Protealysin Targets the Bacterial Housekeeping Proteins FtsZ and RecA

Abstract

Serratia proteamaculans synthesizes the intracellular metalloprotease protealysin. This work was aimed at searching for bacterial substrates of protealysin among the proteins responsible for replication and cell division. We have shown that protealysin unlimitedly cleaves the SOS response protein RecA. Even 20% of the cleaved RecA in solution appears to be incorporated into the polymer of uncleaved monomers, preventing further polymerization and inhibiting RecA ATPase activity. Transformation of Escherichia coli with a plasmid carrying the protealysin gene reduces the bacterial UV survival up to 10 times. In addition, the protealysin substrate is the FtsZ division protein, found in both E. coli and Acholeplasma laidlawii, which is only 51% identical to E. coli FtsZ. Protealysin cleaves FtsZ at the linker between the globular filament-forming domain and the C-terminal peptide that binds proteins on the bacterial membrane. Thus, cleavage of the C-terminal segment by protealysin can lead to the disruption of FtsZ's attachment to the membrane, and thereby inhibit bacterial division. Since the protealysin operon encodes not only the protease, but also its inhibitor, which is typical for the system of interbacterial competition, we assume that in the case of penetration of protealysin into neighboring bacteria that do not synthesize a protealysin inhibitor, cleavage of FtsZ and RecA by protealysin may give S. proteamaculans an advantage in interbacterial competition.

Keywords: FtsZ; RecA; Serratia proteamaculans; interbacterial competition; protealysin.

Figure 1

(A). The actin cleavage by the OMVs components. Purified OMVs were incubated with actin at the volume ratio of 1:1 for 18 h at 4 °C. A—control actin. (B). The FtsZ cleavage by protealysin in solution. Purified E. coli FtsZ and A. laidlawii FtsZ were incubated with protealysin in the mass ratio indicated below for 18 h at 4 °C. C—control (corresponding FtsZ, uncleaved).

Figure 2

The FtsZ cleavage by protealysin in bacteria. (A). Growth curves of S. proteamaculans variants. Values are expressed as a mean of three repetitions ± S.D. (error bars). A difference was considered significant at the * p < 0.05 level. (B). Western blot detection of the expression of pln and m4in genes in S. proteamaculans. M—marker containing purified mature protealysin (Pln), protealysin precursor (proPln), and emfourin (M4in). WT, ∆(pln-m4in) and ∆m4in lanes were loaded with samples prepared from equal amounts of bacterial biomass. (C). Cleavage of actin by extracts of S. proteamaculans variants at 68 h of growth. (D). The number of FtsZ (estimated by Western blotting) in the same number of bacteria (determined by OD₆₀₀) at 16 h of growth. Values are expressed as mean of three repetitions ± S.D. (error bars). A difference was considered significant at the * p < 0.05 level. The insert shows a representative blot. An analysis of three technical replicates is presented. WT—wild-type S. proteamaculans; ∆(pln-m4in)—mutant S. proteamaculans with in-frame deletion of both genes of the protealysin operon; ∆m4in—mutant S. proteamaculans with in-frame deletion of emfourin gene. (E). The number of bacteria (CFU) in the bacterial suspensions at 20 and 47 h of growth normalized to the OD600 is shown in the insert.

Figure 3

The RecA cleavage by protealysin in solution. Purified E. coli RecA was incubated with protealysin in a Pln/RecA mass ratio of 1:20 for 10, 20 and 30 min at 22 °C. (A). The amount of uncleaved RecA was determined by electrophoregram (insert). (B). ATPase activity of RecA after 10, 20, 30 min incubations with protealysin.

Figure 4

UV survivability of bacteria (A). UV survivability of wild-type S. proteamaculans (WT, black circles), and mutant S. proteamaculans with an in-frame deletion of both genes of the protealysin operon (∆(pln-m4in), open circles) and with an in-frame deletion of the emfourin gene (∆m4in, black triangles). (B). UV survivability of E. coli AB1157 transformed with the control plasmid (black circles) and the plasmid carrying the protealysin gene (open circles).