RESUMO
Streptomycetes naturally produce a variety of secondary metabolites, in the process of physiological differentiation. Streptomyces venezuelae differentiates into spores in liquid media, serving as a good model system for differentiation and a host for exogenous gene expression. Here, we report the growth and differentiation properties of S. venezuelae ATCC-15439 in liquid medium, which produces pikromycin, along with genome-wide gene expression profile. Comparison of growth properties on two media (SPA, MYM) revealed that the stationary phase cell viability rapidly decreased in SPA. Submerged spores showed partial resistance to lysozyme and heat, similar to what has been observed for better-characterized S. venezuelae ATCC10712, a chloramphenicol producer. TEM revealed that the differentiated cells in the submerged culture showed larger cell size, thinner cell wall than the aerial spores. We analyzed transcriptome profiles of cells grown in liquid MYM at various growth phases. During transition and/or stationary phases, many differentiationrelated genes were well expressed as judged by RNA level, except some genes forming hydrophobic coats in aerial mycelium. Since submerged spores showed thin cell wall and partial resistance to stresses, we examined cellular expression of MreB protein, an actin-like protein known to be required for spore wall synthesis in Streptomycetes. In contrast to aerial spores where MreB was localized in septa and spore cell wall, submerged spores showed no detectable signal. Therefore, even though the mreB transcripts are abundant in liquid medium, its protein level and/or its interaction with spore wall synthetic complex appear impaired, causing thinner- walled and less sturdy spores in liquid culture.
Assuntos
Macrolídeos/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Streptomyces/crescimento & desenvolvimento , Streptomyces/metabolismo , Parede Celular/fisiologia , Cloranfenicol/biossíntese , Perfilação da Expressão Gênica , Metabolismo Secundário/fisiologia , Streptomyces/citologia , Transcriptoma/genéticaRESUMO
The bacterium Helicobacter pylori infects more than 50% of the world population and causes several gastroduodenal diseases, including gastric cancer. Nevertheless, we still need to explore some protein interactions that may be involved in pathogenesis. MreB, an actin homolog, showed some special characteristics in previous studies, indicating that it could have different functions. Protein functions could be realized via protein-protein interactions. In the present study, the MreB protein from H. pylori 26695 fused with two tags 10×His and GST in tandem was overexpressed and purified from Escherchia coli. The purified recombinant protein was used to perform a pull-down assay with H. pylori 26695 cell lysate. The pulled-down proteins were identified by mass spectrometry (MALDI-TOF), in which the known important proteins related to morphogenesis were absent but several proteins related to pathogenesis process were observed. The bacterial two-hybrid system was further used to evaluate the protein interactions and showed that new interactions of MreB respectively with VacA, UreB, HydB, HylB and AddA were confirmed but the interaction MreB-MreC was not validated. These results indicated that the protein MreB in H. pylori has a distinct interactome, does not participate in cell morphogenesis via MreB-MreC but could be related to pathogenesis.