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

Bioinformatics analysis of the different Rpf proteins

The AlphaFold 3 was used to predict the tertiary structure of Rpf2 and its mutant proteins of R. erythropolis KB1 (Abramson et al., 2024). The protein tertiary structure visualization was employed by VMD software (Humphrey, Dalke & Schulten, 1996). The Interpro was used for domain prediction and DOG was used for visualization (Ren et al., 2009; Paysan-Lafosse et al., 2023).

Bacterial strains and growth conditions

R. erythropolis KB1 (NCBI accession number: CP050124.1) was isolated from petroleum-contaminated soil and preserved in environmental biotechnology laboratory, school of petrochemical engineering, Lanzhou University of Technology. The bacterial cells were cultivated on Lysogeny-broth (LB) solid medium at 30 °C. The E. coli strains were also grown on a solid LB medium at the same temperature. The bacterial growth was monitored at OD₆₀₀ nm using a UV-visible spectrophotometer (Unico UV-5120) at predefined time intervals.

Construction of the expressing plasmids containing the rpf 2 genes with different domain deletions and their expression

We first explored the sequence characteristics of Rpf2 and its variants lacking 1, 2, and 3 DUF348 domains. These sequences of the wild-type Rpf2 and its variants were then synthesized by Nanjing Zhongding Biotechnology Co., Ltd., and the expressing pET28a (+) plasmids containing rpf2 genes with different DUF domain delusions were constructed. The constructed expression vectors were verified by restriction enzyme cleavage and sequencing. The verified plasmids were successfully transformed into E. coli BL21 (DE3) (Studier & Moffatt, 1986).

E. coli BL21 (DE3) cells harboring the pET-28a (+)-rpfs plasmid were inoculated into five mL of LB liquid medium supplemented with 50 µg/mL kanamycin and cultured at 37 °C with shaking overnight. This culture was then transferred to 500 mL of the same medium (containing 50 µg/mL kanamycin). Induction was carried out with 0.6 mmol/L isopropyl- β-D-thiogalactoside (IPTG) at 20 °C for 5.5 h. Subsequently, the Rpf protein was purified using a Ni²⁺ agarose affinity chromatography column and analyzed by SDS-PAGE with Coomassie brilliant blue staining.

Enzymatic and biological activity assays of the recombinant Rpf proteins

The muralytic activities of the recombinant Rpf proteins were evaluated using the fluorescent substrate 4-methylumbelliferyl- β-D-N, N′, N″-triacetyl chitotrioside (Sigma, Germany). The enzymatic activities of the Rpfs were quantified by measuring the production of 4-methylumbelliferone from substrate hydrolysis in a 96-well microplate. In each well, 100 µL of substrate buffer containing the substrate was combined with 5 µL of the purified recombinant Rpf proteins. The reaction mixture was incubated at 37 °C in a water bath for 30 min. The reaction was stopped by adding 100 µL of glycine-sodium hydroxide termination buffer. The fluorescence intensity of the resultant 4-methylumbelliferone was measured using a microplate reader with an excitation wavelength of 355 nm and emission wavelength of 460 nm. The measured values were compared against a standard curve of 4-methylumbelliferone. One unit (U) of the enzyme activity was defined as the amount of enzyme producing 1.0 nmol of product per microgram of the fusion protein per hour under these conditions (Li et al., 2017).

Effect of temperature and metal ions on the muralytic activities of the purified Rpf proteins

To evaluate the effect of temperature on the stability of enzyme activity, The Rpf proteins were incubated at different temperatures of 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, and 80 °C for 30 min, followed by measurement of their muralytic activity.

Individual stock solutions of zinc chloride (ZnCl₂), magnesium chloride (MgCl₂), cobalt chloride (CoCl₂), calcium chloride (CaCl₂), and manganese chloride (MnCl₂) were prepared at a concentration of 10 mmol/L. Subsequently, each metal ion solution was introduced into the reaction system, with a final metal ion concentration of 0.1 mmol/L. The reaction mixtures were then incubated at 37 °C for 30 min. A control sample, containing the Rpf protein reaction system without metal ions was also prepared to establish a baseline for comparison. The muralytic activity of the Rpf protein in each sample was quantified as previously described method.

