SS agar The results from the broiler carcasses examination and the feces examination showed that it is not easy to determine the better bacteriologic routine to search for Salmonella and suggest that the association of two eorichments broth and two plating media may improve the test