The DUF348 domains of resuscitation promoting factor 2 play important roles in the enzymatic and biological activities in <i>Rhodococcus erythropolis</i> KB1

<i>Rhodococcus erythropolis</i> KB1 is a member of the Actinomycetota and a petroleum-degrading bacterium, isolated from soil contaminated with petroleum products. The resuscitation-promoting factors (Rpf) widely exist among Actinomycetota, which revive the viable but nonculturable (VBNC) state cells and facilitate growth of normal cells. The Rpf2 of the <i>R. erythropolis</i> KB1 is the most complex Rpf protein, which consists of the conserved Rpf domain, one G5 domain and three DUF348 domains. The protein demonstrates muralytic activity and growth-promoting and resuscitation effect, but the exact roles of these DUF348 domains in the enzymic and biological activities remain unclear. In this paper, the recombinant plasmids containing <i>rpf</i>2 genes with different DUF348 domain deletion were constructed and expressed in <i>Escherichia coli</i>. The enzymatic and biological activities of the mutated Rpf2 proteins were examined. The results showed that the enzymatic activities of the mutated Rpf2 proteins with 1, 2, and 3 DUF348 deletion decreased by 26.27%, 38.17%, and 42.56% respectively when compared with that of the wild-type Rpf2. A negative correlation between the number of DUF348 deletions and the growth-promoting and resuscitation effect on <i>R. erythropolis</i> KB1 cells were also observed. The muralytic activities of the mutated Rpf2 proteins showed stability at the temperature range of 20 °C to 40 °C, but showed sharp declines at 50 °C, with the activity dropping by 50.07% to 90.06%, and complete loss at 70 °C and 80 °C, underscoring importance of the DUF348 in thermal stability of the Rpf2. Zn^{2 +} and Mn^{2 +} slightly enhanced the muralytic activity, while Mg^{2 +}, Ca^{2 +} and Co^{2 +} had negligible effects. These findings offered significant insights into mechanism of the Rpf action, emphasizing the critical role of the DUF348 domain.