Discovery and Characterization of Three New<i>Escherichia coli</i>Septal Ring Proteins That Contain a SPOR Domain: DamX, DedD, and RlpA

Arends, S J Ryan; Williams, Kyle; Scott, Renada J.; Rolong, Silvana; Popham, David L.; Weiss, David S.

doi:10.1128/jb.01244-09

Cited by 87 publications

(234 citation statements)

References 72 publications

Supporting

Mentioning

218

Contrasting

Order By: Relevance

“…The division apparatus is generally thought of as a multiprotein complex (or a collection of smaller complexes) whose assembly is driven, for the most part, by protein-protein interactions (23,(31)(32)(33)(34)(35)(36). However proteins that contain a small PG-binding domain known as a "SPOR domain" are thought to be recruited by binding to septal PG (15)(16)(17). In particular, at least in E. coli, SPOR domains are hypothesized to bind to denuded glycan strands that accumulate in septal PG because of the intense activity of cell-wall amidases that drive the separation of daughter cells (16,20).…”

Section: Discussionmentioning

confidence: 99%

“…Purified SPOR domains bind to purified PG sacculi in a cosedimentation assay, but whether this binding reflects the specific association of the SPOR domains with septal PG is not known (15,17,20,21). To address this question, we purified hexahistidine (His 6 )-tagged fusions of GFP to the SPOR domains from four E. coli cell-division proteins: DamX, DedD, FtsN, and RlpA.…”

Section: Spor Domains Bind Septal Regions Of Purifiedmentioning

confidence: 99%

“…4A). Because PaRlpA has a SPOR domain and localizes to division sites in vivo (16,17,27), it was necessary to verify that lack of GFPDamX SPOR binding did not result from competition for denuded glycans. We did so in two ways.…”

Section: Binding Of Spor Domains To Sacculi After Treatment With Pgmentioning

confidence: 99%

“…These constructs are referred to hereafter as "GFPDamX SPOR ," "GFP-DedD SPOR ," "GFP-FtsN SPOR ," and "GFPRlpA SPOR ." These various SPOR domains exhibit only ∼18% amino acid identity in pairwise alignments (17). Purified PG sacculi isolated from wild-type E. coli cells were immobilized by adhering them to glass slides coated with poly-L-lysine.…”

Section: Spor Domains Bind Septal Regions Of Purifiedmentioning

confidence: 99%

“…Several years ago it became apparent that a widespread subset of bacterial cell-division proteins is targeted to the division site by a small PG-binding domain known as a "SPOR" domain (15)(16)(17)(18). E. coli has four SPOR domain proteins, all of which are involved in cell division: DamX, DedD, FtsN, and RlpA.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

Yahashiri

Jorgenson

Weiss

2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

102

142

View full text Add to dashboard Cite

Bacterial SPOR domains bind peptidoglycan (PG) and are thought to target proteins to the cell division site by binding to "denuded" glycan strands that lack stem peptides, but uncertainties remain, in part because septal-specific binding has yet to be studied in a purified system. Here we show that fusions of GFP to SPOR domains from the Escherichia coli cell-division proteins DamX, DedD, FtsN, and RlpA all localize to septal regions of purified PG sacculi obtained from E. coli and Bacillus subtilis. Treatment of sacculi with an amidase that removes stem peptides enhanced SPOR domain binding, whereas treatment with a lytic transglycosylase that removes denuded glycans reduced SPOR domain binding. These findings demonstrate unequivocally that SPOR domains localize by binding to septal PG, that the physiologically relevant binding site is indeed a denuded glycan, and that denuded glycans are enriched in septal PG rather than distributed uniformly around the sacculus. Accumulation of denuded glycans in the septal PG of both E. coli and B. subtilis, organisms separated by 1 billion years of evolution, suggests that sequential removal of stem peptides followed by degradation of the glycan backbone is an ancient feature of PG turnover during bacterial cell division. Linking SPOR domain localization to the abundance of a structure (denuded glycans) present only transiently during biogenesis of septal PG provides a mechanism for coordinating the function of SPOR domain proteins with the progress of cell division.A defining feature of bacteria is the peptidoglycan (PG) cell wall or "sacculus" that confers cell shape, protects the cell against lysis caused by turgor pressure, and is the ultimate target of many clinically relevant antibiotics, including β-lactams and vancomycin (1-4). The structure of PG is characterized by a network of glycan strands connected at regular intervals by oligopeptide cross-links (Fig. 1). The glycans are built from a repeating disaccharide of β-1,4-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), from which branch the oligopeptides that mediate cross-linking. During cell division, a new PG wall is assembled between the incipient daughter cells (5, 6), and subsequent cell separation depends on the activity of a collection of PG hydrolases that cleave various bonds in the PG mesh (7).In the model Gram-negative bacterium Escherichia coli, daughter-cell separation is driven primarily by three cell-wall amidases (8-10). These enzymes cleave the amide bond that joins the lactyl group of the MurNAc to the α-amino group of the first amino acid (L-Ala) of the stem peptide, releasing as products free oligopeptides and "denuded" glycan strands (Fig. 1). Lytic transglycosylases (LTs) that cleave the MurNAc-GlcNAc glycosidic bond and endopeptidases that cleave stem peptides make minor but still significant contributions to daughter-cell separation (9, 11). In Bacillus subtilis, a model Gram-positive species, the enzymology of daughter-cell separation is not as well characterized. The...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Spor Domains Bind Septal Regions Of Purifiedmentioning

