Adaptability is an essential property of many sensory systems, enabling maintenance of a sensitive response over a range of background stimulus levels. In bacterial chemotaxis, adaptation to the preset level of pathway activity is achieved through an integral feedback mechanism based on activity-dependent methylation of chemoreceptors. It has been argued that this architecture ensures precise and robust adaptation regardless of the ambient ligand concentration, making perfect adaptation a celebrated property of the chemotaxis system. However, possible deviations from such ideal adaptive behavior and its consequences for chemotaxis have not been explored in detail. Here we show that the chemotaxis pathway in Escherichia coli shows increasingly imprecise adaptation to higher concentrations of attractants, with a clear correlation between the time of adaptation to a step-like stimulus and the extent of imprecision. Our analysis suggests that this imprecision results from a gradual saturation of receptor methylation sites at high levels of stimulation, which prevents full recovery of the pathway activity by violating the conditions required for precise adaptation. We further use computer simulations to show that limited imprecision of adaptation has little effect on the rate of chemotactic drift of a bacterial population in gradients, but hinders precise accumulation at the peak of the gradient. Finally, we show that for two major chemoeffectors, serine and cysteine, failure of adaptation at concentrations above 1 mM might prevent bacteria from accumulating at toxic concentrations of these amino acids.

Deubiquitination of NF-B members by IntroductionCYLD is a tumor suppressor gene that is mutated in familial cylindromatosis, an autosomal dominant predisposition to tumors of skin appendages. 1 CYLD removes lysine-63 polyubiquitin chains from distinct members of the NF-B pathway 2-4 and mutations in CYLD dysregulate NF-B activity.Complete deletion of CYLD in mice (CYLD ko ) renders them susceptible to skin tumors, 5 but the CYLD ko mice do not display alterations of the immune system, as we could show for B-6 and T-cell development (A.W., N.H., S. Reissig, manuscript in preparation). Other knockout mouse models of CYLD point to a role for flCYLD in immunity, including hyperinduction of IFN␣ in virus-infected dendritic cells (DCs) 7 as well as protection from infections in its absence. 8 However, mice exclusively expressing the naturally occurring short splice variant of CYLD, sCYLD, are characterized by lymphomegaly, splenomegaly, and dramatic increases in B-cell numbers 6 caused by aberrant NF-B signaling. Because this pathway is important for the function of DCs, 9 which orchestrate innate and adaptive immune responses, we investigated the effect of sCYLD overexpression on DC function. We found CYLD ex7/8 DCs to be hyperresponsive to LPS, capable of inducing superior T-cell expansion on DC immunization in vivo, and able to suppress tolerance induced by ␣-DEC-205:OVA administration. As a molecular basis for this phenotype, we identified increased nuclear Bcl-3, p50, and p65 in resting and stimulated CYLD ex7/8 bone marrowderived DCs (BMDCs). Methods Mice and BMDC generationC57BL/6 wild-type (WT), CYLD ko , 5 and CYLD ex7/8 6 mice 6 to 8 weeks old were used as recipients and for BMDC generation as previously described. 10 St42 TCR Tg mice have been previously described. 11 OT-I mice 12 were obtained from Christian Kurts (Institute for Molecular Medicine & Experimental Immunology, Bonn, Germany). Approval for these studies was obtained from the review board of the Federal State of Rhineland-Palatinate, Germany. All animal experiments were in accordance with the guidelines of the Central Animal Facility Institution of the University of Mainz. Flow cytometryFACSCanto cytometer and FlowJo software (Tree Star, Ashland, OR) using ␣-CD8 and ␣-CD90.1/CD45.1 (eBioscience, San Diego, CA) was used. ␣-IL-6, IL-10, and TNF-␣ antibodies from Cytometric Bead Array Flex system (Becton Dickinson, Franklin Lakes, NJ) were used and analyzed with FCAP Array Software (BD Biosciences, Mountain View, CA). Immune toleranceRecipient mice were given 10 6 adoptively transferred OT-I cells, immunized with 20 g DEC-205:OVA, and boosted with 50 g OVA protein (Sigma-Aldrich, St Louis, MO) as previously described. 13Western blot analysis and electrophoretic mobility shift assay Western blot analysis was performed as previously described. 8 Nuclear and cytoplasmic fractions were prepared according to standard procedures. 14,15 NF-B Consensus Oligonucleotide was purchased from For personal use only. on May 7, 2018. by guest www.bloodjournal.org ...

Bacteria navigate within inhomogeneous environments by temporally comparing concentrations of chemoeffectors over the course of a few seconds and biasing their rate of reorientations accordingly, thereby drifting towards more favorable conditions. This navigation requires a short-term memory achieved through the sequential methylations and demethylations of several specific glutamate residues on the chemotaxis receptors, which progressively adjusts the receptors’ activity to track the levels of stimulation encountered by the cell with a delay. Such adaptation also tunes the receptors’ sensitivity according to the background ligand concentration, enabling the cells to respond to fractional rather than absolute concentration changes, i.e. to perform logarithmic sensing. Despite the adaptation system being principally well understood, the need for a specific number of methylation sites remains relatively unclear. Here we systematically substituted the four glutamate residues of the Tar receptor of Escherichia coli by non-methylated alanine, creating a set of 16 modified receptors with a varying number of available methylation sites and explored the effect of these substitutions on the performance of the chemotaxis system. Alanine substitutions were found to desensitize the receptors, similarly but to a lesser extent than glutamate methylation, and to affect the methylation and demethylation rates of the remaining sites in a site-specific manner. Each substitution reduces the dynamic range of chemotaxis, by one order of magnitude on average. The substitution of up to two sites could be partly compensated by the adaptation system, but the full set of methylation sites was necessary to achieve efficient logarithmic sensing.