Cell envelope of Neisseria gonorrhoeae: relationship between autolysis in buffer and the hydrolysis of peptidoglycan

Abstract

Neisseria gonorrhoeae readily underwent autolysis when suspended in N-2-'hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at alkaline pH values. Autolysis was inhibited by the addition of Mg2" or other divalent cations. Autolysis was also suppressed at acid pH (pH 6.0). Suspension of cells in buffer was accompanied by the hydrolysis of peptidoglycan. The rate of peptidoglycan hydrolysis in HEPES buffer was maximal at pH 8.5 and was similar in the presence or absence of Mg2+. Therefore, divalent cation stabilization against autolysis is not mediated by inhibition of peptidoglycan hydrolysis. Peptidoglycan hydrolysis occurred in HEPES buffer (pH 6.0), but at a rate that was 50% of the maximum. Incubation of cells with chloramphenicol or rifampin before suspension in HEPES buffer (pH 8.5) partially prevented autolysis; under these conditions, peptidoglycan hydrolysis still occurred, but at a reduced rate. Old and new peptidoglycans were hydrolyzed at similar rates. Peptidoglycan hydrolysis results in solubilization of both the peptide and glycan moieties. When grown in complex media containing glucose, the peptidoglycan (PG) of virulent and avirulent gonococci constitutes 1 to 2% of the dry weight of the cell and contains muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid (DAP) in a ratio of 1:1:2:1:1 (9). A lipoprotein, covalently attached to the PG, has not been demonstrated in Neisseria gonorrhoeae (9, 22). The PG of this organism turns over at a high rate during exponential growth (approximately 50% per generation) (9). Hebeler and Young (10) reported that the enzyme principally responsible for this rapid turnover was N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28), which cleaves the amide linkage between N-acetylmuramic acid and L-alanine. This enzyme, when extracted by salt and detergent from cell walls and assayed in vitro with [3H]DAP-labeled PG as the substrate, exhibited a broad pH optimum and was insensitive to Mg2'. No hexaminidase (endo N-acetylglucosaminidase or endo N-acetylmuramidase), which catalyzes the hydrolysis of the glycan backbone of the PG, has been reported in N. gonorrhoeae. Gonococci resuspended in buffer at an alkaline pH undergo rapid autolysis (8). This autolysis can be prevented by the addition of divalent cations or by osmotic stabilization (3, 4

Phospholipid metabolism inNeisseria gonorrhoeae: Phospholipid hydrolysis in nongrowing cells

“…In several cases, the amount of 3H that was recovered by extraction of the cell pellet was significantly lower than that recovered from zero time cells. The lower recovery of 3H (87%) following incubation of cells at pH 8.5 in the absence of divalent cations correlates with the observation that these conditions promote cell autolysis (6,9). Cell lysis probably results in the release of membrane fragments which are not recovered by centrifugation.…”

“…The properties of these strains have been previously described (7,8). Cells were grown at 37 C in liquid medium containing IsoVitaleX (1% v/v), NaHCO3 (420 mg/1), and 0.5% (w/v) glucose (final pH = 7.2) as described (9). Growth was monitored turbidometrically in nephelometer flasks using a Klett Summerson colorimeter, filter No.…”

A peptidoglycan hydrolase similar to bacteriophage endolysins acts as an autolysin in Neisseria gonorrhoeae

“…The ODs of the cultures show only moderate decreases, and lysed cells still have some shape and size similar to intact cells. One explanation for this stability is that Mg 2þ , which is present in the growth medium, stabilizes the outer membrane lending strength to a broken cell wall (Wegener et al, 1977). Another possibility is that the normal function of AtlA is not to degrade whole strands of PG but rather to produce localized breaks in the PG.…”

“…Previous studies characterizing autolysis in N. gonorrhoeae have measured autolysis by following the decrease of optical density (OD) after the cells were transferred to non-growth conditions (Hebeler and Young, 1975;Elmros et al, 1976a;Wegener et al, 1977). We measured changes in OD after transferring cultures into Tris-buffered solution to determine whether the atlA mutation affected this type of lysis.…”

“…To label the PG, gonococci were grown for 2 h in GCBL containing 0.4% pyruvate and 6-[ 3 H]-glucosamine (New England Nuclear) at 2 Ci ml ¹1 (Wegener et al, 1977). Cells were washed with GCBL to remove unincorporated label.…”