confidence: 99%

Section: Binding Of Spor Domains To Sacculi After Treatment With Pgmentioning

confidence: 99%

Section: Spor Domains Bind Septal Regions Of Purifiedmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

Yahashiri

Jorgenson

Weiss

2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

102

142

View full text Add to dashboard Cite

show abstract

Bacterial cytokinesis: From Z ring to divisome

Lutkenhaus¹,

Pichoff²,

Du³

2012

Cytoskeleton

242

248

View full text Add to dashboard Cite

Ancestral homologues of the major eukaryotic cytoskeletal families, tubulin and actin, play critical roles in cytokinesis of bacterial cells. FtsZ is the ancestral homologue of tubulin and assembles into the Z ring that determines the division plane. FtsA is an ancestral homologue of actin and is involved in coordinating cell wall synthesis during cytokinesis. FtsA assists in the formation of the Z ring and also has a critical role in recruiting downstream division proteins to the Z ring to generate the divisome that divides the cell. Spatial regulation of cytokinesis occurs at the stage of Z ring assembly and regulation of cell size occurs at this stage or during Z ring maturation.

show abstract

Amber‐codon suppression for spatial localization and in vivo photoaffinity capture of the interactome of the Pseudomonas aeruginosa rare lipoprotein A lytic transglycosylase

Avila‐Cobian,

Hoshino,

Horsman

et al. 2023

Protein Science

View full text Add to dashboard Cite

The 11 lytic transglycosylases of Pseudomonas aeruginosa have overlapping activities in the turnover of the cell‐wall peptidoglycan. Rare lipoprotein A (RlpA) is distinct among the 11 by its use of only peptidoglycan lacking peptide stems. The spatial localization of RlpA and its interactome within P. aeruginosa are unknown. We employed suppression of introduced amber codons at sites in the rlpA gene for the introduction of the unnatural‐amino‐acids Νζ‐[(2‐azidoethoxy)carbonyl]‐l‐lysine (compound 1) and Nζ‐[[[3‐(3‐methyl‐3H‐diazirin‐3‐yl)propyl]amino]carbonyl]‐l‐lysine (compound 2). In live P. aeruginosa, full‐length RlpA incorporating compound 1 into its sequence was fluorescently tagged using strained‐promoted alkyne‐azide cycloaddition and examined by fluorescence microscopy. RlpA is present at low levels along the sidewall length of the bacterium, and at higher levels at the nascent septa of replicating bacteria. In intact P. aeruginosa, UV photolysis of full‐length RlpA having compound 2 within its sequence generated a transient reactive carbene, which engaged in photoaffinity capture of neighboring proteins. Thirteen proteins were identified. Three of these proteins—PBP1a, PBP5, and MreB—are members of the bacterial divisome. The use of the complementary methodologies of non‐canonical amino‐acid incorporation, photoaffinity proximity analysis, and fluorescent microscopy confirm a dominant septal location for the RlpA enzyme of P. aeruginosa, as a divisome‐associated activity. This accomplishment adds to the emerging recognition of the value of these methodologies for identification of the intracellular localization of bacterial proteins.

show abstract

Discovery and Characterization of Three NewEscherichia coliSeptal Ring Proteins That Contain a SPOR Domain: DamX, DedD, and RlpA

Cited by 87 publications

References 72 publications

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

Bacterial cytokinesis: From Z ring to divisome

Amber‐codon suppression for spatial localization and in vivo photoaffinity capture of the interactome of the Pseudomonas aeruginosa rare lipoprotein A lytic transglycosylase

Contact Info

Product

Resources

About