Department of Chemistry and Biochemistry

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Browsing Department of Chemistry and Biochemistry by Subject "C-terminal protease"

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    Altered Envelope Structure and Nanomechanical Properties of a C-Terminal Protease A-Deficient Rhizobium leguminosarum
    (Multidisciplinary Digital Publishing Institute, 2020-09-16) Jun, Dong; Idem, Ubong; Dahms, Tanya E. S.
    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).
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    Metabolic adaptation of C-terminal protease A-deficient R. leguminosarum in response to loss of nutrient transport
    (Frontiers Media, 2018-01-04) Jun, Dong; Minic, Zoran; Bhat, Supriya V.; Vanderlinde, Elizabeth M.; Yost, Chris K.; Babu, Mohan; Dahms, Tanya E. S.
    Post-translational modification expands the functionality of the proteome beyond genetic encoding, impacting many cellular processes. Cleavage of the carboxyl terminus is one of the many different ways proteins can be modified for functionality. Gel-electrophoresis and mass spectrometric-based techniques were used to identify proteins impacted by deficiency of a C-terminal protease, CtpA, in Rhizobium leguminosarum bv. viciae 3841. Predicted CtpA substrates from 2D silver stained gels were predominantly outer membrane and transport proteins. Proteins with altered abundance in the wild type and ctpA (RL4692) mutant, separated by 2D difference gel electrophoresis, were selected for analysis by mass spectrometry. Of those identified, 9 were the periplasmic solute-binding components of ABC transporters, 5 were amino acid metabolic enzymes, 2 were proteins involved in sulfur metabolism, and 1 each was related to carbon metabolism, protein folding and signal transduction. Alterations to ABC-binding-cassette transporters, nutrient uptake efficiency and to amino acid metabolism indicated an impact on amino acid metabolism and transport for the ctpA mutant, which was validated by measured amino acid levels.