Expression of the HolC DNA polymerase III chi subunit (ZMO1308) was not detected. A targeted protein-affinity ‘pull-down’ approach such as the one
described here may be used to complement such large scale studies, verifying protein associations inferred by other in silico or experimental approaches. Conclusions Whilst quantitative (real time) PCR approaches have previously been used to establish plasmid copy numbers in microbes, this is the first time it has been used to evaluate plasmid levels in Zymomonas, or closely-related alphaproteobacterial species. Our results indicate that shuttle vectors containing the replicon from the pZMO7 (pZA1003) native plasmid from Z. mobilis NCIMB 11163 may be stably maintained in multi-copy levels for more than 50 generations in the ATCC 29191 and (ATCC 10988-derived)
CU1 Rif2 strains, in the absence Vistusertib cell line of a selectable marker. A selectable marker is required for stable pZMO7-derived NVP-BSK805 mouse shuttle vector maintenance in the parental NCIMB 11163 strain, most probably due to replicative competition with endogenous pZMO7 plasmids. The replication of pZMO7 and pZMO1 plasmids appears to function in an orthologous, and non-competitive manner. The pZMO7 shuttle vector-based expression of N-terminal GST-fusions of ‘bait’ proteins enables the composition of intracellular protein complexes in Z. mobilis to be probed in a convenient and straightforward manner. The co-purification of established and putative functionally-related proteins validates the use of this experimental approach. Taken together, our data suggests that the composition of protein complexes
within Z. mobilis cells may share significant similarity to those found in E. coli, Saccharomyces cerevisae and other microbial species [32–35]. Acknowledgements We are grateful to Prof. Constantin Drainas and Dr. Hideshi Yanase for providing us with Z. mobilis strains and plasmids. We also acknowledge Isoconazole the technical assistance of Mr. Alan Wong and Ms. Becky Cheung, and thank Dr. Tianfan Cheng for his help with the Western blotting experiments. We dedicate this paper to the life and work of Prof. Constantin Drainas. Funding General Research Fund of the Research Grants Council of Hong Kong (704508) to RMW. PROCORE France/Hong Kong Joint Research Scheme (F-HK31/06 T) to RMW and MS. Electronic supplementary material Additional file 1: Primers used in this study. (PDF 81 KB) Additional file 2: Restriction analysis of native plasmid DNA extracted from Z. mobilis NCIMB 11163. (PDF 208 KB) Additional file 3: Predicted positions of open reading frames and putative gene regulatory selleck inhibitor elements on plasmid pZMO7. (PDF 149 KB) Additional file 4: Stability of pZ7C shuttle vector in Z. mobilis NCIMB 11163, CU1 Rif2 and ATCC 29191 strains cultured in media with/without selection agent. (PDF 254 KB) Additional file 5: Quantitative-PCR determination of plasmid copy number for pZMO1A and pZMO7 in Z. mobilis NCIMB 11163 throughout the growth cycle.