The growth-promotion effect of the recombinant Rpf proteins on R. erythropolis

To investigate the promoting effect of the recombinant Rpf proteins on R. erythropolis cells, 5 µL of logarithmic-phase R. erythropolis KB1 cells was added to five mL of LB liquid medium. The recombinant Rpf proteins were then added at a concentration of 10 nM, all the proteins were sterilized through a 0.22 µm filter. Inactivated recombinant Rpf protein was used as the control group (the protein was inactivated in boiling water for 5 min). The cultures were incubated in a shaking incubator at a constant shaking rate of 180 rpm and temperature of 30 °C. At specified time intervals (0, 6, 12, 24, 36, 48, 60, 72, and 84 h), the samples of 0.2 mL were withdrew and optical density of the cultures was measured at 600 nm using a UV-visible spectrophotometer. All the experiments were run in three replications.

Effect of the recombinant Rpf proteins on resuscitation of the VBNC R. erythropolis cells

The method for prepare VBNC cells was described as follow: The R. erythropolis KB1 cells were inoculated into five mL of LB liquid medium and cultured overnight. The culture was centrifuged at 3,500 × g for 5 min, and the resulting pellet was resuspended in physiological saline. The washing procedure was repeated for three times. The washed cells were then inoculated into 500 mL of sterile physiological saline and incubated at 4 °C to induce the VBNC state. The VBNC cells of R. erythropolis KB1 used in this study had maintained at 4 °C for approximately 2,000 days (Luo et al., 2019). In each experiment, five mL of the VBNC R. erythropolis KB1 cells was transferred into a sterile tube. A solution of the recombinant Rpf protein filtered through a 0.22 µm membrane was then added at a concentration of 10 nM, followed by a sterilized yeast extract solution (5 g/L) at a final concentration of 0.025% (v/v). The inoculated tubes were incubated at 180 rpm and 30 °C. Samples of 0.1 mL were withdrew at intervals of 0, 12, 24, 36, 48, 60, 72, and 84 h and measured for optical density with a UV-visible spectrophotometer at 600 nm. The culturable cells were also determined with plate counting method. The inactivated recombinant Rpf2 was used as a negative control.

Statistical analysis

All experiments were performed in three replications and the results were presented as the mean ± standard deviation (SD). One-way ANOVA and comparison of means with Tukey’s honestly significant difference post-hoc test (P < 0.05) was used to analyze the experimental results (Graphpad Prism, La Jolla, CA, USA). The graphs were constructed with Origin 2018 software (OriginLab Corp., Northampton, MA, USA) and Graphpad Prism.

Bioinformatic analysis of the different Rpf proteins of R. erythropolis KB1

The critical domains and tertiary structure of the Rpf2 protein from R. erythropolis KB1 and its variants lacking the DUF348 domain were analyzed (pTM scores from Alphafold3 are shown in Table S2). In the wild-type Rpf2, the catalytic domain (Rpf domain), three tandem DUF348 domains, and the G5 structure were observed (Fig. 1). In the absence of the DUF348 domains, the Rpf domain remained unaltered, whereas the G5 structure exhibited increased flexibility, and the N-terminal conformation was altered. The loss of the DUF348 domains may weaken their direct or indirect interaction with the G5 structure, which could impair overall functional coordination and lead to a reduction in enzymatic activity (Fig. 2). This evidence suggests that the DUF348 domains play a critical role in maintaining protein structure and stability. Furthermore, a comparative structural analysis was performed on Rpf2 from R. erythropolis KB1 and RpfB from Mycobacterium tuberculosis. This structural similarity suggests the conservation of functional domains, potentially indicating conserved roles in bacterial physiology across species.

Expression and enzyme activities of the recombinant Rpf proteins

We successfully constructed, expressed, and purified the full-length Rpf2 protein as well as the different DUF348 domain deleted proteins. The SDS-PAGE gel electrophoresis results of the purified recombinant proteins were show in Fig. 3, and the protein bands in the gel highly matched our expectations.

