The Gonococcal NlpD Protein Facilitates Cell Separation by Activating Peptidoglycan Cleavage by AmiC

Abstract

Key steps in bacterial cell division are the synthesis and subsequent hydrolysis of septal peptidoglycan (PG), which allow efficient separation of daughter cells. Extensive studies in the Gram-negative, rod-shaped bacterium Escherichia coli have revealed that this hydrolysis is highly regulated spatially and temporally. Neisseria gonorrhoeae is an obligate Gram-negative, diplococcal pathogen and is the only causative agent of the sexually transmitted infection gonorrhea. We investigated how cell separation proceeds in this diplococcal organism. We demonstrated that deletion of the nlpD gene in strain FA1090 leads to poor growth and to an altered colony and cell morphology. An isopropyl-beta-D-galactopyranoside (IPTG)-regulated nlpD complemented construct can restore these defects only when IPTG is supplied in the growth medium. Thin-section transmission electron microscopy (TEM) revealed that the nlpD mutant strain grew in large clumps containing live and dead bacteria, which was consistent with deficient cell separation. Biochemical analyses of purified NlpD protein showed that it was able to bind purified PG. Finally, we showed that, although NlpD has no hydrolase activity itself, NlpD potentiates the hydrolytic activity of AmiC. These results indicate that N. gonorrhoeae NlpD is required for proper cell growth and division through its interactions with the amidase AmiC. IMPORTANCE N. gonorrhoeae is the sole causative agent of the sexually transmitted infection gonorrhea. The incidence of antibiotic-resistant gonococcal infections has risen sharply in recent years, and N. gonorrhoeae has been classified as a "superbug" by the CDC. Since there is a dearth of new antibiotics to combat gonococcal infections, elucidating the essential cellular process of N. gonorrhoeae may point to new targets for antimicrobial therapies. Cell division and separation is one such essential process. We identified and characterized the gonococcal nlpD gene and showed that it is essential for cell separation. In contrast to other pathogenic bacteria, the gonococcal system is streamlined and does not appear to have any redundancies.

LytM factor Alr3353 affects filament morphology and cell–cell communication in the multicellular cyanobacterium Anabaena sp. PCC 7120

“…Thus, Alr3353 seems to target the catalytic domain of AmiC, which results in enhanced hydrolase activity. Likewise, in Neisseria gonorrhoeae , NlpD potentiated the activity of AmiC, which had a high basal activity in comparison to E. coli AmiC (Stohl et al ., ).…”

Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae

“…Cloning of His 6 -tagged NlpD for protein purification was performed as described elsewhere (30). Briefly, nlpD lacking its predicted signal sequence was amplified from FA1090 chromosomal DNA by PCR using primers NlpD-10 and NlpD-11 and then cloned into pCR-BLUNT.…”

“…Protein Purification-Overexpression and purification of His 6 -tagged AmiC and NlpD were performed as described elsewhere (30), and AmiC Q316K was purified in essentially the same manner as wild-type AmiC. To overexpress a C-terminal version of AmiC used for metal dependence assays, cells harboring the pT7Htb-AmiC plasmid were incubated at 37°C in LB kan until an A 600 of ϳ0.6 -0.8 was reached.…”

“…Because the Q316K mutation was intended to mimic the activation provided by the LytM domain activator protein NlpD, we also tested a mixture of purified His-AmiC with His 6 -NlpD to determine: 1) whether NlpD augments AmiC activity in N. gonorrhoeae and 2) whether the Q316K mutation and/or the addition of NlpD facilitate production of the same products. NlpD in N. gonorrhoeae contains a predicted M23B peptidase domain but lacks two of the four residues predicted to be necessary for the enzymatic function seen in Gram-positive bacteria (30). Residues Asn-304 and Gln-383 in NlpD of N. gonorrhoeae strain MS11 are the same two residues that are altered from the consensus active site in E. coli, leading to a loss of enzyme activity in E. coli EnvC (25) and NlpD.…”

“…The crystal structure of E. coli AmiC reveals an ␣-helix with a glutamine (Gln-299) in a similar position, predicting a comparable relationship between AmiC and NlpD, which have been shown to bind to each other (21). Along with an AmiC homolog, gonococci contain an NlpD homolog that has an important role in normal cell separation, enhances the activity of AmiC, and binds PG, all without any apparent enzymatic activity (30).…”