Protein Residues and a Novel Motif Involved in the Cellular Localization of CheZ in Azorhizobium caulinodans ORS571

Abstract

Chemotaxis is essential for the competitiveness of motile bacteria in complex and harsh environments. The localization of chemotactic proteins in the cell is critical for coordinating a maximal response to chemotactic signals. One chemotaxis protein with a well-defined subcellular localization is the phosphatase CheZ. CheZ localizes to cell poles by binding with CheA in Escherichia coli and other enteric bacteria, or binding with a poorly understood protein called ChePep in epsilon-Proteobacteria. In alpha-Proteobacteria, CheZ lacks CheA-binding sites, and its cellular localization remains unknown. We therefore determined the localization of CheZ in the alpha-Proteobacteria Azorhizobium caulinodans ORS571. A. caulinodans CheZ, also termed as CheZAC, was found to be located to cell poles independently of CheA, and we suspect that either the N-terminal helix or the four-helix bundle of CheZAC is sufficient to locate to cell poles. We also found a novel motif, AXXFQ, which is adjacent to the phosphatase active motif DXXXQ, which effects the monopolar localization of CheZAC. This novel motif consisting of AXXFQ is conserved in CheZ and widely distributed among Proteobacteria. Finally, we found that the substitution of phosphatase active site affects the polar localization of CheZAC. In total, this work characterized the localization pattern of CheZ containing a novel motif, and we mapped the regions of CheZAC that are critical for its polar localization.

The Hypoxia-Associated Localization of Chemotaxis Protein CheZ in Azorhizorbium caulinodans

“…In this study, we determined CheZ localizes to the poles upon inhibiting the electron transport chain of A. caulinodans, which requires the C-terminus of CheZ and one of the conserved phosphatase active site, D165 but not Q169. These results are consistent with our previous observation that the substitution mutant of CheZ (CheZD165A) showed a low level of localization, while the localization pattern of CheZQ169A was similar to wild-type CheZ (Liu et al, 2020). Recently, we identified that a novel motif AXXF(Y)Q, which is adjacent to the phosphatase active sites, controls the polar localization of CheZ (Liu et al, 2020), and we presumed the role of D165 of CheZ on localization might be achieved by interacting with the motif.…”

“…Furthermore, the polar localization of chemotaxis proteins improves the cooperative interactions between them, which enables bacteria sense nanomolar concentrations of signal molecules over a wide range (Greenfield et al, 2009). We previously identified the residues and domains of CheZ, which are important for the cellular localization of CheZ (Liu et al, 2020). In this study, we further found that the location of CheZ can be regulated by hypoxia.…”

“…We previously reported that CheZ in A. caulinodans can locate to cell poles and the localization pattern of CheZ is diverse, including monopolar, bipolar, and diffuse ( Liu et al, 2020 ). Among them, diffuse localization of CheZ is the most common, observed in about 60% of cells ( Liu et al, 2020 ). Unexpectedly, we observed that the ratio of cells with polar CheZ localization becomes very high after standing over 2days ( Figure 1A ).…”

“…The C-Terminus and a Conserved Active Site D165 of CheZ Are Involved in the Energy-Related Polar Localization Recently, we have mapped the region of CheZ for polar localization and found that the remaining of 70 residues at the N-terminus, CheZN70 (also termed as CheZΔ71-236), is sufficient for polar localization (Liu et al, 2020). To investigate whether the presence of N-terminal region is sufficient for the energy-related localization, the localization pattern of CheZN70 with or without sodium azide was recorded.…”

“…Disruption of cheZ in A. caulinodans abolishes the chemotactic response to attractants and causes an increase in exopolysaccharide production ( Liu et al, 2018b ). Recently, we have determined the localization pattern of CheZ at cell poles and identified a novel motif that is involved in the regulation of localization ( Liu et al, 2020 ).…”