Neisseria gonorrhoeae, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes and their regulation has proven to be elusive. Post-translational modifications (PTMs) of bacterial proteins are now recognized as one mechanism of protein regulation. In the present study, Western blot analyses, with an anti-acetyl-lysine antibody, indicated that a large number of gonococcal proteins are post-translationally modified. Previous work demonstrated that lysine acetylation can occur non-enzymatically with acetyl-phosphate (AcP) as the acetyl donor. In the current study, an acetate kinase mutant (ackA), which accumulates AcP, was generated in N. gonorrhoeae. Broth cultures of N. gonorrhoeae 1291wt and 1291ackA were grown, proteins extracted and digested, and peptides containing acetylated-lysines (K-acetyl) were affinity-enriched from both strains. Mass spectrometric analyses of these samples identified a total of 2686 unique acetylation sites. Label-free relative quantitation of the K-acetyl peptides derived from the ackA and wt samples demonstrated that 109 acetylation sites had an ackA/wt ratio>2 and p-values<0.05 in at least 2/3 of the biological replicates and were designated as “regulated” or AckA-dependent. Regulated K-acetyl sites were found in ribosomal proteins, central metabolism proteins, iron acquisition and regulation proteins, pilus assembly and regulation proteins, and a two-component response regulator. Since AckA is part of a metabolic pathway, comparative growth studies of the ackA mutant and wt strains were performed. The mutant showed a reduced growth rate under aerobic conditions, an inability to grow anaerobically and had a defect in biofilm maturation.

To identify specific lysine acetylation sites that were acetyl-phosphate (Ac-P) dependent, an ackA mutant, that accumulates AcP, was generated. Label-free relative quantitation (MS1 filtering) was used to compare acetylated peptides from wt with ackA samples.

Triplicate biological replicates of Neisseria gonorrhoeae strains 1291wt and 1291ackA were grown in 500 ml cultures of GC-broth supplemented with 1% IsoVitaleX and Kellogg’s supplement at 37°C, rotating at 150 rpm, overnight. Peptides containing acetylated-lysines (K-acetyl) were affinity-enriched from both strains. Two mass spectrometric injection replicates were acquired for each sample.