The C Terminus of the Flagellar Muramidase SltF Modulates the Interaction with FlgJ in Rhodobacter sphaeroides

Mora, Javier; Osorio-Valeriano, Manuel; González‐Pedrajo, Bertha; Ballado, Teresa; Camarena, Laura; Dreyfus, Georges

doi:10.1128/jb.00460-12

Cited by 14 publications

(19 citation statements)

References 39 publications

(43 reference statements)

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“…To further complicate the issue, the monofunctional FlgJs of Rhodobacter sphaeroides (25) and Caulobacter crescentus (26) lack lytic activity and this is compensated for by the production of putative LTs, which for the former bacterium is encoded within its Flg operon. Like other LTs, they too would possess the lysozyme-fold (15).…”

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confidence: 99%

The Essential Protein for Bacterial Flagella Formation FlgJ Functions as a β-N-Acetylglucosaminidase

Herlihey

Moynihan

Clarke

2014

Journal of Biological Chemistry

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Background: FlgJ is required for flagella formation in Salmonella enterica. Results: The lytic activity of FlgJ was determined to be that of a ␤-N-acetylglucosaminidase using a novel assay. Conclusion: FlgJ is a hydrolase and not a lytic transglycosylase. Significance: This information is essential for the search and rational design of inhibitors that may serve as leads to a new class of antibiotics.

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The Essential Protein for Bacterial Flagella Formation FlgJ Functions as a β-N-Acetylglucosaminidase

Herlihey

Moynihan

Clarke

2014

Journal of Biological Chemistry

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“…E. coli M15(pREP4) was transformed with the respective plasmids harboring genes encoding the wild-type (21) and the Glu57Ala (20) forms of SltF (Table 1), and both gene overexpression and isolation of overproduced proteins by immobilized metal affinity chromatography (IMAC) were conducted as described previously (20). Following IMAC on Ni 2ϩ -NTA-agarose and dialysis Freshly prepared IPTG (isopropyl-␤-D-thiogalactopyranoside) was added to final concentrations of 0.1 and 1 mM, and expression was induced for 3 h and 2 h, respectively, at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…It has been shown previously that the lack of FlgJ lytic activity is compensated for by SltF, a PG-lytic enzyme encoded within the flg operon; this enzyme is responsible for flagellar rod penetration in this bacterium (20). During basal body formation, SltF is exported to the periplasm via the Sec pathway, and it is recruited to the basal body near the cell pole by interacting with FlgJ (21).…”

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confidence: 99%

“…A phylogenetic analysis (21) showed that SltF is weakly related to the family 1 LTs proposed by Blackburn and Clarke (22). However, two Glu residues were tentatively identified as being essential for catalytic activity, implying the enzyme may function as a hydrolase (21); LTs use a single catalytic acid/base residue (reviewed in reference 23).…”

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confidence: 99%

“…However, two Glu residues were tentatively identified as being essential for catalytic activity, implying the enzyme may function as a hydrolase (21); LTs use a single catalytic acid/base residue (reviewed in reference 23). No biochemical analysis has been conducted on SltF, and consequently confusion exists as to whether this enzyme functions as a hydrolase (i.e., muramidase or ␤-N-acetylglucosaminidase) or an LT (20,21).…”

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confidence: 99%

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

et al. 2016

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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...

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Controlling Autolysis During Flagella Insertion in Gram-Negative Bacteria

Herlihey

Clarke

2016

Advances in Experimental Medicine and Biology

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The flagellum is an important macromolecular machine for many pathogenic bacteria. It is a hetero-oligomeric structure comprised of three major sub-structures: basal body, hook and thin helical filament. An important step during flagellum assembly is the localized and controlled degradation of the peptidoglycan sacculus to allow for the insertion of the rod as well as to facilitate anchoring for proper motor function. The peptidoglycan lysis events require specialized lytic enzymes, β-N-acetylglucosaminidases and lytic transglycosylases, which differ in flagellated proteobacteria. Due to their autolytic activity, these enzymes need to be controlled in order to prevent cellular lysis. This review summarizes are current understanding of the peptidoglycan lysis events required for flagellum assembly and motility with a main focus on Gram-negative bacteria.

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The C Terminus of the Flagellar Muramidase SltF Modulates the Interaction with FlgJ in Rhodobacter sphaeroides

Cited by 14 publications

References 39 publications

The Essential Protein for Bacterial Flagella Formation FlgJ Functions as a β-N-Acetylglucosaminidase

The Essential Protein for Bacterial Flagella Formation FlgJ Functions as a β-N-Acetylglucosaminidase

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

Controlling Autolysis During Flagella Insertion in Gram-Negative Bacteria

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