Modulation of the enzymatic activity of the flagellar lytic transglycosylase SltF by rod components, and the scaffolding protein FlgJ in Rhodobacter sphaeroides .

2021 ◽  
Author(s):  
Mariela García-Ramos ◽  
Javier de la Mora ◽  
Teresa Ballado ◽  
Laura Camarena ◽  
Georges Dreyfus
Keyword(s):  
Cell Wall ◽  
Enzymatic Activity ◽  
Rhodobacter Sphaeroides ◽  
Cell Envelope ◽  
Scaffold Protein ◽  
Scaffolding Protein ◽  
Lytic Enzyme ◽  
Periplasmic Space ◽  
Bacterial Flagellum ◽  
Lytic Transglycosylase

Macromolecular cell-envelope-spanning structures such as the bacterial flagellum must traverse the cell wall. Lytic transglycosylases enzymes are capable of enlarging gaps in the peptidoglycan meshwork to allow the efficient assembly of supramolecular complexes. In the periplasmic space, the assembly of the flagellar rod requires the scaffold protein FlgJ, which includes a muramidase domain in the canonical models Salmonella enterica and Escherichia coli . In contrast, in Rhodobacter sphaeroides , FlgJ and the dedicated flagellar lytic transglycosylase SltF are separate entities that interact in the periplasm. In this study we show that sltF is expressed along with the genes encoding the early components of the flagellar hierarchy that include the hook-basal body proteins, making SltF available during the rod assembly. Protein-protein interaction experiments demonstrated that SltF interacts with the rod proteins FliE, FlgB, FlgC, FlgF and FlgG through its C-terminal region. A deletion analysis that divides the C-terminus in two halves revealed that the interacting regions for most of the rod proteins are not redundant. Our results also show that the presence of the rod proteins FliE, FlgB, FlgC, and FlgF displace the previously reported SltF-FlgJ interaction. In addition, we observed modulation of the transglycosylase activity of SltF mediated by FlgB and FlgJ that could be relevant to coordinate rod assembly with cell wall remodeling. In summary, different mechanisms regulate the flagellar lytic transglycosylase, SltF ensuring a timely transcription, a proper localization and a controlled enzymatic activity. Importance Several mechanisms participate in the assembly of cell-envelope-spanning macromolecular structures. The sequential expression of substrates to be exported, selective export, and a specific order of incorporation are some of the mechanisms that stand out to drive an efficient assembly process. In this work we analyze how the structural rod proteins, the scaffold protein FlgJ and the flagellar lytic enzyme SltF, interact in an orderly fashion to assemble the flagellar rod into the periplasmic space. A complex arrangement of transient interactions directs a dedicated flagellar muramidase towards the flagellar rod. All these interactions bring this protein to the proximity of the peptidoglycan wall while also modulating its enzymatic activity. This study suggests how a dynamic network of interactions participates in controlling SltF, a prominent component for flagellar formation.

scholarly journals Direct Interaction of Polar Scaffolding Protein Wag31 with Nucleoid-Associated Protein Rv3852 Regulates Its Polar Localization

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1558
Author(s):  
Rajni Garg ◽  
Chinmay Anand ◽  
Sohini Ganguly ◽  
Sandhya Rao ◽  
Rinkee Verma ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Envelope ◽  
Transmembrane Helix ◽  
Ectopic Expression ◽  
Direct Interaction ◽  
Fine Tuning ◽  
Scaffolding Protein ◽  
Physical Interaction ◽  
Cell Wall Synthesis ◽  
Interaction Approach

