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Title: Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

Abstract

ABSTRACT Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions, including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport the S-layer protein from the cytoplasm to the cell exterior.Caulobacter crescentusis unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaF aand RsaF b, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology toEscherichia coliTolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaF aand RsaF bare not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaF aand RsaF bare required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaF aand RsaF bled to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and transcriptome sequencing (RNA-seq), we show that loss of both RsaF aand RsaF bled to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involvedmore » in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaF aand RsaF bin cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels inC. crescentus. IMPORTANCEDecreased growth rate and reduced cell fitness are common side effects of protein production in overexpression systems. Inclusion bodies typically form inside the cell, largely due to a lack of sufficient export machinery to transport the overexpressed proteins to the extracellular environment. This phenomenon can conceivably also occur in natural systems. As one example of a system evolved to prevent intracellular protein accumulation, our study demonstrates thatCaulobacter crescentushas two homologous outer membrane transporter proteins that are involved in S-layer export. This is an interesting case study that demonstrates how bacteria can evolve redundancy to ensure adequate protein export functionality and maintain high cellular fitness. Moreover, we provide evidence that these two outer membrane proteins, although being the closestC. crescentushomologs to TolC inE. coli, do not process TolC functionality inC. crescentus.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE
OSTI Identifier:
1339856
Report Number(s):
PNNL-SA-119098
Journal ID: ISSN 0099-2240; 48829; KP1704020
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Applied and Environmental Microbiology; Journal Volume: 82; Journal Issue: 23
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Environmental Molecular Sciences Laboratory

Citation Formats

Overton, K. Wesley, Park, Dan M., Yung, Mimi C., Dohnalkova, Alice C., Smit, John, Jiao, Yongqin, and Parales, R. E. Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation. United States: N. p., 2016. Web. doi:10.1128/AEM.02479-16.
Overton, K. Wesley, Park, Dan M., Yung, Mimi C., Dohnalkova, Alice C., Smit, John, Jiao, Yongqin, & Parales, R. E. Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation. United States. doi:10.1128/AEM.02479-16.
Overton, K. Wesley, Park, Dan M., Yung, Mimi C., Dohnalkova, Alice C., Smit, John, Jiao, Yongqin, and Parales, R. E. Fri . "Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation". United States. doi:10.1128/AEM.02479-16.
@article{osti_1339856,
title = {Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation},
author = {Overton, K. Wesley and Park, Dan M. and Yung, Mimi C. and Dohnalkova, Alice C. and Smit, John and Jiao, Yongqin and Parales, R. E.},
abstractNote = {ABSTRACT Surface layers, or S-layers, are two-dimensional protein arrays that form the outermost layer of many bacteria and archaea. They serve several functions, including physical protection of the cell from environmental threats. The high abundance of S-layer proteins necessitates a highly efficient export mechanism to transport the S-layer protein from the cytoplasm to the cell exterior.Caulobacter crescentusis unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFaand RsaFb, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology toEscherichia coliTolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFaand RsaFbare not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFaand RsaFbare required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFaand RsaFbled to a general growth defect and lowered cellular fitness. Using Western blotting, transmission electron microscopy, and transcriptome sequencing (RNA-seq), we show that loss of both RsaFaand RsaFbled to accumulation of insoluble RsaA in the cytoplasm, which in turn caused upregulation of a number of genes involved in protein misfolding and degradation pathways. These findings provide new insight into the requirement for RsaFaand RsaFbin cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels inC. crescentus. IMPORTANCEDecreased growth rate and reduced cell fitness are common side effects of protein production in overexpression systems. Inclusion bodies typically form inside the cell, largely due to a lack of sufficient export machinery to transport the overexpressed proteins to the extracellular environment. This phenomenon can conceivably also occur in natural systems. As one example of a system evolved to prevent intracellular protein accumulation, our study demonstrates thatCaulobacter crescentushas two homologous outer membrane transporter proteins that are involved in S-layer export. This is an interesting case study that demonstrates how bacteria can evolve redundancy to ensure adequate protein export functionality and maintain high cellular fitness. Moreover, we provide evidence that these two outer membrane proteins, although being the closestC. crescentushomologs to TolC inE. coli, do not process TolC functionality inC. crescentus.},
doi = {10.1128/AEM.02479-16},
journal = {Applied and Environmental Microbiology},
number = 23,
volume = 82,
place = {United States},
year = {Fri Sep 23 00:00:00 EDT 2016},
month = {Fri Sep 23 00:00:00 EDT 2016}
}