RESUMO
Bacillus subtilis 168 EpsC is annotated as "Probable polysaccharide biosynthesis protein" in the SwissProt database. epsC is part of the eps operon, thought to be involved in the biosynthesis of exopolymeric substances (EPS). The present study was undertaken to determine the molecular function of EpsC. Sequence analysis of EpsC suggested the presence of a transmembrane domain. Two N-terminal deletion mutants in which residues 1-89 (EpsC89) and 1-115 (EpsC115) are deleted were cloned and overexpressed. Enzyme activity and substrate preferences were investigated by reverse phase HPLC, surface plasmon resonance (SPR) spectroscopy and absorption spectroscopy. These data show that EpsC has UDP-GlcNAc 4,6-dehydratase activity in vitro. Purified recombinant proteins were found to utilise UDP-Glc and TDP-Glc also as substrates. In addition, EpsC115 could utilise UDP-Gal and UDP-GalNAc as substrates whereas EpsC89 could only bind these two sugar nucleotides. These results show that deletion of a longer N-terminal region broadens substrate specificity. These broadened specificity is perhaps an outcome of the deletion of the putative transmembrane domain and may not be present in vivo. EpsC, together with the aminotransferase EpsN (Kaundinya CR et al., Glycobiology, 2018) and acetyltransferase EpsM (unpublished data), appears to be involved in the biosynthesis of N,N'-diacetylbacillosamine.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Hidroliases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Ensaios Enzimáticos , Escherichia coli/genética , Hidroliases/genética , Hidroliases/isolamento & purificação , Cinética , Mutação , Açúcares de Nucleosídeo Difosfato/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Domínios Proteicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , Ressonância de Plasmônio de SuperfícieRESUMO
In this study we have investigated the role of Epr, a minor extracellular serine protease, in the swarming motility of Bacillus subtilis 168. We identified that the protease activity of Epr was dispensable for swarming. Since the protease activity of Epr was confined to its N-terminal domain, we hypothesized instead that its C-terminal domain (CTD) could be critical for swarming. Our study showed that not only the expression of Epr-CTD was necessary, but also its secretion was crucial for the swarming motility of B. subtilis 168.