Rv3852 is a unique nucleoid-associated protein (NAP) found exclusively in Mycobacterium tuberculosis (Mtb) and closely related species. Although annotated as H-NS, we showed previously that it is very different from H-NS in its properties and is distinct from other NAPs, anchoring to cell membrane by virtue of possessing a C-terminal transmembrane helix. Here, we investigated the role of Rv3852 in Mtb in organizing architecture or synthesis machinery of cell wall by protein–protein interaction approach. We demonstrated a direct physical interaction of Rv3852 with Wag31, an important cell shape and cell wall integrity determinant essential in Mtb. Wag31 localizes to the cell poles and possibly acts as a scaffold for cell wall synthesis proteins, resulting in polar cell growth in Mtb. Ectopic expression of Rv3852 in M. smegmatis resulted in its interaction with Wag31 orthologue DivIVAMsm. Binding of the NAP to Wag31 appears to be necessary for fine-tuning Wag31 localization to the cell poles, enabling complex cell wall synthesis in Mtb. In Rv3852 knockout background, Wag31 is mislocalized resulting in disturbed nascent peptidoglycan synthesis, suggesting that the NAP acts as a driver for localization of Wag31 to the cell poles. While this novel association between these two proteins presents one of the mechanisms to structure the elaborate multi-layered cell envelope of Mtb, it also exemplifies a new function for a NAP in mycobacteria.

scholarly journals Native Cell Wall Organization Shown by Cryo-Electron Microscopy Confirms the Existence of a Periplasmic Space in Staphylococcus aureus

2006 ◽  
Vol 188 (3) ◽  
pp. 1011-1021 ◽  
Author(s):  
Valério R. F. Matias ◽  
Terry J. Beveridge
Keyword(s):  
Electron Microscopy ◽  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Cell Envelope ◽  
High Density ◽  
Low Density ◽  
Periplasmic Space ◽  
Wall Zone ◽  
Cell Envelopes

ABSTRACT The current perception of the ultrastructure of gram-positive cell envelopes relies mainly on electron microscopy of thin sections and on sample preparation. Freezing of cells into a matrix of amorphous ice (i.e., vitrification) results in optimal specimen preservation and allows the observation of cell envelope boundary layers in their (frozen) hydrated state. In this report, cryo-transmission electron microscopy of frozen-hydrated sections of Staphylococcus aureus D2C was used to examine cell envelope organization. A bipartite wall was positioned above the plasma membrane and consisted of a 16-nm low-density inner wall zone (IWZ), followed by a 19-nm high-density outer wall zone (OWZ). Observation of plasmolyzed cells, which were used to artificially separate the membrane from the wall, showed membrane vesicles within the space associated with the IWZ in native cells and a large gap between the membrane and OWZ, suggesting that the IWZ was devoid of a cross-linked polymeric cell wall network. Isolated wall fragments possessed only one zone of high density, with a constant level of density throughout their thickness, as was previously seen with the OWZs of intact cells. These results strongly indicate that the IWZ represents a periplasmic space, composed mostly of soluble low-density constituents confined between the plasma membrane and OWZ, and that the OWZ represents the peptidoglycan-teichoic acid cell wall network with its associated proteins. Cell wall differentiation was also seen at the septum of dividing cells. Here, two high-density zones were sandwiched between three low-density zones. It appeared that the septum consisted of an extension of the IWZ and OWZ from the outside peripheral wall, plus a low-density middle zone that separated adjacent septal cross walls, which could contribute to cell separation during division.

scholarly journals The Flagellar Muramidase from the Photosynthetic Bacterium Rhodobacter sphaeroides

2007 ◽  
Vol 189 (22) ◽  
pp. 7998-8004 ◽  
Author(s):  
Javier de la Mora ◽  
Teresa Ballado ◽  
Bertha González-Pedrajo ◽  
Laura Camarena ◽  
Georges Dreyfus
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Micrococcus Luteus ◽  
Photosynthetic Bacterium ◽  
Soft Agar ◽  
Lytic Activity ◽  
Scaffolding Protein ◽  
Amino Acid Sequence Analysis ◽  
Reading Frame ◽  
Lytic Transglycosylase ◽  
Mutant Cells

