Solubilization and characterization of extracellular proteins from anammox granular sludge

Boleij, Marissa; Seviour, Thomas; Wong, Lan Li; Loosdrecht, Mark C.M. van; Lin, Yuemei

doi:10.1016/j.watres.2019.114952

Cited by 75 publications

(32 citation statements)

References 42 publications

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“…Progress in identifying extracellular protein targets in environmental systems such as anammox biofilms is limited compared to that for single-species model biofilm systems (e.g., Pseudomonas aeruginosa and Bacillus subtilis ), which likely reflects the fact that anammox bacteria are unavailable in pure culture and genetically intractable ( 11 ). This makes applying classical microbiological approaches to functional assignment challenging.…”

Section: Introductionmentioning

confidence: 99%

“…Nonetheless, no direct functional characterization has been undertaken on a known or isolated anammox biofilm extracellular protein. In the absence of characterized reference extracellular proteins for important industrial and environmental anammox biofilms (11), direct functional characterization on isolated exoproteins is the only strategy available to explain how they mediate anammox biofilm structure and function.…”

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confidence: 99%

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Phase Transitions by an Abundant Protein in the Anammox Extracellular Matrix Mediate Cell-to-Cell Aggregation and Biofilm Formation

Seviour

Wong

Lü

et al. 2020

mBio

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This study describes the first direct functional assignment of a highly abundant extracellular protein from a key environmental and biotechnological biofilm performing an anaerobic ammonium oxidation (anammox) process. Expression levels of Brosi_A1236, belonging to a class of proteins previously suggested to be cell surface associated, were in the top one percentile of all genes in the “Candidatus Brocadia sinica”-enriched biofilm. The Brosi_A1236 structure was computationally predicted to consist of immunoglobulin-like anti-parallel β-strands, and circular dichroism conducted on the isolated surface protein indicated that β-strands are the dominant higher-order structure. The isolated protein was stained positively by the β-sheet-specific stain thioflavin T, along with cell surface- and matrix-associated regions of the biofilm. The surface protein has a large unstructured content, including two highly disordered domains at its C terminus. The disordered domains bound to the substratum and thereby facilitated the adhesion of negatively charged latex microspheres, which were used as a proxy for cells. The disordered domains and isolated whole surface protein also underwent liquid-liquid phase separation to form liquid droplets in suspension. Liquid droplets of disordered protein wet the surfaces of microspheres and bacterial cells and facilitated their coalescence. Furthermore, the surface layer protein formed gels as well as ordered crystalline structures. These observations suggest that biophysical remodeling through phase transitions promotes aggregation and biofilm formation. IMPORTANCE By employing biophysical and liquid-liquid phase separation concepts, this study revealed how a highly abundant extracellular protein enhances the key environmental and industrial bioprocess of anaerobic ammonium oxidation (anammox). Extracellular proteins of environmental biofilms are understudied and poorly annotated in public databases. Understanding the function of extracellular proteins is also increasingly important for improving bioprocesses and resource recovery. Here, protein functions were assessed based on theoretical predictions of intrinsically disordered domains, known to promote adhesion and liquid-liquid phase separation, and available surface layer protein properties. A model is thus proposed to explain how the protein promotes aggregation and biofilm formation by extracellular matrix remodeling and phase transitions. This work provides a strong foundation for functional investigations of extracellular proteins involved in biofilm development.

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Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

Phase Transitions by an Abundant Protein in the Anammox Extracellular Matrix Mediate Cell-to-Cell Aggregation and Biofilm Formation

Seviour

Wong

Lü

et al. 2020

mBio

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“…After electrophoresis, gels were washed twice with ddH 2 O, fixed (10% acetic acid, 25% ethanol, 4 9 15 min) (Boleij et al, 2019), washed again with ddH 2 O twice, and stained overnight in a Alcian Blue solution at pH = 2.5 (0.1% w/v Alcian Blue, 3% acetic acid, 0.5% v/ v Tween-20) or pH = 1.0 (0.1 % w/v Alcian Blue, 0.1 M HCl, 0.5% v/v Tween-20). At pH = 2.5, the cationic dye Alcian Blue stains dissociated (ionic) acidic groups (Boleij et al, 2019) including carboxylated and sulfated mucins, whereas at pH = 1.0 the dye is specific to sulfated mucopolysaccharides. Gels were destained in solutions of the same composition as the respective staining solutions, except for Alcian Blue, and documented.…”

