| Summary: | (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 <i>Rhizobium leguminosarum</i> bv. <i>viciae</i> 3841 and the C-terminal protease A (<i>ctpA</i>) 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 <i>ctpA</i> 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 <i>ctpA</i> mutant, indicative of increased crosslinking. The <i>ctpA</i> 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 <i>ctpA</i> mutant was indicative of a viscoelastic cell envelope, with best fit to the four-element Burgers model and generating values for viscoelastic parameters k<sub>1</sub>, k<sub>2</sub>, η<sub>1</sub>, and η<sub>2</sub>; (4) Conclusions: The viscoelastic response of the <i>ctpA</i> mutant is consistent with both its compromised outer membrane (TEM) and fortified peptidoglycan layer (HPLC/MS).
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