Increasing evidence points to a dynamical compartmentalization of the cell nucleus, yet the mechanisms by which interphase chromatin moves and is positioned within nuclei remain unclear. Here, we study the dynamics of chromatin in vivo applying a novel particle-tracking method in a two-photon microscope that provides approximately 10-fold higher spatial and temporal resolutions than previous measurements. We followed the motion of a chromatin sequence containing a lac-operator repeat in cells stably expressing lac repressor fused with enhanced green fluorescent protein, observing long periods of apparent constrained diffusion interrupted by relatively abrupt jumps of approximately 150 nm lasting 0.3-2 s. During these jumps, the particle moved an average of four times faster than in the periods between jumps and in paths more rectilinear than predicted for random diffusion motion. Additionally, the jumps were sensitive to the temperature and absent after ATP depletion. These experimental results point to an energy-dependent mechanism driving fast motion of chromatin in interphase cells.

Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggest that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation.

Large-scale chromatin decondensation has been observed after the targeting of certain acidic activators to heterochromatic chromatin domains. Acidic activators are often modular, with two or more separable transcriptional activation domains. Whether these smaller regions are sufficient for all functions of the activators has not been demonstrated. We adapted an inducible heterodimerization system to allow systematic dissection of the function of acidic activators, individual subdomains within these activators, and short acidic-hydrophobic peptide motifs within these subdomains. Here, we demonstrate that large-scale chromatin decondensation activity is a general property of acidic activators. Moreover, this activity maps to the same acidic activator subdomains and acidic-hydrophobic peptide motifs that are responsible for transcriptional activation. Two copies of a mutant peptide motif of VP16 (viral protein 16) possess large-scale chromatin decondensation activity but minimal transcriptional activity, and a synthetic acidic-hydrophobic peptide motif had large-scale chromatin decondensation activity comparable to the strongest full-length acidic activator but no transcriptional activity. Therefore, the general property of large-scale chromatin decondensation shared by most acidic activators is not simply a direct result of transcription per se but is most likely the result of the concerted action of coactivator proteins recruited by the activators' short acidic-hydrophobic peptide motifs.

We investigated pH taxis in Bacillus subtilis. This bacterium was found to perform bidirectional taxis in response to external pH gradients, enabling it to preferentially migrate to neutral environments. We next investigated the chemoreceptors involved in sensing pH gradients. We identified four chemoreceptors involved in sensing pH: McpA and TlpA for sensing acidic environments and McpB and TlpB for sensing alkaline ones. In addition, TlpA was found to also weakly sense alkaline environments. By analyzing chimeras between McpA and TlpB, the principal acid- and base-sensing chemoreceptors, we identified four critical amino acid residues—Thr199, Gln200, His273, and Glu274 on McpA and Lys199, Glu200, Gln273, and Asp274 on TlpB—involved in sensing pH. Swapping these four residues between McpA and TlpB converted the former into a base receptor and the latter into an acid receptor. Based on the results, we propose that disruption of hydrogen bonding between the adjacent residues upon pH changes induces signaling. Collectively, our results further our understanding of chemotaxis in B. subtilis and provide a new model for pH sensing in bacteria. IMPORTANCE Many bacteria can sense the pH in their environment and then use this information to direct their movement toward more favorable locations. In this study, we investigated the pH sensing mechanism in Bacillus subtilis. This bacterium preferentially migrates to neutral environments. It employs four chemoreceptors to sense pH. Two are involved in sensing acidic environments, and two are involved in sensing alkaline ones. To identify the mechanism for pH sensing, we constructed receptor chimeras of acid- and base-sensing chemoreceptors. By analyzing the responses of these chimeric receptors, we were able to identify four critical amino acid residues involved in pH sensing and propose a model for the pH sensing mechanism in B. subtilis.

Summary Chemotaxis by Bacillus subtilis requires the CheD protein for proper function. In a cheD mutant when McpB was the sole chemoreceptor in B. subtilis, chemotaxis to asparagine was quite good. When McpC was the sole chemoreceptor in a cheD mutant, chemotaxis to proline was very poor. The reason for the difference between the chemoreceptors is because CheD deamidates Q609 in McpC and does not deamidate McpB. When mcpC-Q609E is expressed as the sole chemoreceptor in a cheD background, chemotaxis is almost fully restored. Concomitantly, in vitro McpC activates the CheA kinase poorly, whereas McpC-Q609E activates it much more. Moreover, CheD, which activates chemoreceptors, binds better to McpC-Q609E compared with unmodified McpC. Using hydroxyl radical susceptibility in the presence or absence of CheD, the most likely sites of CheD binding were the modification sites where CheD, CheB, and CheR carry out their catalytic activities. Thus, CheD appears to have two separate roles in B. subtilis chemotaxis - to bind to chemoreceptors to activate them as part of the CheC/CheD/CheYp adaptation system and to deamidate selected residues to activate the chemoreceptors and enable them to mediate amino acid chemotaxis.