Development of an intergeneric conjugal transfer system for rimocidin‐producing Streptomyces rimosus

Aims: To develop an intergeneric conjugation system for rimocidin‐producing Streptomyces rimosus. Methods and Results: High efficiencies of conjugation [10^?2–10^?3 transconjugants/recipient colony forming units (CFU)] were obtained when spores of S. rimosus were heat treated at 40°C for 10 min prior to mixing with E. coli ET12567(pUZ8002/pIJ8600) as donor. Mycelium from liquid grown cultures of S. rimosus could also be used as recipient instead of spores, with 24‐h cultures giving optimal results. TSA (Oxoid) medium containing 10 m mol l^?1 MgCl₂ was the preferred medium for conjugation. Southern hybridization was used to confirm that transconjugants of S. rimosus contained a single copy of pIJ8600 integrated at a unique chromosomal attachment site (attB). The transconjugants exhibited a high stability of plasmid integration and showed strong expression of green fluorescent protein when using pIJ8655 as the conjugative vector. Conclusion: Intergeneric conjugation between E. coli and S. rimosus was achieved at high efficiency using both spores and mycelium. Significance and Impact of the Study: The conjugation system developed provides a convenient gene expression system for S. rimosus R7 and will enable the genetic manipulation of the rimocidin gene cluster.     

Non-native,N-terminal Hsp70 Molecular Motor Recognition Elements in Transit Peptides Support Plastid Protein Translocation

Previously, we identified the N-terminal domain of transit peptides (TPs) as a major determinant for the translocation step in plastid protein import. Analysis of Arabidopsis TP dataset revealed that this domain has two overlapping characteristics, highly uncharged and Hsp70-interacting. To investigate these two properties, we replaced the N-terminal domains of the TP of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its reverse peptide with a series of unrelated peptides whose affinities to the chloroplast stromal Hsp70 have been determined. Bioinformatic analysis indicated that eight out of nine peptides in this series are not similar to the TP N terminus. Using in vivo and in vitro protein import assays, the majority of the precursors containing Hsp70-binding elements were targeted to plastids, whereas none of the chimeric precursors lacking an N-terminal Hsp70-binding element were targeted to the plastids. Moreover, a pulse-chase assay showed that two chimeric precursors with the most uncharged peptides failed to translocate into the stroma. The ability of multiple unrelated Hsp70-binding elements to support protein import verified that the majority of TPs utilize an N-terminal Hsp70-binding domain during translocation and expand the mechanistic view of the import process. This work also indicates that synthetic biology may be utilized to create de novo TPs that exceed the targeting activity of naturally occurring sequences.     

Differential Transit Peptide Recognition during Preprotein Binding and Translocation into Flowering Plant Plastids

Despite the availability of thousands of transit peptide (TP) primary sequences, the structural and/or physicochemical properties that determine TP recognition by components of the chloroplast translocon are not well understood. By combining a series of in vitro and in vivo experiments, we reveal that TP recognition is determined by sequence-independent interactions and vectorial-specific recognition domains. Using both native and reversed TPs for two well-studied precursors, small subunit of ribulose-1,5-bis-phosphate carboxylase/oxygenase, and ferredoxin, we exposed these two modes of recognition. Toc34 receptor (34-kD subunit of the translocon of the outer envelope) recognition in vitro, preprotein binding in organellar, precursor binding in vivo, and the recognition of TPs by the major stromal molecular motor Hsp70 are specific for the physicochemical properties of the TP. However, translocation in organellar and in vivo demonstrates strong specificity to recognition domain organization. This organization specificity correlates with the N-terminal placement of a strong Hsp70 recognition element. These results are discussed in light of how individual translocon components sequentially interact with the precursor during binding and translocation and helps explain the apparent lack of sequence conservation in chloroplast TPs.