Development of a Highly Sensitive Luciferase-Based Reporter System To Study Two-Step Protein Secretion in Cyanobacteria
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Development of a Highly Sensitive Luciferase-Based Reporter System To Study Two-Step Protein Secretion in Cyanobacteria. / Russo, David A.; Zedler, Julie A.Z.; Conradi, Fabian D.; Schuergers, Nils; Jensen, Poul Erik; Mullineaux, Conrad W.; Wilde, Annegret; Pohnert, Georg.
In: Journal of Bacteriology, Vol. 204, No. 2, e0050421, 2022.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Development of a Highly Sensitive Luciferase-Based Reporter System To Study Two-Step Protein Secretion in Cyanobacteria
AU - Russo, David A.
AU - Zedler, Julie A.Z.
AU - Conradi, Fabian D.
AU - Schuergers, Nils
AU - Jensen, Poul Erik
AU - Mullineaux, Conrad W.
AU - Wilde, Annegret
AU - Pohnert, Georg
PY - 2022
Y1 - 2022
N2 - 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 multiprotein 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 nonpilin proteins, we developed a NanoLuc (NLuc)-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. strain PCC 6803. The NLuc reporter presented a wide dynamic range with at least 1 order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that the two-step secretion of NLuc 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 exploring protein secretion in cyanobacteria further. IMPORTANCE Protein 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 an 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 of cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.
AB - 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 multiprotein 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 nonpilin proteins, we developed a NanoLuc (NLuc)-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. strain PCC 6803. The NLuc reporter presented a wide dynamic range with at least 1 order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that the two-step secretion of NLuc 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 exploring protein secretion in cyanobacteria further. IMPORTANCE Protein 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 an 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 of cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.
KW - cyanobacteria
KW - general secretory pathway
KW - NanoLuc luciferase
KW - protein secretion
KW - Synechocystis
KW - type IV pili
U2 - 10.1128/JB.00504-21
DO - 10.1128/JB.00504-21
M3 - Journal article
C2 - 34898262
AN - SCOPUS:85124633512
VL - 204
JO - Journal of Bacteriology
JF - Journal of Bacteriology
SN - 0021-9193
IS - 2
M1 - e0050421
ER -
ID: 298034470