Self-generated covalent cross-links in the cell-surface adhesins of Gram-positive bacteria

2015 ◽  
Vol 43 (5) ◽  
pp. 787-794 ◽  
Author(s):  
Edward N. Baker ◽  
Christopher J. Squire ◽  
Paul G. Young
Keyword(s):  
Cell Surface ◽  
Mechanical Strength ◽  
Cell Walls ◽  
Side Chains ◽  
Structural Studies ◽  
Gram Positive Bacteria ◽  
Intramolecular Reactions ◽  
Isopeptide Bonds ◽  
Cross Links ◽  
Surface Adhesins

The ability of bacteria to adhere to other cells or to surfaces depends on long, thin adhesive structures that are anchored to their cell walls. These structures include extended protein oligomers known as pili and single, multi-domain polypeptides, mostly based on multiple tandem Ig-like domains. Recent structural studies have revealed the widespread presence of covalent cross-links, not previously seen within proteins, which stabilize these domains. The cross-links discovered so far are either isopeptide bonds that link lysine side chains to the side chains of asparagine or aspartic acid residues or ester bonds between threonine and glutamine side chains. These bonds appear to be formed by spontaneous intramolecular reactions as the proteins fold and are strategically placed so as to impart considerable mechanical strength.

scholarly journals Preliminary crystallographic study of theStreptococcus agalactiaesortases, sortase A and sortase C1

2010 ◽  
Vol 66 (9) ◽  
pp. 1096-1100 ◽  
Author(s):  
Baldeep Khare ◽  
Alexandra Samal ◽  
Krishnan Vengadesan ◽  
K. R. Rajashankar ◽  
Xin Ma ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Substrate Specificity ◽  
Streptococcus Agalactiae ◽  
Crystal Form ◽  
Sortase A ◽  
Crystal Forms ◽  
Gram Positive Bacteria ◽  
Crystallographic Study ◽  
Surface Adhesins

Sortases are cysteine transpeptidases that are essential for the assembly and anchoring of cell-surface adhesins in Gram-positive bacteria. InStreptococcus agalactiae(GBS), the pilin-specific sortase SrtC1 catalyzes the polymerization of pilins encoded by pilus island 1 (PI-1) and the housekeeping sortase SrtA is necessary for cell-wall anchoring of the resulting pilus polymers. These sortases are known to utilize different substrates for pilus polymerization and cell-wall anchoring; however, the structural correlates that dictate their substrate specificity have not yet been clearly defined. This report presents the expression, purification and crystallization of SrtC1 (SAG0647) and SrtA (SAG0961) fromS. agalactiaestrain 2603V/R. The GBS SrtC1 has been crystallized in three crystal forms and the GBS SrtA has been crystallized in one crystal form.

scholarly journals Involvement of CesA4, CesA7-A/B and CesA8-A/B in secondary wall formation in Populus trichocarpa wood

2019 ◽  
Vol 40 (1) ◽  
pp. 73-89 ◽  
Author(s):  
Manzar Abbas ◽  
Ilona Peszlen ◽  
Rui Shi ◽  
Hoon Kim ◽  
Rui Katahira ◽  
...  
Keyword(s):  
Solid State ◽  
Mechanical Strength ◽  
Cell Walls ◽  
Secondary Wall ◽  
Populus Trichocarpa ◽  
Wood Formation ◽  
Fiber Cell ◽  
Modulus Of Rupture ◽  
Cellulose Content ◽  
Wall Formation

Abstract Cellulose synthase A genes (CesAs) are responsible for cellulose biosynthesis in plant cell walls. In this study, functions of secondary wall cellulose synthases PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B were characterized during wood formation in Populus trichocarpa (Torr. & Gray). CesA RNAi knockdown transgenic plants exhibited stunted growth, narrow leaves, early necrosis, reduced stature, collapsed vessels, thinner fiber cell walls and extended fiber lumen diameters. In the RNAi knockdown transgenics, stems exhibited reduced mechanical strength, with reduced modulus of rupture (MOR) and modulus of elasticity (MOE). The reduced mechanical strength may be due to thinner fiber cell walls. Vessels in the xylem of the transgenics were collapsed, indicating that water transport in xylem may be affected and thus causing early necrosis in leaves. A dramatic decrease in cellulose content was observed in the RNAi knockdown transgenics. Compared with wildtype, the cellulose content was significantly decreased in the PtrCesA4, PtrCesA7 and PtrCesA8 RNAi knockdown transgenics. As a result, lignin and xylem contents were proportionally increased. The wood composition changes were confirmed by solid-state NMR, two-dimensional solution-state NMR and sum-frequency-generation vibration (SFG) analyses. Both solid-state nuclear magnetic resonance (NMR) and SFG analyses demonstrated that knockdown of PtrCesAs did not affect cellulose crystallinity index. Our results provided the evidence for the involvement of PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B in secondary cell wall formation in wood and demonstrated the pleiotropic effects of their perturbations on wood formation.

scholarly journals Structural studies on the neutral polysaccharide of Bacillus subtilis AHU 1219 cell walls

1989 ◽  
Vol 178 (3) ◽  
pp. 635-641 ◽  
Author(s):  
Hiroyoshi IWASAKI ◽  
Yoshio ARAKI ◽  
Shunji KAYA ◽  
Eiji ITO
Keyword(s):  
Bacillus Subtilis ◽  
Cell Walls ◽  
Structural Studies

scholarly journals Cooperativity of folding in multidomain surface adhesins containing intramolecular cross-links

