Modulation of the Lytic Activity of the Dedicated Autolysin for Flagellum Formation SltF by Flagellar Rod Proteins FlgB and FlgF

Abstract

SltF was identified previously as an autolysin required for the assembly of flagella in the alphaproteobacteria, but the nature of its peptidoglycan lytic activity remained unknown. Sequence alignment analyses suggest that it could function as either a muramidase, lytic transglycosylase, or ␤-N-acetylglucosaminidase. Recombinant SltF from Rhodobacter sphaeroides was purified to apparent homogeneity, and it was demonstrated to function as a lytic transglycosylase based on enzymatic assays involving mass spectrometric analyses. Circular dichroism (CD) analysis determined that it is composed of 83.4% ␣-structure and 1.48% ␤-structure and thus is similar to family 1A lytic transglycosylases. However, alignment of apparent SltF homologs identified in the genome database defined a new subfamily of the family 1 lytic transglycosylases. SltF was demonstrated to be endo-acting, cleaving within chains of peptidoglycan, with optimal activity at pH 7.0. Its activity is modulated by two flagellar rod proteins, FlgB and FlgF: FlgB both stabilizes and stimulates SltF activity, while FlgF inhibits it. Invariant Glu57 was confirmed as the sole catalytic acid/base residue of SltF. IMPORTANCEThe bacterial flagellum is comprised of a basal body, hook, and helical filament, which are connected by a rod structure. With a diameter of approximately 4 nm, the rod is larger than the estimated pore size within the peptidoglycan sacculus, and hence its insertion requires the localized and controlled lysis of this essential cell wall component. In many beta-and gammaproteobacteria, this lysis is catalyzed by the ␤-N-acetylglucosaminidase domain of FlgJ. However, FlgJ of the alphaproteobacteria lacks this activity and instead it recruits a separate enzyme, SltF, for this purpose. In this study, we demonstrate that SltF functions as a newly identified class of lytic transglycosylases and that its autolytic activity is uniquely modulated by two rod proteins, FlgB and FlgF.T he photosynthetic bacterium Rhodobacter sphaeroides is motile through the use of a single, subpolar flagellum (1). This locomotive organelle is tightly regulated and comprised of approximately 25 different proteins arranged into three major substructures: a basal body, hook, and thin helical filament. The basal body spans the bacterial cell envelope and contains the motor, a flagellum-specific type III export apparatus and at least four ringlike structures which are all connected by a filamentous rod (2). The rod assembles into a proximal rod that lies between the MS ring and the cell wall, which is composed of nine subunits of FliE (3) and six subunits of FlgB, FlgC, and FlgF (4), as well as a distal rod that is composed of 26 subunits of FlgG (5).During flagellum assembly, extensive modifications need to occur to the peptidoglycan (PG) sacculus to accommodate the insertion of the secretion apparatus, as well as to stabilize the function of this system by acting as an assembly scaffold (6, 7). PG is a heteropolymer of glycan strands and peptide chains forming a rig...

The lytic transglycosylase MltB connects membrane homeostasis and in vivo fitness of Acinetobacter baumannii

“…In addition to being involved in membrane homeostasis, it is postulated that peptidoglycan degrading enzymes, such as LTs and endopeptidases, act as bacterial 'space-making' autolysins (Scheurwater et al, 2008;Stohl et al, 2013). Accordingly, it was observed they are required for assembly of flagella and/or pili and in Caulobacter crescentus, Neisseria gonorrhoeae, and Rhodobacter sphaeroides (Viollier and Shapiro, 2003;Stohl et al, 2013;Herlihey et al, 2016), the type III secretion system in Xanthomonas campestris pv. Vesicatoria (Hausner et al, 2017) and the type VI secretion system in E. coli and A. baumannii (Weber et al, 2016;Santin and Cascales, 2017).…”

Lytic transglycosylases: concinnity in concision of the bacterial cell wall

“…Moreover, a homology model of SltF displayed the classic Family 1 LT catalytic fold. More recently, its catalytic activity was elucidated (confirming it as an endolytic LT) through MS analysis of the products released from the sacculus by a C-terminal SLT domain construct (Herlihey et al , 2016). Its LT activity is regulated by two proteins (FlgB and FlgF), which stabilize SltF and increase its catalytic activity.…”

From Genome to Proteome to Elucidation of Reactions for All Eleven Known Lytic Transglycosylases from Pseudomonas aeruginosa

“…TheL Ts of E. coli were classified on the basis of sequence, [5,18] but continuing study of the P. aeruginosa and Escherichia coli genomes and proteomes has increased the number and the diversity of the LT superfamily. [5,7,19] This study-the first comprehensive comparison of substrate recognition and processing by the individual members of the P. aeruginosa LT superfamily-provides important insight toward functional LT classification. Akey discovery is the requirement of some LTsf or a + 2a nhNAM residue for substrate recognition.…”