Cloning and expression of Micrococcus luteus IAM 14879 Rpf and its role in the recovery of the VBNC state in Rhodococcus sp.DS471

Abstract:

Abstract:［Objective］The purpose of the present study was to produce the Rpf (resuscitation promoting factor) protein by cloning and expressing the rpf gene，secreted by Micrococcus luteus IAM 14879，in Escherichia coli and to evaluate its role in the recovery of the VBNC (viable but non-culturable) state in high-GC Gram-positive bacteria.［Methods］Genomic DNA was extracted from Micrococcus luteus IAM 14879 and the rpf gene was amplified by PCR using specific primers． The PCR products was purified，cloned into a pET15b expression vector，and transformed into Escherichia coli BL21(DE3)．Then the pET15b plasmid expression vector was used to confirm the purification of the recombinant proteins via SDS-PAGE． The VBNC state cells from the high-GC Gram-positive bacteria，Rhodococcus sp. DS471，were used to confirm the promotion and recovery of growth capacity． Rhodococcus sp． DS471 were isolated from soil and closely related to Micrococcus luteus IAM 14879． ［Results］The gene sequences confirmed that the rpf gene from Micrococcus luteus IAM 14879 that was expressed in Escherichia coli，was 672 bp． SDS-PAGE analysis showed that the recombinant Rpf protein was obtained successfully，and further studies showed it capable of promoting the recovery of the VBNC state by about 100-fold relative to the control．［Conclusion］Rpf of Micrococus luteus IAM 14879 can be successfully cloned and expressed in Escherichia coli and shows a strong ability to promote the recovery of the VBNC state of cells of Rhodococcus sp． DS471.