Biophysical traits of surface (S-) layer protein involved in biofilm matrix assembly and function

Extracellular proteins play crucial roles in bacterial biofilm formation, serving as cellular adhesives, structural scaffolds, and matrix stabilizers. However, many of these proteins remain uncharacterized due to a lack of reference protein databases, challenges in extracellular polymer extraction,...

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Main Author: Wong, Lan Li
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Format: Thesis-Doctor of Philosophy
Language:English
Published: Nanyang Technological University 2024
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Online Access:https://hdl.handle.net/10356/175712
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spelling sg-ntu-dr.10356-1757122024-06-03T06:51:19Z Biophysical traits of surface (S-) layer protein involved in biofilm matrix assembly and function Wong, Lan Li - School of Biological Sciences Singapore Centre for Environmental Life Sciences and Engineering (SCELSE) Staffan Kjelleberg laskjelleberg@ntu.edu.sg Medicine, Health and Life Sciences Anammox Extracellular polymeric substances (EPS) Extracellular proteins play crucial roles in bacterial biofilm formation, serving as cellular adhesives, structural scaffolds, and matrix stabilizers. However, many of these proteins remain uncharacterized due to a lack of reference protein databases, challenges in extracellular polymer extraction, and disagreement on the best biophysical methods for resolving functions. Direct functional characterization of isolated exoproteins remains the only strategy available for understanding their role in biofilm matrix formation in different microbial systems. In this study, the ionic liquid (IL), 1-ethyl-3-methylimidazolium acetate, was employed to extract the major extracellular protein from anaerobic ammonium oxidation (anammox) bacteria biofilms. The extract contained a glycoprotein which was homologous to a putative surface (S-) layer protein identified from Candidatus Kuenenia stuttgartiensis, KUSTD 1514. Structural characterization demonstrated that the exoprotein adopted an anti-parallel β-sheet secondary structure with an unstructured C-terminus containing two highly disordered domains. The recombinantly produced disordered domains and biofilm exoprotein isolate underwent liquid-liquid phase separation (LLPS) to form liquid condensates in suspension. Additionally, the disordered domains bound to and facilitated the aggregation of negatively charged latex microspheres and bacteria, indicating their molecular adhesive property. Immunofluorescence and fluorescence in situ hybridization (FISH) staining methods demonstrated that the extracellular glycoproteins form envelopes around the anammox cells, supporting their identity as a S-layer protein. The S-layer protein also coated a member of filamentous non-S-layer protein producing Chloroflexi bacteria, surrounding the anammox bacteria in the biofilm granules. Therefore, the S-layer protein is likely transported through the matrix as an extracellular polymeric substance (EPS) to serve as an anammox biofilm structural scaffold stabilizer. Collectively, the anammox exoprotein isolates formed gels and ordered crystalline structures, and the observation of various forms of S-layer protein on anammox cells suggests that the exoprotein can exist in multiple phases for biofilm formation. Doctor of Philosophy 2024-05-06T02:44:57Z 2024-05-06T02:44:57Z 2023 Thesis-Doctor of Philosophy Wong, L. L. (2023). Biophysical traits of surface (S-) layer protein involved in biofilm matrix assembly and function. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/175712 https://hdl.handle.net/10356/175712 10.32657/10356/175712 en 1301-IRIS-59 This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). application/pdf Nanyang Technological University
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Medicine, Health and Life Sciences
Anammox
Extracellular polymeric substances (EPS)
spellingShingle Medicine, Health and Life Sciences
Anammox
Extracellular polymeric substances (EPS)
Wong, Lan Li
Biophysical traits of surface (S-) layer protein involved in biofilm matrix assembly and function
description Extracellular proteins play crucial roles in bacterial biofilm formation, serving as cellular adhesives, structural scaffolds, and matrix stabilizers. However, many of these proteins remain uncharacterized due to a lack of reference protein databases, challenges in extracellular polymer extraction, and disagreement on the best biophysical methods for resolving functions. Direct functional characterization of isolated exoproteins remains the only strategy available for understanding their role in biofilm matrix formation in different microbial systems. In this study, the ionic liquid (IL), 1-ethyl-3-methylimidazolium acetate, was employed to extract the major extracellular protein from anaerobic ammonium oxidation (anammox) bacteria biofilms. The extract contained a glycoprotein which was homologous to a putative surface (S-) layer protein identified from Candidatus Kuenenia stuttgartiensis, KUSTD 1514. Structural characterization demonstrated that the exoprotein adopted an anti-parallel β-sheet secondary structure with an unstructured C-terminus containing two highly disordered domains. The recombinantly produced disordered domains and biofilm exoprotein isolate underwent liquid-liquid phase separation (LLPS) to form liquid condensates in suspension. Additionally, the disordered domains bound to and facilitated the aggregation of negatively charged latex microspheres and bacteria, indicating their molecular adhesive property. Immunofluorescence and fluorescence in situ hybridization (FISH) staining methods demonstrated that the extracellular glycoproteins form envelopes around the anammox cells, supporting their identity as a S-layer protein. The S-layer protein also coated a member of filamentous non-S-layer protein producing Chloroflexi bacteria, surrounding the anammox bacteria in the biofilm granules. Therefore, the S-layer protein is likely transported through the matrix as an extracellular polymeric substance (EPS) to serve as an anammox biofilm structural scaffold stabilizer. Collectively, the anammox exoprotein isolates formed gels and ordered crystalline structures, and the observation of various forms of S-layer protein on anammox cells suggests that the exoprotein can exist in multiple phases for biofilm formation.
author2 -
author_facet -
Wong, Lan Li
format Thesis-Doctor of Philosophy
author Wong, Lan Li
author_sort Wong, Lan Li
title Biophysical traits of surface (S-) layer protein involved in biofilm matrix assembly and function
title_short Biophysical traits of surface (S-) layer protein involved in biofilm matrix assembly and function
title_full Biophysical traits of surface (S-) layer protein involved in biofilm matrix assembly and function
title_fullStr Biophysical traits of surface (S-) layer protein involved in biofilm matrix assembly and function
title_full_unstemmed Biophysical traits of surface (S-) layer protein involved in biofilm matrix assembly and function
title_sort biophysical traits of surface (s-) layer protein involved in biofilm matrix assembly and function
publisher Nanyang Technological University
publishDate 2024
url https://hdl.handle.net/10356/175712
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