scholarly journals Lytic transglycosylases RlpA and MltC assist inVibrio choleraedaughter cell separation

2019 ◽  
Author(s):  
Anna I. Weaver ◽  
Valeria Jiménez-Ruiz ◽  
Srikar R. Tallavajhalla ◽  
Brett P. Ransegnola ◽  
Kimberly Q. Wong ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Separation ◽  
Specific Activity ◽  
Dynamic Structure ◽  
Bacterial Survival ◽  
Network Cell ◽  
Lytic Transglycosylases ◽  
Daughter Cells ◽  
Daughter Cell Separation ◽  
Normal Laboratory

ABSTRACTThe cell wall is a crucial structural feature in the vast majority of bacteria and comprises a rigid, covalently closed, mesh-like network of peptidoglycan (PG) strands. While PG synthesis is important for bacterial survival under many conditions, the cell wall is also a dynamic structure, undergoing degradation and remodeling by so-called “autolysins”, enzymes that break bonds in the PG network. Cell division, for example, requires extensive PG remodeling and separation of daughter cells, which depends primarily upon the activity of amidases. However, inV. cholerae, we have found that amidase activity alone is insufficient for daughter cell separation and that the lytic transglycosylases RlpA and MltC both contribute to this process. MltC and RlpA both localize to the septum and are functionally redundant under normal laboratory conditions; however, only RlpA can support normal cell separation in low salt media. The division-specific activity of lytic transglycosylases has implications for the local structure of septal PG, suggesting that there may be glycan bridges between daughter cells that cannot be resolved by amidases. We propose that lytic transglycosylases at the septum serve as a back-up mechanism to cleave rare, stochastically produced PG strands that are crosslinked beyond the reach of the highly spatio-temporally limited activity of the amidase and to clear PG debris that may block the completion of outer-membrane invagination.

scholarly journals Daughter Cell Separation by Penicillin-Binding Proteins and Peptidoglycan Amidases in Escherichia coli

2006 ◽  
Vol 188 (15) ◽  
pp. 5345-5355 ◽  
Author(s):  
Richa Priyadarshini ◽  
David L. Popham ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Cell Separation ◽  
Growth Cycle ◽  
Lytic Transglycosylases ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Daughter Cells ◽  
Substrate Preferences ◽  
Peptidoglycan Hydrolases ◽  
Daughter Cell Separation

ABSTRACT As one of the final steps in the bacterial growth cycle, daughter cells must be released from one another by cutting the shared peptidoglycan wall that separates them. In Escherichia coli, this delicate operation is performed by several peptidoglycan hydrolases, consisting of multiple amidases, lytic transglycosylases, and endopeptidases. The interactions among these enzymes and the molecular mechanics of how separation occurs without lysis are unknown. We show here that deleting the endopeptidase PBP 4 from strains lacking AmiC produces long chains of unseparated cells, indicating that PBP 4 collaborates with the major peptidoglycan amidases during cell separation. Another endopeptidase, PBP 7, fulfills a secondary role. These functions may be responsible for the contributions of PBPs 4 and 7 to the generation of regular cell shape and the production of normal biofilms. In addition, we find that the E. coli peptidoglycan amidases may have different substrate preferences. When the dd-carboxypeptidase PBP 5 was deleted, thereby producing cells with higher levels of pentapeptides, mutants carrying only AmiC produced a higher percentage of cells in chains, while mutants with active AmiA or AmiB were unaffected. The results suggest that AmiC prefers to remove tetrapeptides from peptidoglycan and that AmiA and AmiB either have no preference or prefer pentapeptides. Muropeptide compositions of the mutants corroborated this latter conclusion. Unexpectedly, amidase mutants lacking PBP 5 grew in long twisted chains instead of straight filaments, indicating that overall septal morphology was also defective in these strains.

scholarly journals Characterization of IsaA and SceD, Two Putative Lytic Transglycosylases of Staphylococcus aureus

2007 ◽  
Vol 189 (20) ◽  
pp. 7316-7325 ◽  
Author(s):  
Melanie R. Stapleton ◽  
Malcolm J. Horsburgh ◽  
Emma J. Hayhurst ◽  
Lynda Wright ◽  
Ing-Marie Jonsson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Separation ◽  
Dynamic Structure ◽  
Lytic Transglycosylases ◽  
Peptidoglycan Hydrolases ◽  
Cotton Rats ◽  
Autolytic Activity ◽  
Wall Growth

