期刊
PEERJ
卷 7, 期 -, 页码 -出版社
PEERJ INC
DOI: 10.7717/peerj.6951
关键词
Resuscitation promoting factor; Rhodococcus erythropolis; VBNC; Muralytic activity; Resuscitation
资金
- National Natural Science Foundation of China [31272694, 31760028]
Resuscitation-promoting factors (Rpf) are a class of muralytic enzymes, which participate in recovery of dormant cells and promoting bacteria growth in poor media. In the present study the expression vector of the rpf-1 gene from an oil-degrading bacterium Rhodococcus erythropolis KB1 was constructed and expressed in Escherichia coli. The expressed protein was purified by Ni2+-affinity chromatography, and showed muralytic activity when measured with 4-methylumbelliferyl-beta-D-N,N',N ''-triacetyl chitotrioside as substrate. Addition of purified Rpf-1 to R. erythropolis culture efficiently improved bacterial cell growth. The purified protein also increased resuscitation of viable but nonculturable cells of R. erythropolis to culturable state. The conserved amino acid residues including Asp(45), Glu(51), Cys(50), Thr(60), Gln(69), Thr(74), Trp(75) and Cys(114) of the Rpf-1 were replaced with different amino acids. The mutant proteins were also expressed and purified with Ni2+-affinity chromatography. The muralytic activities of the mutant proteins decreased to different extents when compared with that of the wild type Rpf-1. Gln(69) was found to play the most important role in the enzyme activity, substitution of Gln(69) with lysine (Q69K) resulted in the greatest decrease of muralytic activity. The other amino acid residues such as Asp(45), Glu(51), Cys(50 )and Cys(114) were also found to be very important in maintaining muralytic activity and biological function of the Rpf-1. Our results indicated that Rpf- 1 from R. erythropolis showed muralytic activities and weak protease activity, but the muralytic activity was responsible for its growth promotion and resuscitation activity.
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