ABSTRACT We have characterized open reading frame RSP0072, which is located within the flgG operon in Rhodobacter sphaeroides. The amino acid sequence analysis of this gene product showed the presence of a soluble lytic transglycosylase domain. The deletion of the N-terminal region (90 amino acids) of the product of RSP0072 yields a leaky nonmotile phenotype, as determined by swarm assays in soft agar. Electron micrographs revealed the lack of flagella in mutant cells. The purified wild-type protein showed lytic activity on extracts of Micrococcus luteus. In contrast, no lytic activity was observed when the residues E57 or E83 were replaced by alanine. Affinity blotting suggests that the protein encoded by RSP0072 interacts with the flagellar rod-scaffolding protein FlgJ, which lacks the muramidase domain present in FlgJ of many bacteria. We propose that the product of RSP0072 is a flagellar muramidase that is exported to the periplasm via the Sec pathway, where it interacts with FlgJ to open a gap in the peptidoglycan layer for the subsequent penetration of the nascent flagellar structure.

Microanatomy of the Periplasm of Bacillus Licheniformis

1971 ◽  
Vol 29 ◽  
pp. 262-263
Author(s):  
B.K. Ghosh
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Bacillus Licheniformis ◽  
External Environment ◽  
Permeability Barrier ◽  
Periplasmic Space ◽  
Modified Technique ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria

Periplasm of bacteria is the space outside the permeability barrier of plasma membrane but enclosed by the cell wall. The contents of this special milieu exterior could be regulated by the plasma membrane from the internal, and by the cell wall from the external environment of the cell. Unlike the gram-negative organism, the presence of this space in gram-positive bacteria is still controversial because it cannot be clearly demonstrated. We have shown the importance of some periplasmic bodies in the secretion of penicillinase from Bacillus licheniformis.In negatively stained specimens prepared by a modified technique (Figs. 1 and 2), periplasmic space (PS) contained two kinds of structures: (i) fibrils (F, 100 Å) running perpendicular to the cell wall from the protoplast and (ii) an array of vesicles of various sizes (V), which seem to have evaginated from the protoplast.

In vitro assembly of 2-D crystals from partially purified EA1 protein secreted by Bacillus anthracis strain Delta Sterne-1

1994 ◽  
Vol 52 ◽  
pp. 276-277
Author(s):  
Mary Beth Downs ◽  
Wilson Ribot ◽  
Joseph W. Farchaus
Keyword(s):  
Cell Wall ◽  
Molecular Sieves ◽  
Cell Envelope ◽  
Single Species ◽  
Supporting Cell ◽  
Surface Layers ◽  
Rabbit Igg ◽  
In Vitro Assembly ◽  
Covalently Linked

Many bacteria possess surface layers (S-layers) that consist of a two-dimensional protein lattice external to the cell envelope. These S-layer arrays are usually composed of a single species of protein or glycoprotein and are not covalently linked to the underlying cell wall. When removed from the cell, S-layer proteins often reassemble into a lattice identical to that found on the cell, even without supporting cell wall fragments. S-layers exist at the interface between the cell and its environment and probably serve as molecular sieves that exclude destructive macromolecules while allowing passage of small nutrients and secreted proteins. Some S-layers are refractory to ingestion by macrophages and, generally, bacteria are more virulent when S-layers are present.When grown in rich medium under aerobic conditions, B. anthracis strain Delta Sterne-1 secretes large amounts of a proteinaceous extractable antigen 1 (EA1) into the growth medium. Immunocytochemistry with rabbit polyclonal anti-EAl antibody made against the secreted protein and gold-conjugated goat anti-rabbit IgG showed that EAI was localized at the cell surface (fig 1), which suggests its role as an S-layer protein.

A Computational Method for the Identification of Endolysins and Autolysins

2020 ◽  
Vol 27 (4) ◽  
pp. 329-336 ◽  
Author(s):  
Lei Xu ◽  
Guangmin Liang ◽  
Baowen Chen ◽  
Xu Tan ◽  
Huaikun Xiang ◽  
...  
Keyword(s):  
Support Vector Machine ◽  
Cell Wall ◽  
Experimental Results ◽  
Computational Method ◽  
Lytic Enzyme ◽  
Support Vector ◽  
Lytic Enzymes ◽  
Data Set ◽  
Optimal Feature ◽  
Better Than

