Peptidoglycan-Hydrolyzing Activity of the FlgJ Protein, Essential for Flagellar Rod Formation in Salmonella typhimurium

Abstract

Because the rod structure of the flagellar basal body crosses the inner membrane, the periplasmic space, and the outer membrane, its formation must involve hydrolysis of the peptidoglycan layer. So far, more than 10 genes have been shown to be required for rod formation inSalmonella typhimurium. Some of them encode the component proteins of the rod structure, and most of the remaining genes are believed to encode proteins involved in the export process of the component proteins. Although FlgJ has also been known to be involved in rod formation, its exact role has not been understood. Recently, it was suggested that the C-terminal half of the FlgJ protein has homology to the active center of some muramidase enzymes from gram-positive bacteria. In this study, we showed that the purified FlgJ protein fromS. typhimurium has a peptidoglycan-hydrolyzing activity and that this activity is localized in its C-terminal half. Through oligonucleotide-directed mutagenesis, we constructed flgJmutants with amino acid substitutions in the putative active center of the muramidase. The resulting mutants produced FlgJ proteins with reduced enzymatic activity and showed poor motility. These results indicate that the muramidase activity of FlgJ is essential for flagellar formation. Immunoblotting analysis with the fractionated cell extracts revealed that FlgJ is exported to the periplasmic space, where the peptidoglycan layer is localized. On the basis of these results, we conclude that FlgJ is the flagellum-specific muramidase which hydrolyzes the peptidoglycan layer to assemble the rod structure in the periplasmic space.

AcmA of Lactococcus lactis is an N‐acetylglucosaminidase with an optimal number of LysM domains for proper functioning

“…Taken together, these results indicate that, although the N-terminus of AcmA contains the active site, the presence of at least one complete repeat is needed for the enzyme to retain appreciable activity in vitro, whereas optimal activity is obtained with three repeats. A similar result was obtained for the active site domain of the FlgJ protein of S. typhimurium, a muramidase-like enzyme involved in flagellar rod formation [10]. The N-terminal half of FlgJ is dispensable for peptidoglycan-hydrolyzing activity in vitro, but the truncated protein does not support cellular flagellation.…”

“…However, the AcmA homologs AcmB [7], AcmC [8] and LytG [9] have been shown to be glucosaminidases. Amino acid substitutions in the AcmA homolog FlgJ of Salmonella typhimurium have shown that two conserved amino acid residues, aspartyl and glutamyl, which are also preserved in AcmA, muramidase-2 and LytG, are part of the putative active center of this peptidoglycan hydrolase that is essential for flagellar rod formation [10]. In the sequence of the genome of L. lactis IL1403 genes putatively encoding cell wall hydrolases with an active site homologous to that of AcmA are present (acmB, acmC and acmD).…”

Mur1, a Streptococcus thermophilus peptidoglycan hydrolase devoid of a specific cell wall binding domain

“…Comparison of the predicted amino acid sequence of Mur1 with protein sequence databases revealed sequence identity, in its 72^218 amino acid region, with E. hirae muramidase-2 (55%) [8], E. faecalis autolysin (54%) [2] and L. lactis AcmA (52%) [5]. Sequence identity was also found with the ORF39 of the B. subtilis phage P105 (sequence accession number AB016282) (42.7%) and the B. subtilis hypothetical muramidase YubE (41%) (sequence accession number O32083), which functions are unknown, and with the Salmonella typhimurium £agellar speci¢c muramidase FlgJ (30.3%) [14] (Fig. 1A).…”

“…1B). Mur1 encompasses exactly the sequence corresponding to the catalytic domain of the enzymes of the muramidase family [5,11,14]. Especially, the Asp and Glu residues, which are conserved in the putative active centre of these enzymes [11], are also present in the corresponding positions of Mur1 (Glu-135 and Asp-161) ( Fig.…”

Protein export and drug efflux through bacterial channel-tunnels

“…review the peptidoglycan layer negotiated? Perhaps cellular enzymes that degrade the peptidoglycan might have a function in TolC assembly, analagous to the dedicated muraminidase needed for assembly of flagella on the cell surface (Nambu et al, 1999). Another question relates to how the channel-tunnels are located by substrate-laden IM complexes.…”