Altered Envelope Structure and Nanomechanical Properties of a C-Terminal Protease A-Deficient Rhizobium leguminosarum

Abstract

(1) Background: Many factors can impact bacterial mechanical properties, which play an important role in survival and adaptation. This study characterizes the ultrastructural phenotype, elastic and viscoelastic properties of Rhizobium leguminosarum bv. viciae 3841 and the C-terminal protease A (ctpA) null mutant strain predicted to have a compromised cell envelope; (2) Methods: To probe the cell envelope, we used transmission electron microscopy (TEM), high performance liquid chromatography (HPLC), mass spectrometry (MS), atomic force microscopy (AFM) force spectroscopy, and time-dependent AFM creep deformation; (3) Results: TEM images show a compromised and often detached outer membrane for the ctpA mutant. Muropeptide characterization by HPLC and MS showed an increase in peptidoglycan dimeric peptide (GlcNAc-MurNAc-Ala-Glu-meso-DAP-Ala-meso-DAP-Glu-Ala-MurNAc-GlcNAc) for the ctpA mutant, indicative of increased crosslinking. The ctpA mutant had significantly larger spring constants than wild type under all hydrated conditions, attributable to more highly crosslinked peptidoglycan. Time-dependent AFM creep deformation for both the wild type and ctpA mutant was indicative of a viscoelastic cell envelope, with best fit to the four-element Burgers model and generating values for viscoelastic parameters k₁, k₂, η₁, and η₂; (4) Conclusions: The viscoelastic response of the ctpA mutant is consistent with both its compromised outer membrane (TEM) and fortified peptidoglycan layer (HPLC/MS).

Keywords: C-terminal protease; Rhizobium leguminosarum; atomic force microscopy; cell envelope; force spectroscopy; viscoelasticity.

Figure 1

Representative atomic force microscopy (AFM) topography images and corresponding force curves of live Rhizobium leguminosarum bv. viciae 3841 (wild type, top row) and 3845 (ctpA mutant, bottom row). Shown are low (A,C) and high (B,D) resolution AFM images of the live wild type (A,B) and ctpA mutant (C,D) rhizobia. Bar for A and C is 500 nm. Red dots on A, C indicate the approximate locations at the top center of the live cells, for collecting corresponding force approach curves (E) with solid (wild type) and dashed (ctpA mutant) lines, and subsequent creep experiments.

Figure 2

TEM images of Rhizobium leguminosarum bv. viciae 3841 (wild type, top row), the majority of which show clearly defined inner and outer membranes, and 3845 (ctpA mutant, bottom row). Red arrows indicate detached outer membranes, which are more numerous in the mutant.

Figure 3

Bacterial spring constant of Rhizobium leguminsarum bv. viciae 3841 (wt) and 3845 (ctpA mutant) as a function of applied force. Spring constants were calculated from the ratio between the loading force and indentation depth during the linear response of a cell to the loading force. Values at 2 and 10 nN are statistically different (p < 0.05).

Figure 4

Histograms of viscoelastic parameters k₁, k₂, η₂, 1/η₁ for Rhizobium leguminsarum bv. viciae 3841 (wt, grey) and 3845 (ctpA mutant, pink) at low loading forces (2, 4 nN) during creep deformation experiments. k₁ and k₂ are elastic spring constants, and η₁ and η₂ are viscosity parameters from the Burgers model, in which the Maxwell and Kelvin–Voigt models are connected in series.