Automated genomic context analysis and experimental validation platform for discovery of prokaryote transcriptional regulator functions

Martí‐Arbona, Ricardo; Mu, Fangping; Nowak‐Lovato, Kristy; Wren, Melinda; Ünkefer, Clifford J.; Unkefer, Pat J.

doi:10.1186/1471-2164-15-1142

Cited by 3 publications

(1 citation statement)

References 51 publications

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“…To further explore the role of Ms6243, we carried out an extensive search of the literature and found many homologs of Ms6243 in different organism, adapting various functions. One of them we found is Bxe_B3017 from Burkholderia xenovorans , the function of which is unknown, that shares 78% sequence identity with Ms6243 [31]. bxe_B3017 is the neighbouring gene of bxe_B3018 , a TetR family transcriptional regulator that shares its 39% of amino acid sequence with that of Ms6244.…”

Section: Resultsmentioning

confidence: 99%

Ms6244 is a novel Mycobacterium smegmatisTetR family transcriptional repressor that regulates cell growth and morphophysiology

2023

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Transcriptional factors such as the TetR family of transcriptional regulators (TFTRs) are widely found amongst bacteria, including mycobacteria, and are accountable for their survival. Here, we characterized a novel TFTR, Ms6244, from Mycobacterium smegmatis that negatively autoregulates its expression and represses its neighbouring gene, Ms6243. We also report the binding of Ms6244 to the inverted repeats in the intergenic region of Ms6244 and Ms6243. Further, an Ms6244-deleted strain shows various morphophysiological differences compared to the wild type. We further confirmed that the deletion of Ms6244 itself and not the resultant Ms6243 overexpression is the cause of the altered physiology. Our data thus suggest that Ms6244 is an essential regulator, having far-reaching effects on M. smegmatis physiology.

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Section: Resultsmentioning

confidence: 99%

Ms6244 is a novel Mycobacterium smegmatisTetR family transcriptional repressor that regulates cell growth and morphophysiology

2023

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Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

2015

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FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is thus of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl-CoA, we predicted amino acid positions within the effector binding pocket that would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.

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Microbial biosensors for discovery and engineering of enzymes and metabolism

Borzyskowski

Costa

Moritz

et al. 2021

Microbial Cell Factories Engineering for Production of Biomolecules

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Automated genomic context analysis and experimental validation platform for discovery of prokaryote transcriptional regulator functions

Cited by 3 publications

References 51 publications

Ms6244 is a novel Mycobacterium smegmatisTetR family transcriptional repressor that regulates cell growth and morphophysiology

Ms6244 is a novel Mycobacterium smegmatisTetR family transcriptional repressor that regulates cell growth and morphophysiology

Modulation of FadR Binding Capacity for Acyl-CoA Fatty Acids Through Structure-Guided Mutagenesis

Microbial biosensors for discovery and engineering of enzymes and metabolism

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