Isolation of Extracellular Polymeric Substances from Biofilms of the Thermoacidophilic Archaeon Sulfolobus acidocaldarius.

Silke Jachlewski,Bettina Siebers,Christopher Bräsen,Uwe Linne,Jost Wingender,Witold D Jachlewski

doi:10.3389/fbioe.2015.00123

Abstract

Extracellular polymeric substances (EPS) are the major structural and functional components of microbial biofilms. The aim of this study was to establish a method for EPS isolation from biofilms of the thermoacidophilic archaeon, Sulfolobus acidocaldarius, as a basis for EPS analysis. Biofilms of S. acidocaldarius were cultivated on the surface of gellan gum-solidified Brock medium at 78°C for 4 days. Five EPS extraction methods were compared, including shaking of biofilm suspensions in phosphate buffer, cation-exchange resin (CER) extraction, and stirring with addition of EDTA, crown ether, or NaOH. With respect to EPS yield, impact on cell viability, and compatibility with subsequent biochemical analysis, the CER extraction method was found to be the best suited isolation procedure resulting in the detection of carbohydrates and proteins as the major constituents and DNA as a minor component of the EPS. Culturability of CER-treated cells was not impaired. Analysis of the extracellular proteome using two-dimensional gel electrophoresis resulted in the detection of several hundreds of protein spots, mainly with molecular masses of 25–116 kDa and pI values of 5–8. Identification of proteins suggested a cytoplasmic origin for many of these proteins, possibly released via membrane vesicles or biofilm-inherent cell lysis during biofilm maturation. Functional analysis of EPS proteins, using fluorogenic substrates as well as zymography, demonstrated the activity of diverse enzyme classes, such as proteases, lipases, esterases, phosphatases, and glucosidases. In conclusion, the CER extraction method, as previously applied to bacterial biofilms, also represents a suitable method for isolation of water soluble EPS from the archaeal biofilms of S. acidocaldarius, allowing the investigation of composition and function of EPS components in these types of biofilms.

Highlights

Microorganisms in the biofilm mode of life predominate, numerically and metabolically, in a wide variety of natural, technical, and medical environments (Costerton et al, 1995)
Composition of S. acidocaldarius Biofilm Biofilms of S. acidocaldarius grown at 78°C for 4 days on gellan gum-solidified Brock medium contained a total biomass of approximately 0.6 g per Petri dish (100 mm in diameter)
Extracellular polymeric substances were isolated from biofilms of S. acidocaldarius scraped from the medium plates using five different procedures, including shaking, shaking in the presence of cationexchange resin (CER), stirring with added NaOH, EDTA, and crown ether, respectively

Summary

Introduction

Microorganisms in the biofilm mode of life predominate, numerically and metabolically, in a wide variety of natural, technical, and medical environments (Costerton et al, 1995). Microbial biofilms are single- or multi-species communities that accumulate at interfaces (solid–liquid or solid–air), where the microorganisms live at high cell densities in a matrix of hydrated extracellular polymeric substances (EPS) (Hall-Stoodley et al, 2004). EPS are mainly polysaccharides, proteins, extracellular DNA (eDNA), and lipids. They mediate biofilm adhesion to surfaces and form a cohesive, three-dimensional polymer network interconnecting and immobilizing biofilm cells, and they provide mechanical stability to biofilms (Flemming and Wingender, 2010). Bacterial biofilms have been intensively studied because of their ecological importance, application in biotechnology, and waste water treatment and due to their potential role in human infections and function as environmental reservoirs for pathogens (Hall-Stoodley et al, 2004). It has become evident that environmental biofilm communities often contain both bacterial and archaeal species; in addition, laboratory investigations have shown that single archaeal species are able to form biofilms on diverse biotic and abiotic surfaces (Fröls, 2013; Orell et al, 2013)

Objectives

Methods

Results

Discussion

Conclusion