Section: Electrophoretic Analysis Of Biofilm Epsmentioning

confidence: 99%

Regulation of colony morphology and biofilm formation in Shewanella algae

Martín‐Rodríguez

Villion

Yilmaz-Turan

et al. 2021

Microbial Biotechnology

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Bacterial colony morphology can reflect different physiological stages such as virulence or biofilm formation. In this work we used transposon mutagenesis to identify genes that alter colony morphology and cause differential Congo Red (CR) and Brilliant Blue G (BBG) binding in Shewanella algae, a marine indigenous bacterium and occasional human pathogen. Microscopic analysis of colonies formed by the wild-type strain S. algae CECT 5071 and three transposon integration mutants representing the diversity of colony morphotypes showed production of biofilm extracellular polymeric substances (EPS) and distinctive morphological alterations. Electrophoretic and chemical analyses of extracted EPS showed differential patterns between strains, although the targets of CR and BBG binding remain to be identified. Galactose and galactosamine were the preponderant sugars in the colony biofilm EPS of S. algae. Surface-associated biofilm formation of transposon integration mutants was not directly correlated with a distinct colony morphotype. The hybrid sensor histidine kinase BarA abrogated surface-associated biofilm formation. Ectopic expression of the kinase and mutants in the phosphorelay cascade partially recovered biofilm formation. Altogether, this work provides the basic analysis to subsequently address the complex and intertwined networks regulating colony morphology and biofilm formation in this poorly understood species.

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“…A relatively higher phosphorus content (Figure 3d) was observed in the EPS of the non-P-limited CT (0.62 ± 0.02% compared with 0.37 ± 0.06% and 0.35 ± 0.01% in the reference and P-limited). The FTIR spectra (Supporting Information Figure S3) of the extracted EPS display the characteristic carbohydrate band at 940-1,200 cm À1 (Zhu et al 2012;Boleij et al 2019) and protein band at 1,500-1,700 cm À1 with amide I and amide II corresponding peaks (Barth 2007). The ratio of the intensity of the protein band compared with the carbohydrate band is lower in the reference and Plimited samples than in the non-P-limited sample, indicating a preference for carbohydrate production rather than protein when phosphorus is limited.…”

Section: Compositions Of Basin Biofilmsmentioning

confidence: 99%

Effect of phosphate availability on biofilm formation in cooling towers

et al. 2020

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Phosphate limitation has been suggested as a preventive method against biofilms. P-limited feed water was studied as a preventive strategy against biofouling in cooling towers (CTs). Three pilot-scale open recirculating CTs were operated in parallel for five weeks. RO permeate was fed to the CTs (1) without supplementation (reference), (2) with supplementation by biodegradable carbon (P-limited) and (3) with supplementation of all nutrients (non-P-limited). The P-limited water contained 10 mg PO 4 l À1. Investigating the CT-basins and coupons showed that P-limited water (1) did not prevent biofilm formation and (2) resulted in a higher volume of organic matter per unit of active biomass compared with the other CTs. Exposure to external conditions and cycle of concentration were likely factors that allowed a P concentration sufficient to cause extensive biofouling despite being the limiting compound. In conclusion, phosphate limitation in cooling water is not a suitable strategy for CT biofouling control.

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Solubilization and characterization of extracellular proteins from anammox granular sludge

Cited by 75 publications

References 42 publications

Phase Transitions by an Abundant Protein in the Anammox Extracellular Matrix Mediate Cell-to-Cell Aggregation and Biofilm Formation

Phase Transitions by an Abundant Protein in the Anammox Extracellular Matrix Mediate Cell-to-Cell Aggregation and Biofilm Formation

Regulation of colony morphology and biofilm formation in Shewanella algae

Effect of phosphate availability on biofilm formation in cooling towers

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