2017 ◽  
Vol 73 (a2) ◽  
pp. C650-C650
Author(s):  
Christopher John Squire ◽  
Yuliana Yosaatmadja ◽  
Edward Baker ◽  
Paul Young
Keyword(s):  
Cross Links ◽  
Surface Adhesins

Cation exchange in cell walls of gram-positive bacteria

1976 ◽  
Vol 22 (7) ◽  
pp. 975-982 ◽  
Author(s):  
Robert E. Marquis ◽  
Kathleen Mayzel ◽  
Edwin L. Carstensen
Keyword(s):  
Cation Exchange ◽  
Cell Walls ◽  
Divalent Cations ◽  
Teichoic Acid ◽  
Dry Weight ◽  
High Ionic Strength ◽  
Monovalent Cations ◽  
Anionic Sites ◽  
Gram Positive Bacteria ◽  
Anionic Groups

The relative affinities of various cations for anionic sites in isolated, bacterial cell walls were assessed by means of a technique involving displacement of one cation by another. The affinity series determined was [Formula: see text]. High affinity was correlated with low mobility of the bound ions in an electric field. The net cation-exchange capacities of walls isolated from a variety of bacteria were estimated by preparing the magnesium forms of the walls, washing them well with deionized water to remove supernumerary ions, and then completely displacing the magnesium with Na+ or H+. Total amounts of magnesium displaced varied from 73 μmol per gram dry weight, for walls of the teichoic acid-deficient 52A5 strain of Staphylococcus aureus to about 520 μmol per gram for Bacillus megaterium KM walls. The amount of displacable magnesium was inversely related to the physical compactness of the walls, except for walls of Streptococcus mutans GS-5. It was found that magnesium or calcium ions can each neutralize, or pair with, two anionic groups in walls suspended in ion-deficient media. Previous work had indicated that these ions may pair with only one anionic group at high ionic strength. Therefore, it appeared that there is a great deal of flexibility in the arrangement of charged groups in the wall. It was concluded also that for cells growing in commonly used laboratory media, which generally contain large excesses of monovalent versus divalent cations, there is a mix of small, cationic counterions in the wall and that monovalent cations may predominate even though the wall has higher affinity for divalent ions.

scholarly journals In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking

2018 ◽  
Vol 115 (24) ◽  
pp. E5477-E5486 ◽  
Author(s):  
Chungyu Chang ◽  
Brendan R. Amer ◽  
Jerzy Osipiuk ◽  
Scott A. McConnell ◽  
I-Hsiu Huang ◽  
...  
Keyword(s):  
Cross Linking ◽  
Structural Elements ◽  
Gram Positive Bacteria ◽  
Activating Mutations ◽  
In Vitro Reconstitution ◽  
Isopeptide Bonds ◽  
Signature Sequence ◽  
Class C ◽  
Enzyme Intermediate

Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction, first cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate and then joining the terminal Thr to the nucleophilic Lys residue residing within the pilin motif of another pilin protomer. To date, the determinants of class C enzymes that uniquely enable them to construct pili remain unknown. Here, informed by high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and utilizing a structural variant of the enzyme (SrtA2M), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Mass spectrometry, electron microscopy, and biochemical experiments authenticated that SrtA2Msynthesizes pilus fibers with correct Lys–Thr isopeptide bonds linking individual pilins via a thioacyl intermediate. Structural modeling of the SpaA–SrtA–SpaA polymerization intermediate depicts SrtA2Msandwiched between the N- and C-terminal domains of SpaA harboring the reactive pilin and LPXTG motifs, respectively. Remarkably, the model uncovered a conserved TP(Y/L)XIN(S/T)H signature sequence following the catalytic Cys, in which the alanine substitutions abrogated cross-linking activity but not cleavage of LPXTG. These insights and our evidence that SrtA2Mcan terminate pilus polymerization by joining the terminal pilin SpaB to SpaA and catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile platform for protein engineering and bio-conjugation that has major implications for biotechnology.

scholarly journals Two DD-Carboxypeptidases fromMycobacterium smegmatisAffect Cell Surface Properties through Regulation of Peptidoglycan Cross-Linking and Glycopeptidolipids

2018 ◽  
Vol 200 (14) ◽  
Author(s):  
Satya Deo Pandey ◽  
Shilpa Pal ◽  
Ganesh Kumar N ◽  
Ankita Bansal ◽  
Sathi Mallick ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Cross Linking ◽  
Surface Lipid ◽  
Cross Link ◽  
Content Type ◽  
Murine Macrophages ◽  
Cross Links ◽  
Link Formation

ABSTRACTDuring the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between twomeso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (betweend-Ala andmeso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. Thedd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used byld-transpeptidases as substrates to form 3-3 cross-links. Therefore,dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology ofdd-CPases in mycobacteria is relatively unexplored. In this study, we deleted twodd-CPase genes,msmeg_2433andmsmeg_2432, both individually and in combination, fromMycobacterium smegmatismc2155. Though the singledd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant,viz., a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to β-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of thedd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCEThe glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. Thedd-CPases generate tetrapeptides by acting on the pentapeptides, andld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of twodd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, includingMycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology ofdd-CPases might help us design antimycobacterial therapeutics in the future.

Sign in / Sign up

Export Citation Format

Share Document