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Absolute Measurements of mRNA Translation in Caulobacter crescentus Reveal Important Fitness Costs of Vitamin B12 Scavenging.

ABSTRACT: Caulobacter crescentus is a model for the bacterial cell cycle which culminates in asymmetric cell division, yet little is known about the absolute levels of protein synthesis of the cellular parts needed to complete the cell cycle. Here we utilize ribosome profiling to provide absolute measurements of mRNA translation in C. crescentus, providing an important resource with quantitative genome-wide measurements of protein output across individual genes. Analysis of protein synthesis rates revealed ?4.5% of cellular protein synthesis is for genes related to vitamin B12 import (btuB) and B12-independent methionine biosynthesis (metE) when grown in common growth media lacking B12 While its facultative B12 lifestyle provides a fitness advantage in the absence of B12, we find that it provides a fitness disadvantage of the cells in the presence of B12, potentially explaining why many Caulobacter species have lost the metE gene and become obligates for B12 IMPORTANCE Caulobacter crescentus is a model system of the bacterial cell cycle culminating in asymmetric cell division, with each daughter cell inheriting a distinct set of proteins. While a genetic network of master transcription factors coordinates the cell cycle timing of transcription for nearly 20% of Caulobacter genes, we lack knowledge of how many of each protein "part" encoded in the genome are synthesized. Therefore, to determine the absolute production rates across the genome, we performed ribosome profiling, providing, for the first time, a quantitative resource with measurements of each protein "part" needed to generate daughter cells. This resource furthers the goal of a systems-level understanding of the genetic network controlling asymmetric cell division. To highlight the utility of this data set, we probe the protein synthesis cost of a B12 utilization pathway and provide new insights into Caulobacter's adaptation to its natural environments.

SUBMITTER: Aretakis JR

PROVIDER: S-EPMC6538847 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Absolute Measurements of mRNA Translation in Caulobacter crescentus Reveal Important Fitness Costs of Vitamin B12 Scavenging.

Aretakis James R JR Gega Alisa A Schrader Jared M JM

mSystems 20190528 4

Caulobacter crescentus is a model for the bacterial cell cycle which culminates in asymmetric cell division, yet little is known about the absolute levels of protein synthesis of the cellular parts needed to complete the cell cycle. Here we utilize ribosome profiling to provide absolute measurements of mRNA translation in C. crescentus, providing an important resource with quantitative genome-wide measurements of protein output across individual genes. Analysis of protein synthesis ...[more]

PMID: 31138672

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Project description: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 crescentus is unique in that it has two homologous, seemingly redundant outer membrane proteins, RsaFa and RsaFb, which together with other components form a type I protein translocation pathway for S-layer export. These proteins have homology to Escherichia coli TolC, the outer membrane channel of multidrug efflux pumps. Here we provide evidence that, unlike TolC, RsaFa and RsaFb are not involved in either the maintenance of membrane stability or the active export of antimicrobial compounds. Rather, RsaFa and RsaFb are required to prevent intracellular accumulation and aggregation of the S-layer protein RsaA; deletion of RsaFa and RsaFb led 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 RsaFa and RsaFb led 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 RsaFa and RsaFb in cellular fitness and tolerance to antimicrobial agents and further our understanding of the S-layer export mechanism on both the transcriptional and translational levels in C. crescentusIMPORTANCE Decreased 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 that Caulobacter crescentus has 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 closest C. crescentus homologs to TolC in E. coli, do not process TolC functionality in C. crescentus.

Dataset Information

Absolute Measurements of mRNA Translation in Caulobacter crescentus Reveal Important Fitness Costs of Vitamin B12 Scavenging.

Publications

Absolute Measurements of mRNA Translation in Caulobacter crescentus Reveal Important Fitness Costs of Vitamin B<sub>12</sub> Scavenging.

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