Biology subjects

Schuergers, N.

Publications and source records attributed to Schuergers, N..

4 recordsLinked to original sources

bioRxiv2021

Development of a highly sensitive luciferase-based reporter system to study two-step protein secretion in cyanobacteria

Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multi-protein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however little is known regarding their function, regulation and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of non-pilin proteins, we developed a NanoLuc-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. PCC 6803. The NLuc reporter presented a wide dynamic range with at least one order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that two-step protein secretion in cyanobacteria is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to explore protein secretion in cyanobacteria further. ImportanceProtein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here we introduce a NanoLuc (NLuc)-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge on cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.

microbiology↗

bioRxiv2021

Tailored extracellular electron transfer pathways enhance the electroactivity of Escherichia coli

Extracellular electron transfer (EET) engineering in Escherichia coli holds great potential for bioremediation, energy and electrosynthesis applications fueled by readily available organic substrates. Due to its vast metabolic capabilities and availability of synthetic biology tools to adapt strains to specific applications, E. coli is of advantage over native exoelectrogens, but limited in electron transfer rates. We enhanced EET in engineered strains through systematic expression of electron transfer pathways differing in cytochrome composition, localization and origin. While a hybrid pathway harboring components of an E. coli nitrate reductase and the Mtr complex from the exoelectrogen Shewanella oneidensis MR-1 enhanced EET, the highest efficiency was achieved by implementing the complete Mtr pathway from S. oneidensis MR1 in E. coli. We show periplasmic electron shuttling through overexpression of a small tetraheme cytochrome to be central to the electroactivity of this strain, leading to enhanced degradation of the pollutant methyl orange and significantly increased electrical current to graphite electrodes.

bioengineering↗

bioRxiv2021

PATAN-domain response regulators interact with the Type IV pilus motor to control phototactic orientation in the cyanobacterium Synechocystis sp. PCC 6803

Many prokaryotes show complex behaviors that require the intricate spatial and temporal organization of cellular protein machineries, leading to asymmetrical protein distribution and cell polarity. One such behavior is cyanobacterial phototaxis which relies on the dynamic localization of the Type IV pilus motor proteins in response to light. In the cyanobacterium Synechocystis, various signaling systems encompassing chemotaxis-related CheY- and PatA-like response regulators are critical players in switching between positive and negative phototaxis depending on the light intensity and wavelength. In this study, we show that PatA-type regulators evolved from chemosensory systems. Using fluorescence microscopy and yeast-two-hybrid analysis, we demonstrate that they localize to the inner membrane, where they interact with the N-terminal cytoplasmic domain of PilC and the pilus assembly ATPase PilB1. By separately expressing the subdomains of the response regulator PixE, we confirm that only the N-terminal PATAN domain interacts with PilB1, localizes to the membrane, and is sufficient to reverse phototactic orientation. These experiments established that the PATAN domain is the principal output domain of PatA-type regulators which we presume to modulate pilus extension by binding to the pilus motor components.

microbiology↗

bioRxiv2020

Minor pilin genes are involved in motility and natural competence in Synechocystis sp. PCC 6803

Cyanobacteria synthesize type IV pili, which are known to be essential for motility, adhesion and natural competence. They consist of long flexible fibres that are primarily composed of the major pilin PilA1 in Synechocystis sp. PCC 6803. In addition, Synechocystis encodes less abundant pilin-like proteins, which are known as minor pilins. The transcription of the minor pilin genes pilA5, pilA6 and pilA9-pilA11 is inversely regulated in response to different conditions. In this study, we show that the minor pilin PilA5 is essential for natural transformation but is dispensable for motility and flocculation. In contrast, a set of minor pilins encoded by the pilA9-slr2019 transcriptional unit are necessary for motility but are dispensable for natural transformation. Neither pilA5-pilA6 nor pilA9-slr2019 are essential for pilus assembly as mutant strains showed type IV pili on the cell surface. Microarray analysis demonstrated that the transcription levels of known and newly predicted minor pilin genes change in response to surface contact. A total of 120 genes were determined to have altered transcription between planktonic and surface growth. Among these genes, 13 are located on the pSYSM plasmid. The results of our study indicate that different minor pilins facilitate distinct pilus functions.

microbiology↗