Background: Cell lytic enzyme is a kind of highly evolved protein, which can destroy the cell structure and kill the bacteria. Compared with antibiotics, cell lytic enzyme will not cause serious problem of drug resistance of pathogenic bacteria. Thus, the study of cell wall lytic enzymes aims at finding an efficient way for curing bacteria infectious. Compared with using antibiotics, the problem of drug resistance becomes more serious. Therefore, it is a good choice for curing bacterial infections by using cell lytic enzymes. Cell lytic enzyme includes endolysin and autolysin and the difference between them is the purpose of the break of cell wall. The identification of the type of cell lytic enzymes is meaningful for the study of cell wall enzymes. Objective: In this article, our motivation is to predict the type of cell lytic enzyme. Cell lytic enzyme is helpful for killing bacteria, so it is meaningful for study the type of cell lytic enzyme. However, it is time consuming to detect the type of cell lytic enzyme by experimental methods. Thus, an efficient computational method for the type of cell lytic enzyme prediction is proposed in our work. Method: We propose a computational method for the prediction of endolysin and autolysin. First, a data set containing 27 endolysins and 41 autolysins is built. Then the protein is represented by tripeptides composition. The features are selected with larger confidence degree. At last, the classifier is trained by the labeled vectors based on support vector machine. The learned classifier is used to predict the type of cell lytic enzyme. Results: Following the proposed method, the experimental results show that the overall accuracy can attain 97.06%, when 44 features are selected. Compared with Ding's method, our method improves the overall accuracy by nearly 4.5% ((97.06-92.9)/92.9%). The performance of our proposed method is stable, when the selected feature number is from 40 to 70. The overall accuracy of tripeptides optimal feature set is 94.12%, and the overall accuracy of Chou's amphiphilic PseAAC method is 76.2%. The experimental results also demonstrate that the overall accuracy is improved by nearly 18% when using the tripeptides optimal feature set. Conclusion: The paper proposed an efficient method for identifying endolysin and autolysin. In this paper, support vector machine is used to predict the type of cell lytic enzyme. The experimental results show that the overall accuracy of the proposed method is 94.12%, which is better than some existing methods. In conclusion, the selected 44 features can improve the overall accuracy for identification of the type of cell lytic enzyme. Support vector machine performs better than other classifiers when using the selected feature set on the benchmark data set.

A Simple Method for Removal of the Chlamydomonas reinhardtii Cell Wall Using a Commercially Available Subtilisin (Alcalase)

2018 ◽  
Vol 28 (4) ◽  
pp. 169-178 ◽  
Author(s):  
Hyun-Ju Hwang ◽  
Yong Tae Kim ◽  
Nam Seon Kang ◽  
Jong Won Han
Keyword(s):  
Cell Wall ◽  
Chlamydomonas Reinhardtii ◽  
Algal Cell ◽  
Chemical Properties ◽  
Transformation Method ◽  
Lytic Enzyme ◽  
Higher Plant ◽  
Time Saving ◽  
Simple Method ◽  
Cell Wall Disruption

The algal cell wall is a potent barrier for delivery of transgenes for genetic engineering. Conventional methods developed for higher plant systems are often unable to penetrate or remove algal cell walls owing to their unique physical and chemical properties. Therefore, we developed a simple transformation method for <i>Chlamydomonas reinhardtii</i> using commercially available enzymes. Out of 7 enzymes screened for cell wall disruption, a commercial form of subtilisin (Alcalase) was the most effective at a low concentration (0.3 Anson units/mL). The efficiency was comparable to that of gamete lytic enzyme, a protease commonly used for the genetic transformation of <i>C. reinhardtii</i>. The transformation efficiency of our noninvasive method was similar to that of previous methods using autolysin as a cell wall-degrading enzyme in conjunction with glass bead transformation. Subtilisin showed approximately 35% sequence identity with sporangin, a hatching enzyme of <i>C. reinhardtii</i>, and shared conserved active domains, which may explain the effective cell wall degradation. Our trans­formation method using commercial subtilisin is more reliable and time saving than the conventional method using autolysin released from gametes for cell wall lysis.

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