O-Acetylation of Peptidoglycan in Gram-negative Bacteria

Abstract

The ape2 gene encoding a hypothetical O-acetylpeptidoglycan esterase was amplified from genomic DNA of Neisseria gonorrhoeae FA1090 and cloned to encode either the full-length protein or a truncated version lacking its hypothetical signal sequence. Expression trials revealed that production of the fulllength version possessing either an N-terminal or C-terminal His 6 tag was toxic to Escherichia coli transformants and that the host rapidly degraded the small amount of protein that was produced. An N-terminally truncated protein could be produced in sufficient yields for purification only if it possessed an N-terminal His 6 tag. This form of the protein was isolated and purified to apparent homogeneity, and its enzymatic properties were characterized. Whereas the protein could bind to insoluble peptidoglycan, it did not function as an esterase. Phenotypic characterization of E. coli transformants producing various forms of the protein revealed that it functions instead to O-acetylate peptidoglycan within the periplasm, and it was thus renamed peptidoglycan O-acetyltransferase B. This activity was found to be dependent upon a second protein, which functions to translocate acetate from the cytoplasm to the periplasm, demonstrating that the O-acetylation of peptidoglycan in N. gonorrhoeae, and other Gram-negative bacteria, requires a two component system. The bacterial cell wall heteropolymer peptidoglycan (PG)2 is comprised of alternating N-acetylglucosaminyl-␤-1,4-Nacetylmuramoyl residues with attached stem peptides. The existence of the peptides permits interpeptide cross-linking between neighboring strands of PG to provide a continuous three-dimensional macromolecule, the PG sacculus. This sacculus completely surrounds the bacterial cell over its cytoplasmic membrane to withstand the internal turgor pressure of the cell and thereby providing structural rigidity and shape to the cells. Given the essential role in the vitality of bacterial cells, the PG sacculus is the target of lytic enzymes such as lysozyme (muramidases), which are produced and released by eukaryotic hosts as the first line of defense against invasion. As found with the xylans of plant cell walls (reviewed in Ref.

Characterization of O-Acetylation of N-Acetylglucosamine

“…Both domains exhibit sequence identity with PatA (7 and 9% for OatA and OatB, respectively) and PatB (17 and 20%), which were identified, respectively, as the acyl donor transporter and O-acetyltransferase in Neisseria gonorrhoeae (supplemental Fig. S3) (16). Both acetyltransferase domains of OatA and OatB were assigned to the family of SGNH/GDSL hydrolases (LOMETS prediction) as reported for PatB with a conserved Ser-Asp-His predicted catalytic triad ( Fig.…”

“…However, there is no clear clue to assign any of these conservations regarding to substrate specificity. The globular C-terminal domain, predicted to be surface exposed and assigned to the family of SGNH/GDSL hydrolases, displays a similar conservation level between OatA, OatB, and PatB (32-33% of similarity), the latter being reported to display a MurNAc-specific O-acetyltransferase activity (16). The two most conserved regions GDSV (509 -512 amino acids in OatA Lp ) and DxxH (634 -637 amino acids in OatA Lp ) are supposed to contain the predicted Ser-Asp-His catalytic triad (underlined amino acids) (supplemental Fig.…”

“…MurNAc O-acetylation is described in Gram-negative bacteria as well as in Gram-positive bacteria but involves a different set of dedicated proteins (16,20). Moynihan and Clarke (16) have recently demonstrated that two different proteins are involved in this process in Gram-negative bacteria: 1) an integral membrane protein called PatA is dedicated to the transport of the acetyl-donor and 2) a periplasmic membrane-anchored protein called PatB catalyzes the acetylation reaction.…”

The Vertebrate Lysozyme Inhibitor Ivy Functions to Inhibit the Activity of Lytic Transglycosylase

“…Indeed, of the many bacteria that have been examined for the presence of O-acetylated PG (6 -8), only E. coli and P. aeruginosa (7) have been found to be devoid of the modification. Thus, we conducted a search of each of the genomes of the Ivy-producing bacteria for the presence of either oatA (18) or the poa cluster (19,20), genes known to encode the enzymes responsible for PG O-acetylation in Gram-positive and Gram-negative bacteria, respectively. Thus, S. aureus OatA (ZP_06334688), N. gonorrhoeae PatB (AAW89271), and B. anthracis Ape3 (NP843402) were used in these BLASTP searches.…”

Peptidoglycan Acetylation of Campylobacter jejuni Is Essential for Maintaining Cell Wall Integrity and Colonization in Chicken Intestines

“…It was subsequently reported that Ape2 of Neisseria gonorrhoeae does not exhibit esterase activity but functions as a periplasmic protein for the O-acetylation of PG. Thus, the protein was renamed PG O-acetyltransferase B (PatB) (14). PatA and PatB have recently been proposed to function together in translocating acetate from cytoplasmic pools to the periplasm, where it is transferred to peptidoglycan in Gram-negative bacteria (15) (Fig.…”