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      Mechanosensitive recruitment of stator units promotes binding of the response regulator CheY-P to the flagellar motor

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          Abstract

          Reversible switching of the bacterial flagellar motor between clockwise (CW) and counterclockwise (CCW) rotation is necessary for chemotaxis, which enables cells to swim towards favorable chemical habitats. Increase in the viscous resistance to the rotation of the motor (mechanical load) inhibits switching. However, cells must maintain homeostasis in switching to navigate within environments of different viscosities. The mechanism by which the cell maintains optimal chemotactic function under varying loads is not understood. Here, we show that the flagellar motor allosterically controls the binding affinity of the chemotaxis response regulator, CheY-P, to the flagellar switch complex by modulating the mechanical forces acting on the rotor. Mechanosensitive CheY-P binding compensates for the load-induced loss of switching by precisely adapting the switch response to a mechanical stimulus. The interplay between mechanical forces and CheY-P binding tunes the chemotactic function to match the load. This adaptive response of the chemotaxis output to mechanical stimuli resembles the proprioceptive feedback in the neuromuscular systems of insects and vertebrates.

          Abstract

          It is unclear how bacterial cells adapt the reversible switching of flagellar motor rotation to environments of different viscosities. Here, Antani et al. show that flagellar mechanosensors allosterically control the motor’s binding affinity for the chemotaxis response regulator, CheY-P, to adapt flagellar switching over varying viscous loads.

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          Most cited references37

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          One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.

          We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.
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            Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.

            We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.
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              An ultrasensitive bacterial motor revealed by monitoring signaling proteins in single cells.

              Understanding biology at the single-cell level requires simultaneous measurements of biochemical parameters and behavioral characteristics in individual cells. Here, the output of individual flagellar motors in Escherichia coli was measured as a function of the intracellular concentration of the chemotactic signaling protein. The concentration of this molecule, fused to green fluorescent protein, was monitored with fluorescence correlation spectroscopy. Motors from different bacteria exhibited an identical steep input-output relation, suggesting that they actively contribute to signal amplification in chemotaxis. This experimental approach can be extended to quantitative in vivo studies of other biochemical networks.
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                Author and article information

                Contributors
                plele@tamu.edu
                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group UK (London )
                2041-1723
                14 September 2021
                14 September 2021
                2021
                : 12
                : 5442
                Affiliations
                [1 ]GRID grid.264756.4, ISNI 0000 0004 4687 2082, Artie McFerrin Department of Chemical Engineering, , Texas A&M University, ; College Station, TX 77843-3122 USA
                [2 ]GRID grid.264756.4, ISNI 0000 0004 4687 2082, Department of Biology, , Texas A&M University, ; College Station, TX 77843-3258 USA
                [3 ]GRID grid.47100.32, ISNI 0000000419368710, Present Address: Department of Ecology & Evolutionary Biology, , Yale University, ; New Haven, CT 06520-8106 USA
                [4 ]GRID grid.47100.32, ISNI 0000000419368710, Present Address: Department of Molecular, Cellular, and Developmental Biology, , Yale University, ; New Haven, CT 06520-8103 USA
                Author information
                http://orcid.org/0000-0002-7402-983X
                http://orcid.org/0000-0003-4681-6374
                http://orcid.org/0000-0001-9183-3021
                http://orcid.org/0000-0002-2894-3487
                Article
                25774
                10.1038/s41467-021-25774-2
                8440544
                34521846
                f78650eb-cced-4563-b0f9-88c91706c503
                © The Author(s) 2021

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 30 April 2021
                : 25 August 2021
                Funding
                Funded by: FundRef https://doi.org/10.13039/100000057, U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS);
                Award ID: R01-GM123085
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/100006751, United States Department of Defense | U.S. Army (United States Army);
                Award ID: W911NF1810353
                Award Recipient :
                Categories
                Article
                Custom metadata
                © The Author(s) 2021

                Uncategorized
                motor protein function,chemotaxis,bacterial physiology,cellular microbiology
                Uncategorized
                motor protein function, chemotaxis, bacterial physiology, cellular microbiology

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