Insights into the cell envelope of Paracoccus denitrificans, a member of the α-subdivision of purple bacteria, through studies of its lysozyme susceptibility

Exponential-phase Paracoccus denitrificans cells grown in a complex medium are known to be lysozyme susceptible without pretreatment with outer membrane perturbing agents. Stationary-phase cells are more lysozyme resistant, but simple washing of these cells with moderate concentrations (154 mM) of monovalent inorganic cations (NaCl, KCl, or LiCl), or 100 mM Tris(hydroxymethyl)aminomethane-HCl (Tris–HCl) rendered them lysozyme susceptible. Subsequent washing with divalent cations reversed the enhanced lysozyme susceptibility. Cells grown in succinate–salts medium were not rendered lysozyme susceptible by NaCl washing. Supplementation of complex medium with various salts used in succinate–salts medium showed that Mg2+ and Ca2+ supplementation resulted in increased growth yields and in cells that were not rendered lysozyme susceptible by NaCl washing. Measurement of the Mg2+ and Ca2+ content of peptone and yeast extract revealed Mg2+ and Ca2+ were present at 50–80 and 15–30 μM concentrations, respectively, in complex medium. Omission of Ca2+ or reduction of Mg2+ from 810 to 50 μM in succinate–salts medium resulted in cells that became lysozyme susceptible after NaCl washing. Incubation of cells grown in succinate–salts medium with Tris–HCl – ethylenediaminetetraacetate – sucrose caused them to become lysozyme susceptible, indicating that their lack of lysozyme susceptibility was due to a more effective outer membrane barrier than that of complex medium grown cells, rather than a lysozyme-resistant peptidoglycan. The protein composition of outer membranes from complex and complex + Mg2+ + Ca2+ grown cells was similar, suggesting that divalent cations did not have a major influence on protein production related to outer membrane stability. The results indicate that certain complex bacteriological media may be deficient in divalent cations for maximum cell envelope stability and growth yield. This might be particularly significant for soil bacteria such as P. denitrificans, which may have evolved to have outer membranes more dependent on divalent cations for structural integrity than animal host adapted bacteria such as enteric bacteria. The results also draw attention to NaCl as an outer membrane perturbing agent.