ABSTRACT Bacterial cell wall peptidoglycan is a dynamic structure requiring hydrolysis to allow cell wall growth and division. Staphylococcus aureus has many known and putative peptidoglycan hydrolases, including two likely lytic transglycosylases. These two proteins, IsaA and SceD, were both found to have autolytic activity. Regulatory studies showed that the isaA and sceD genes are partially mutually compensatory and that the production of SceD is upregulated in an isaA mutant. The expression of sceD is also greatly upregulated by the presence of NaCl. Several regulators of isaA and sceD expression were identified. Inactivation of sceD resulted in impaired cell separation, as shown by light microscopy, and “clumping” of bacterial cultures. An isaA sceD mutant is attenuated for virulence, while SceD is essential for nasal colonization in cotton rats, thus demonstrating the importance of cell wall dynamics in host-pathogen interactions.

scholarly journals Growth of Escherichia coli: Significance of Peptidoglycan Degradation during Elongation and Septation

2008 ◽  
Vol 190 (11) ◽  
pp. 3914-3922 ◽  
Author(s):  
Tsuyoshi Uehara ◽  
James T. Park
Keyword(s):  
Escherichia Coli ◽  
Cell Elongation ◽  
Cell Separation ◽  
New Method ◽  
Striking Difference ◽  
Penicillin Binding Protein ◽  
Rapid Degradation ◽  
Lytic Transglycosylases ◽  
Daughter Cells ◽  
Rapid Removal

ABSTRACT We have found a striking difference between the modes of action of amdinocillin (mecillinam) and compound A22, both of which inhibit cell elongation. This was made possible by employment of a new method using an Escherichia coli peptidoglycan (PG)-recycling mutant, lacking ampD, to analyze PG degradation during cell elongation and septation. Using this method, we have found that A22, which is known to prevent MreB function, strongly inhibited PG synthesis during elongation. In contrast, treatment of elongating cells with amdinocillin, which inhibits penicillin-binding protein 2 (PBP2), allowed PG glycan synthesis to proceed at a nearly normal rate with concomitant rapid degradation of the new glycan strands. By treating cells with A22 to inhibit sidewall synthesis, the method could also be applied to study septum synthesis. To our surprise, over 30% of newly synthesized septal PG was degraded during septation. Thus, excess PG sufficient to form at least one additional pole was being synthesized and rapidly degraded during septation. We propose that during cell division, rapid removal of the excess PG serves to separate the new poles of the daughter cells. We have also employed this new method to demonstrate that PBP2 and RodA are required for the synthesis of glycan strands during elongation and that the periplasmic amidases that aid in cell separation are minor players, cleaving only one-sixth of the PG that is turned over by the lytic transglycosylases.

scholarly journals The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Atsushi Yahashiri ◽  
Matthew A. Jorgenson ◽  
David S. Weiss
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Protein Interactions ◽  
Cell Separation ◽  
Protein Protein Interactions ◽  
Target Proteins ◽  
Bacterial Proteins ◽  
Binding Domains ◽  
Division Site ◽  
Daughter Cell Separation

ABSTRACT Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name “SPOR domain” stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides (“denuded” glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites.

scholarly journals Lytic transglycosylases mitigate periplasmic crowding by degrading soluble cell wall turnover products

2021 ◽  
Author(s):  
Anna I Weaver ◽  
Laura Alvarez ◽  
Kelly M Rosch ◽  
Asraa Ahmed ◽  
Garrett S Wang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Critical Role ◽  
Compositional Analysis ◽  
Dynamic Structure ◽  
Lytic Transglycosylases ◽  
E Coli ◽  
Peptidoglycan Synthesis ◽  
Soluble Cell ◽  
Peptidoglycan Cell Wall ◽  
New Material

The peptidoglycan cell wall is a predominant defining structure of bacteria, determining cell shape and supporting survival in diverse conditions. As a single, macromolecular sacculus enveloping the bacterial cell during growth and division, peptidoglycan is necessarily a dynamic structure that requires highly regulated synthesis of new material, remodeling, and turnover, or autolysis, of old material. Despite ubiquitous clinical exploitation of peptidoglycan synthesis as an antibiotic target, much remains unknown about how bacteria modulate synthetic and autolytic processes. Here, we couple bacterial genetics in <em>Vibrio cholerae</em> with compositional analysis of soluble pools of peptidoglycan turnover products to uncover a critical role for a widely misunderstood class of autolytic enzymes, the lytic transglycosylases (LTGs). We demonstrate that LTG activity is specifically required for vegetative growth. The vast majority of LTGs, however, are dispensable for growth, and defects that are ultimately lethal accumulate due to generally inadequate LTG activity, rather than the absence of specific individual enzymes. Consistent with this, we found that a heterologously expressed <em>E. coli</em> LTG, MltE, is capable of sustaining <em>V. cholerae</em> growth in the absence of endogenous LTGs. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains accumulate in the WT, and, to a higher degree, in LTG mutants, and that LTG mutants are hyper-susceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that a key function of LTGs is to prevent toxic crowding of the periplasm with synthesis-derived PG fragments. Contrary to prevailing models, our data further suggest that this process can be temporally separate from peptidoglycan synthesis.