The standard muralytic activity assay method was used to evaluate the catalytic function of the recombinant proteins. All of the purified Rpf proteins showed significant muralytic activities (Fig. 4), but an inverse relationship between muralytic activity and the number of deleted DUF348 domains was observed, indicating the pivotal roles of the DUF348 domains in catalytic activity of the Rpf protein.

Muralytic activity of the recombinant Rpf proteins at different temperatures and metal ions

The muralytic activities of the recombinant Rpf2 protein and its three variants (1 ΔDUF348, 2 ΔDUF348, and 3 ΔDUF348) were evaluated at a temperature range of 20 °C to 80 °C (Fig. 5). The optimal temperature for maximal muralytic activity of the Rpf2 proteins was observed at 30 °C, the activity of native Rpf2 reached its peak activity of 755 U, while the activities of the different Rpf2 variants were lower than that of the Rpf2. The activities of all proteins decreased with the increasing of temperature. The native Rpf2 consistently exhibited higher muralytic activity than its variants at each temperature point. As the temperature increased to 40 °C and 50  °C, all proteins displayed a significant decline in activities, with native Rpf2 retaining relatively higher activity levels (approximately 724 U and 377 U, respectively) compared to its variants, which indicated the important role of the DUF348 domains in the structural and functional stability of Rpf2, especially under optimal and elevated temperatures.

We also assessed the effect of different metal ions on the muralytic activity of the recombinant Rpf proteins. We found that addition of Zn²⁺ and Mn²⁺led to a statistically significant enhancement of the muralytic activity of Rpf proteins (p < 0.05), indicating that these ions play a crucial role as potent cofactors. The enhancement of muralytic activity by Zn²⁺ and Mn²⁺was less pronounced in the variants compared to the native Rpf protein, suggesting that the DUF348 domain may play a role in optimizing the binding and interaction of these metal ions with the enzyme. Mg²⁺, Co²⁺, and Ca²⁺ showed little increases in enzymic activities (p > 0.05) (Fig. 6).

Effects of the recombinant Rpf proteins on the growth of R. erythropolis KB1

We evaluated the effects of the recombinant proteins on the growth of R. erythropolis KB1 cells. It was found that the R. erythropolis populations in the groups without Rpf2 or containing the inactivated Rpf2 kept the same. In contrast, addition of the recombinant Rpf2 protein and its mutants significantly promoted growth of the R. erythropolis KB1 cells. Notably, the growth promotion effect was positively correlated with the number of DUF348 domains in these proteins (Fig. 7). The wild-type Rpf2 and mutants lacking different amounts of DUF348 had significant differences in their growth-promoting effects on R. erythropolis KB1 (Table S3). The intact Rpf2 protein has the most pronounced growth-promoting effect at the same molar amount, this effect is diminished as the number of DUF348 structural domains decreases. This observation is consistent with our muralytic activity assay and previous experiments, confirming a positive correlation between the muralytic activities of the Rpf proteins and their abilities to promote cell growth (Luo et al., 2019).

Resuscitation effect of the recombinant Rpf proteins on the VBNC state cells of R. erythropolis KB1

Resuscitation effect of the different recombinant Rpf proteins on the VBNC state cells was observed by addition of the recombinant Rpf proteins in the VBNC R. erythropolis KB1 cells. Our results showed that the growth patterns of the experimental groups (without recombinant Rpf protein) and the control group (with inactivated protein) were similar, neither demonstrated a significant resuscitation effect on VBNC R. erythropolis KB1 cells (Fig. 8). The experimental groups with the recombinant Rpf proteins showed pronounced resuscitation and growth promotion effects on the VBNC cells. Further analysis indicated an inverse relationship between the number of missing DUF348 domains and the resuscitation level, highlighting the importance of DUF348 domains in Rpf protein functionality. Deletion of these domains significantly reduced the promoting capabilities of the mutant proteins when compared with the wild-type Rpf2. Additionally, the promoting effect on the VBNC cells was also correlated with their muralytic activities.