Dataset Information

Extracytoplasmic Function ? Factors Can Be Implemented as Robust Heterologous Genetic Switches in Bacillus subtilis.

ABSTRACT: In bacteria, the promoter specificity of RNA polymerase is determined by interchangeable ? subunits. Extracytoplasmic function ? factors (ECFs) form the largest and most diverse family of alternative ? factors, and their suitability for constructing genetic switches and circuits was already demonstrated. However, a systematic study on how genetically determined perturbations affect the behavior of these switches is still lacking, which impairs our ability to predict their behavior in complex circuitry. Here, we implemented four ECF switches in Bacillus subtilis and comprehensively characterized their robustness toward genetic perturbations, including changes in copy number, protein stability, or antisense transcription. All switches show characteristic dose-response behavior that varies depending on the individual ECF-promoter pair. Most perturbations had performance costs. Although some general design rules could be derived, a detailed characterization of each ECF switch before implementation is recommended to understand and thereby accommodate its individual behavior.

SUBMITTER: Pinto D

PROVIDER: S-EPMC6426705 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Extracytoplasmic Function σ Factors Can Be Implemented as Robust Heterologous Genetic Switches in Bacillus subtilis.

Pinto Daniela D Dürr Franziska F Froriep Friederike F Araújo Dayane D Liu Qiang Q Mascher Thorsten T

iScience 20190305

In bacteria, the promoter specificity of RNA polymerase is determined by interchangeable σ subunits. Extracytoplasmic function σ factors (ECFs) form the largest and most diverse family of alternative σ factors, and their suitability for constructing genetic switches and circuits was already demonstrated. However, a systematic study on how genetically determined perturbations affect the behavior of these switches is still lacking, which impairs our ability to predict their behavior in complex cir ...[more]

PMID: 30897511

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Project description:Bacteria must sense alterations in their environment and respond with changes in function and/or structure in order to cope. Extracytoplasmic function sigma factors (ECF ?s) modulate transcription in response to cellular and environmental signals. The symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti carries genes for 11 ECF-like ?s (RpoE1 to -E10 and FecI). We hypothesized that some of these play a role in mediating the interaction between the bacterium and its plant symbiotic partner. The bacterium senses changes in its immediate environment as it establishes contact with the plant root, initiates invasion of the plant as the root nodule is formed, traverses several root cell layers, and enters plant cortical cells via endocytosis. We used genetics, transcriptomics, and functionality to characterize the entire S. meliloti cohort of ECF ?s. We discovered new targets for individual ?s, confirmed others by overexpressing individual ECF ?s, and identified or confirmed putative promoter motifs for nine of them. We constructed precise deletions of each ECF ? gene and its demonstrated or putative anti-? gene and also a strain in which all 11 ECF ? and anti-? genes were deleted. This all-ECF ? deletion strain showed no major defects in free-living growth, in Biolog Phenotype MicroArray assays, or in response to multiple stresses. None of the ECF ?s were required for symbiosis on the host plants Medicago sativa and Medicago truncatula: the strain deleted for all ECF ? and anti-? genes was symbiotically normal.IMPORTANCE Fixed (reduced) soil nitrogen plays a critical role in soil fertility and successful food growth. Much soil fertility relies on symbiotic nitrogen fixation: the bacterial partner infects the host plant roots and reduces atmospheric dinitrogen in exchange for host metabolic fuel, a process that involves complex interactions between the partners mediated by changes in gene expression in each partner. Here we test the roles of a family of 11 extracytoplasmic function (ECF) gene regulatory proteins (sigma factors [?s]) that interact with RNA polymerase to determine if they play a significant role in establishing a nitrogen-fixing symbiosis or in responding to various stresses, including cell envelope stress. We discovered that symbiotic nitrogen fixation occurs even when all 11 of these regulatory genes are deleted, that most ECF sigma factors control accessory functions, and that none of the ECF sigma factors are required to survive envelope stress.

Project description:The Gram-positive, anaerobic, cellulolytic, thermophile Clostridium (Ruminiclostridium) thermocellum secretes a multi-enzyme system called the cellulosome to solubilize plant cell wall polysaccharides. During the saccharolytic process, the enzymatic composition of the cellulosome is modulated according to the type of polysaccharide(s) present in the environment. C. thermocellum has a set of eight alternative RNA polymerase sigma (?) factors that are activated in response to extracellular polysaccharides and share sequence similarity to the Bacillus subtilis ?I factor. The aim of the present work was to demonstrate whether individual C. thermocellum ?I-like factors regulate specific cellulosomal genes, focusing on C. thermocellum ?I6 and ?I3 factors. To search for putative ?I6- and ?I3-dependent promoters, bioinformatic analysis of the upstream regions of the cellulosomal genes was performed. Because of the limited genetic tools available for C. thermocellum, the functionality of the predicted ?I6- and ?I3-dependent promoters was studied in B. subtilis as a heterologous host. This system enabled observation of the activation of 10 predicted ?I6-dependent promoters associated with the C. thermocellum genes: sigI6 (itself, Clo1313_2778), xyn11B (Clo1313_0522), xyn10D (Clo1313_0177), xyn10Z (Clo1313_2635), xyn10Y (Clo1313_1305), cel9V (Clo1313_0349), cseP (Clo1313_2188), sigI1 (Clo1313_2174), cipA (Clo1313_0627), and rsgI5 (Clo1313_0985). Additionally, we observed the activation of 4 predicted ?I3-dependent promoters associated with the C. thermocellum genes: sigI3 (itself, Clo1313_1911), pl11 (Clo1313_1983), ce12 (Clo1313_0693) and cipA. Our results suggest possible regulons of ?I6 and ?I3 in C. thermocellum, as well as the ?I6 and ?I3 promoter consensus sequences. The proposed -35 and -10 promoter consensus elements of ?I6 are CNNAAA and CGAA, respectively. Additionally, a less conserved CGA sequence next to the C in the -35 element and a highly conserved AT sequence three bases downstream of the -10 element were also identified as important nucleotides for promoter recognition. Regarding ?I3, the proposed -35 and -10 promoter consensus elements are CCCYYAAA and CGWA, respectively. The present study provides new clues for understanding these recently discovered alternative ?I factors.

Dataset Information

Extracytoplasmic Function ? Factors Can Be Implemented as Robust Heterologous Genetic Switches in Bacillus subtilis.

Publications

Extracytoplasmic Function σ Factors Can Be Implemented as Robust Heterologous Genetic Switches in Bacillus subtilis.

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