Polar Localization of the Serine Chemoreceptor of Escherichia coli Is Nucleoid Exclusion-Dependent.

Article Polar Localization of the Serine Chemoreceptor of Escherichia coli Is Nucleoid Exclusion-Dependent Ramakanth Neeli-Venkata,1 Sofia Startceva,1 Teppo Annila,1 and Andre S. Ribeiro1,* 1 Laboratory of Biosystem Dynamics, Department of Signal Processing, Tampere University of Technology, Tampere, Finland ABSTRACT We studied whether nucleoid exclusion contributes to the segregation and retention of Tsr chemoreceptor clusters at the cell poles. Using live time-lapse, single-cell microscopy measurements, we show that the single-cell spatial distributions of Tsr clusters have heterogeneities and asymmetries that are consistent with nucleoid exclusion and cannot be explained by the diffusion-and-capture mechanism supported by Tol-Pal complexes at the poles. Also, in cells subjected to ampicillin, which enhances relative nucleoid lengths, Tsr clusters locate relatively closer to the cell extremities, whereas in anucleated cells (deletion mutants for mukB), the Tsr clusters are closer to midcell. In addition, we find that the fraction of Tsr clusters at the poles is smaller in deletion mutants for Tol-Pal than in wild-type cells, although it is still larger than would be expected by chance. Also in deletion mutants, the distribution of Tsr clusters differs widely between cells with relatively small and large nucleoids, in a manner consistent with nucleoid exclusion from midcell. This comparison further showed that diffusion-and-capture by TolPal complexes and nucleoid exclusion from the midcell have complementary effects. Subsequently, we subjected deletion mutants to suboptimal temperatures that are known to enhance cytoplasm viscosity, which hampers nucleoid exclusion effects. As the temperature was lowered, the fraction of clusters at the poles decreased linearly. Finally, a stochastic model including nucleoid exclusion at midcell and diffusion-and-capture due to Tol-Pal at the poles is shown to exhibit a cluster dynamics that is consistent with the empirical data. We conclude that nucleoid exclusion also contributes to the preference of Tsr clusters for polar localization. INTRODUCTION Escherichia coli chemoreceptor proteins perform multiple tasks, including assessing chemical gradients (1), thermosensing (2), and aerotaxis (3). These proteins are organized in trimer-of- dimers that form large clusters whose structure is further stabilized by the adaptor protein CheW and the histidine kinase CheA (1,4,5). The purpose of clustering is likely signal-processing enhancement of the receptor system (6–9). The clustering process is robust, as receptors can assemble via their cytoplasmic domains even in the absence of some chemotaxis-associated proteins, such as CheW (10). Most studies agree that cluster formation occurs via an energy-free, self-assembly process known as stochastic nucleation (11–14). Chemotaxis-associated clusters preferentially locate at the cell poles (15–17), but the means by which this occurs remain unclear, given the lack of evidence for active transport mechanisms. Studies have suggested various mechanisms by which this may occur. For example, it has been Submitted June 6, 2016, and accepted for publication October 19, 2016. *Correspondence: Editor: Zemer Gitai. http://dx.doi.org/10.1016/j.bpj.2016.10.024 Ó 2016 Biophysical Society. 2512 Biophysical Journal 111, 2512–2522, December 6, 2016 suggested that the clusters first form at midcell and then attach to the cell membranes, and are dragged to the poles by cell growth after a few rounds of cell division (11,12). It has also been suggested that the clusters diffuse freely in the cell membranes and that polar accumulation is caused by the curved shape of the poles and the ability of the clusters to match this curvature (7,18). Recent studies suggested that instead a diffusion-andcapture process (19) is responsible for the spatial distribution of this and several other polar proteins (20–23). One study in particular (24) identified the trans-envelope TolPal complex, a widely conserved component of the cell envelope of Gram-negative bacteria (25), as being responsible for capturing the clusters at the poles, since in deletion mutants for Tol-Pal this process is impaired. The existence of a diffusion-and-capture mechanism is further supported by the observation that a fairly constant fraction (~7%) of Tsr proteins exhibit free diffusion over the entire cell surface at any given time (26). Tsr, one of the methyl-accepting chemoreceptor proteins of the E. coli chemotaxis system (2), is a serine chemotaxis receptor protein that preferentially forms heterotrimeric membrane complexes at the poles. The mobility of Tsr Nucleoid Exclusion of Tsr Clusters labeled with fluorescent Venus proteins was recently investigated and found to be similar to that of the natural system (26). These proteins can diffuse over the entire cell surface but usually exhibit restricted diffusion, particularly at the poles, where they appear to move freely except for being restricted to the same pole for several generations (12). When the cytoskeletal protein MreB is disrupted and the cell becomes rounded, Tsr clusters at the poles tend to fragment and the fraction of mobile Tsr increases (26). This suggests that, aside from the diffusion-and-capture process made possible by Tol-Pal complexes (24), one or more additional mechanisms may contribute to the preference of the chemoreceptor clusters for a polar location. In E. coli, the nucleoid is usually at midcell and confined within the cell cylinder. Among other components, it contains most of the DNA, RNA, and nucleoid-associated proteins of the cell. Major nucleoid-associated proteins include H-NS, HU, Fis, IHF, and StpA. The dimeric histone-like protein HU in particular is highly abundant and involved in DNA compaction, and thus can be used to assess the nucleoid’s morphology and positioning in vivo when fused with fluorescent proteins such as mCherry (27). The partitioning of replicated nucleoids in cell division involves the structural maintenance of chromosome complex MukBEF (28). The deletion of mukB causes a temperature-sensitive lethal phenotype that fails to partition the chromosome, resulting in the formation of anucleate cells. Recent studies have reported that in addition to Tsr clusters, other types of large biomolecules in E. coli are segregated to and then retained at the poles. This is due to an energy-free volume exclusion caused by the presence of the nucleoid at midcell (29,30) that affects plasmids (31,32) and other large complexes (33,34). A possible contribution of nucleoid exclusion to the distribution of chemoreceptor clusters associated with chemotaxis has not yet been considered. Here, we explored whether nucleoid exclusion contributes to the segregation and retention of Tsr chemoreceptor clusters at the cell poles. In addition, we evaluated the contribution from other, previously proposed mechanisms, namely, Tol-Pal diffusion-and-capture and draggi (...truncated)