Transcriptomic Analysis Followed by the Isolation of Extracellular Bacteriolytic Proteases from Lysobacter capsici VKM B-2533T

Abstract

The aim of the study was to search for, isolate and characterize new bacteriolytic enzymes that show promising potential for their use in medicine, agriculture and veterinary. Using a transcriptomic analysis, we annotated in Lysobacter capsici VKM B-2533^T the genes of known bacteriolytic and antifungal enzymes, as well as of antibiotics, whose expression levels increased when cultivated on media conducive to the production of antimicrobial agents. The genes of the secreted putative bacteriolytic proteases were also annotated. Two new bacteriolytic proteases, Serp and Serp3, were isolated and characterized. The maximum bacteriolytic activities of Serp and Serp3 were exhibited at low ionic strength of 10 mM Tris-HCl, and high temperatures of, respectively, 80 °C and 70 °C. The pH optimum for Serp was 8.0; for Serp3, it was slightly acidic, at 6.0. Both enzymes hydrolyzed autoclaved cells of Micrococcus luteus Ac-2230^T, Proteus vulgaris H-19, Pseudomonas aeruginosa and Staphylococcus aureus 209P. Serp also digested cells of Bacillus cereus 217. Both enzymes hydrolyzed casein and azofibrin. The newly discovered enzymes are promising for developing proteolytic antimicrobial drugs on their basis.

Keywords: Lysobacter capsici; RNA-seq; antimicrobial potency; extracellular bacteriolytic enzymes; homologous expression system.

Figure 1

Dynamics of growth and bacteriolytic activity of Lysobacter capsici VKM B-2533^T cultivated on media RM (a), SYM (b) and 5/5 (c) for 32 h. Blue bars indicate the total bacteriolytic activity of the culture fluid with respect to autoclaved cells of Staphylococcus aureus 209P; red bars indicate the same with respect to living S. aureus 209P cells. The antifungal activity of the culture fluid after cultivation on media RM (1), 5/5 (2) and SYM (3) for 19 h with respect to Fusarium solani (d) and Sclerotinia sclerotiorum (e). Medium 5/5 was used as a negative control. d, Lysis zone diameter measured from the center of the well to the edge of the lysis zone.

Figure 2

Volcano plots of gene expression for RM (a) and SYM (b) media. Genes that significantly changed their expression levels on both media are highlighted in black. 1, Locus tag IEQ11_03495 (UOF15745.1); 2, IEQ11_04180 (UOF15870.1); 3, IEQ11_06885 (UOF16369.1); 4, IEQ11_09745 (UOF16892.1); 5, IEQ11_12530 (UOF17397.1); 6, IEQ11_14225 (UOF12917.1); 7, IEQ11_15570 (UOF13168.1); 8, IEQ11_15580 (UOF13170.1); 9, IEQ11_17420 (UOF13513.1); 10, IEQ11_22400 (UOF14439.1); 11, IEQ11_23755 (UOF14691.1); 12, IEQ11_08595 (UOF16681.1); 13, IEQ11_14490 (UOF12968.1). Blue and red triangles, the genes of interest. Red triangles, the genes chosen to be studied.

Figure 3

Area-proportional Venn diagrams showing the number of DEGs that overlapped between RM (red) and SYM (blue) media. The diagrams show the number of induced (a) and repressed (b) genes, respectively.

Figure 4

Purification of bacteriolytic enzymes Serp and Serp3 from the culture fluid of L. capsici expression strains. (a) Serp and Serp3 purification schemes. Red arrows indicate the Serp purification scheme; green arrows indicate the Serp3 purification scheme. (b) SDS-PAGE: 1, 4, 6 and 8, markers; 2, the culture fluid of wild-type L. capsici (Supplementary File S1 Figure S5); 3, the culture fluid of L. capsici P_Gro(A)–serp3(6his) (Supplementary File S1 Figure S5); 5, the culture fluid of L. capsici P_T5–serp(6his) (Supplementary File S1 Figure S6); 7, purified Serp3 of L. capsici P_Gro(A)–serp3(6his) (0.18 μg) (Supplementary File S1 Figure S7); 9, purified Serp of L. capsici P_T5–serp (0.35 μg) (Supplementary File S1 Figure S8). A 12 μL amount of the preparations was applied to the electrophoresis.

Figure 5

Optimal conditions of hydrolysis of autoclaved S. aureus 209P cells by bacteriolytic enzymes Serp3 (a–c) and Serp (d–f).

Figure 6

Specificity of action of the bacteriolytic enzymes Serp and Serp3 with respect to protein: (a) azofibrin; (b) casein. K, 10 mM Tris-HCl, pH 8.0. The diameter of the zones was measured from the center of the well to the edge of the proteolysis zone.