Surface display of roGFP for monitoring redox status of extracellular microenvironments in Shewanella oneidensis biofilms

Abstract

Biofilms are the most ubiquitous and resilient form of microbial life on earth. One most important feature of a biofilm is the presence of a self-produced matrix, which creates highly heterogeneous and dynamic microenvironments within biofilms. Redox status in biofilm microenvironments plays a critical role in biofilm development and function. However, there is a lack of non-intrusive tools to quantify extracellular redox status of microenvironments within a biofilm matrix. In this study, using Shewanella oneidensis as a model organism, we demonstrated a novel approach to monitor extracellular redox status in biofilm microenvironments. Specifically, we displayed a redox sensitive fluorescence protein roGFP onto the cell surface of S. oneidensis by fusing it to the C-terminus of BpfA, a large surface protein, and used the surface displayed roGFP as a sensor to quantify the extracellular redox status in the matrix of S. oneidensis biofilms. The fusion of roGFP into BpfA has no negative impacts on cell growth and biofilm formation. Upon exposure to oxidizing agents such as H2 O2 , Ag(+) , and SeO3 (2-) , S. oneidensis BpfA-roGFP cells exhibited a characteristic fluorescence of roGFP. Proteinase treatment assay and super-resolution structured illumination microscopy confirmed the surface localization of BpfA-roGFP. We further used the surface displayed roGFP monitored the extracellular redox status in the matrix at different depths of a biofilm exposed to H2 O2 . This study provides a novel approach to non-invasively monitor extracellular redox status in microenvironments within biofilms, which can be used to understand redox responses of biofilms to environmental perturbations.

A microfluidic gradient mixer‐flow chamber as a new tool to study biofilm development under defined solute gradients

“…Moreover, Ca 2+ ‐binding proteins play an important role in bacterial surface adhesion as attachment factors (Porsch et al, ). In S. oneidensis , Ca 2+ ‐binding proteins have been reported to be involved in initial attachment, where the Ca 2+ stimulates auto aggregation of MR‐1 cells (Satchell, ; Sivakumar et al, ; Theunissen et al, ).…”

Construction of controllable and reproducible biofilms through hydrogel based biofilm mimics

“…Calcium regulates extracellular polymeric substance production during biofilm formation and development in many species (Patrauchan et al 2005;Sarkisova et al 2005), and calcium-binding proteins play important role in bacterial surface adhesion (Porsch et al 2013). Specifically, in S. oneidensis MR-1, calcium-binding proteins have been reported to be involved in initial attachment, and calcium stimulates autoaggregation of MR-1 cells significantly (Satchell 2011;Sivakumar et al 2015;Theunissen et al 2010). Hence, the positive relation between biofilm formation and calcium supply in substratum was as expected, indicating the practicability of this gradient generator flow chamber.…”

Enhancing extracellular biocatalysis through biofilm engineering and mutualistic interactions

“…The bacterial strains, plasmids and primers used in the current study are shown in Table 6.1. The growth media and culture incubation conditions for bacterial growth in both planktonic and biofilm mode were described in detail previously (Ding et al 2014a;Sivakumar et al 2015). Unless otherwise stated, the bacterial cultures in planktonic growth mode were incubated under aerobic conditions in a rotary shaker (200 rpm) at 30 o C in LB medium or highly defined modified M1 minimal medium (MM1) with 20 mM lactate as the carbon source.…”

“…The composition of the MM1 medium was described in detail in Section 3.2.1 (Chapter 3) (Ding et al 2014a;Sivakumar et al 2015). For biofilm growth, diluted LB medium (10 × dilution) was used (Zhang et al 2014).…”

“…The Man5C expressing strain was created essentially as described previously (Sivakumar et al 2015). The Man5C expression vector pUC57-Man5C-T5-APH-loxP-H6-aggC contains a DNA cassette that encodes the β-1,3glucomannanases, Man5C (man5C); codon optimized kanamycin resistant aminoglycoside phosphotransferase Aph (aph) of Escherichia coli, and the 305 bp aggC gene fragment derived from S. oneidensis MR-1 chromosome DNA (Bagdasarian et al 1983;Heidelberg et al 2002).…”