scholarly journals The Sle1 Cell Wall Amidase Controls Daughter Cell Splitting, Cell Size, and β-Lactam Resistance in Community Acquired Methicillin Resistant Staphylococcus aureus USA300

2019 ◽  
Author(s):  
Ida Thalsø-Madsen ◽  
Fernando Ruiz Torrubia ◽  
Lijuan Xu ◽  
Andreas Petersen ◽  
Camilla Jensen ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Size ◽  
Cell Separation ◽  
Methicillin Resistant ◽  
Daughter Cells ◽  
Fast Spreading ◽  
A Cell ◽  
Usa300 Clone ◽  
Highly Virulent

SummaryMost clinically relevant methicillin resistant Staphylococcus aureus (MRSA) strains have become resistant to β-lactams antibiotics through horizontal acquisition of the mecA gene encoding PBP2a, a peptidoglycan transpeptidase with low affinity for β-lactams. The level of resistance conferred by mecA is, however, strain dependent and the mechanisms underlying this phenomenon remain poorly understood. We here show that β-lactam resistance correlates to expression of the Sle1 cell wall amidase in the fast spreading and highly virulent community-acquired MRSA USA300 clone. Sle1 is a substrate of the ClpXP protease, and while the high Sle1 levels in cells lacking ClpXP activity confer β-lactam hyper-resistance, USA300 cells lacking Sle1 are as sensitive to β-lactams as cells lacking mecA. This finding prompted us to assess the cellular roles of Sle1 in more detail, and we demonstrate that high Sle1 levels accelerate the onset of daughter cells splitting and decrease cell size. Vice versa, oxacillin decreases the Sle1 level, and imposes a cell-separation defect that is antagonized by high Sle1 levels, suggesting that high Sle1 levels increase tolerance to oxacillin by promoting cell separation. In contrast, increased oxacillin sensitivity of sle1 cells appears linked to a synthetical lethal effect on septum synthesis. In conclusion, this study demonstrates that Sle1 is a key factor in resistance to β-lactam antibiotics in the JE2 USA300 model strain, and that PBP2a is required for expression of Sle1 in JE2 cells exposed to oxacillin.ImportanceThe bacterium Staphylococcus aureus is a major cause of human disease, and the global spread of S. aureus resistant to β-lactam antibiotics (MRSA) has made treatment increasingly difficult. β-lactams interfere with cross-linking of the bacterial cell wall, however, the killing mechanism of this important class of antibiotics is still not fully understood. Here we provide novel insight into this topic by showing that β-lactam resistance is controlled by the Sle1 cell wall amidase in the fast spreading and highly virulent MRSA USA300 clone. We show that Sle1 high levels accelerate the onset of daughter cells splitting and decrease cell size. Vice versa, oxacillin decreases the Sle1 level, and imposes a cell-separation defect that is antagonized Sle1. The key finding that resistance to β-lactams correlates positively to expression of Sle1 indicates that, in S. aureus, the detrimental effects of β-lactam antibiotics are linked to inhibition of daughter cells splitting.

scholarly journals Daughter cell separation is controlled by cytokinetic ring-activated cell wall hydrolysis

2010 ◽  
Vol 29 (8) ◽  
pp. 1412-1422 ◽  
Author(s):  
Tsuyoshi Uehara ◽  
Katherine R Parzych ◽  
Thuy Dinh ◽  
Thomas G Bernhardt
Keyword(s):  
Cell Wall ◽  
Cell Separation ◽  
Daughter Cell ◽  
Daughter Cell Separation ◽  
Cell Wall Hydrolysis

scholarly journals Lytic transglycosylases RlpA and MltC assist in Vibrio cholerae daughter cell separation

2019 ◽  
Vol 112 (4) ◽  
pp. 1100-1115 ◽  
Author(s):  
Anna I. Weaver ◽  
Valeria Jiménez‐Ruiz ◽  
Srikar R. Tallavajhala ◽  
Brett P. Ransegnola ◽  
Kimberly Q. Wong ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Cell Separation ◽  
Daughter Cell ◽  
Lytic Transglycosylases ◽  
Daughter Cell Separation
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