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

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.


INTRODUCTION
In harsh and complex environments, bacteria must adapt and respond to external changes quickly. Chemotaxis systems are one-way bacteria have envolved to do this. Chemotaxis enables bacteria to regulate their motility in response to environmental signals. The chemotaxis pathway has been well studied in Escherichia coli. External signals or nutrient molecules are sensed by chemoreceptors. Upon binding with attractant signals, conformational changes of chemoreceptors inhibit the autokinase activity of the associated histidine kinase CheA. In the presents of a repellent signal, CheA can phosphorylate the response regulator CheY, and CheY-P diffuses and binds with the flagellar motor proteins FliM and FliN, causing the flagella to change rotational direction from counterclockwise to clockwise (Szurmant and Ordal, 2004). The phosphatase CheZ promotes the intrinsic dephosphorylation of CheY-P to terminate the signal transduction (Blat and Eisenbach, 1994;Silversmith et al., 2008;Silversmith, 2010).
The spatial organization of chemotaxis proteins is critical for bacterial chemotaxis to adapt to environments. Chemotaxis proteins are localized to cellular poles using multiple strategies, including the nucleoid occlusion, Tol/Pal complex, membrane curvature, and protein-protein interactions (Laloux and Jacobs-Wagner, 2014). Transmembrane E. coli chemoreceptors maintain polar localization through the Tol/Pal complex, strong membrane curvature, or nucleoid exclusion (Santos et al., 2014;Neeli-Venkata et al., 2016;Saaki et al., 2018) The Tol/Pal complex is a conserved component of bacterial cell envelope, which is involved in the maintenance of cell wall integrity (Bernadac et al., 1998). Other chemotaxis proteins including CheA, CheW, CheY, and CheZ locate to cellular poles based on the interaction with other chemotaxis proteins (Sourjik and Berg, 2000). CheA and CheW can bind to chemoreceptor forming polar chemotaxis complexes (Pinas et al., 2016), and the localization of CheZ and CheY depends on the presence of CheA in E. coli (Sourjik and Berg, 2000).
CheZ is encoded in around 40% of bacterial genomes (Wuichet and Zhulin, 2010), and the localization mechanism of CheZ has been well studied in E. coli. E. coli CheZ, termed as CheZ EC , locates to cellular poles with the help of CheA-short (CheAs), a short form of CheA lacking the first 97 amino residues of full length CheA, called CheA-long. CheZ EC interacts with CheA using a small region of amino acids with most interactions coming from the apical hairpin loop consisting of two aromatic residues, Phe-97 and Trp-98 (Cantwell and Manson, 2009). For CheAs, two hydrophobic residues Leu-123 and Leu-126 in the N-terminus of CheA are responsible for CheZ EC interactions (Cantwell et al., 2003;Hao et al., 2009).
Azorhizobium caulinodans ORS571 is a rhizobium belonging to alpha-Proteobacteria uses chemotaxis for plant colonization. It fixes nitrogen with the host Sesbania rostrata by forming stem or root nodules (Dreyfus et al., 1983). A. caulinodans ORS571 has only one chemotaxis pathway including one gene cluster (cheA, cheW, cheY2, cheB, and cheR) and two orphan genes (cheY1 and cheZ) (Jiang et al., 2016). Deletion of one or several genes within the A. caulinodans ORS571 chemotaxis cluster reduces or abolishes the chemotaxis of A. caulinodans ORS571, confirming the role of these genes in chemotaxis . Deletion of cheZ causes A. caulinodans non-chemotactic, while in contrast to other chemotaxis proteins which are important for host plant colonization, CheZ plays negative roles on early colonization (Liu et al., 2019). We previously found that a soluble heme-binding chemotaxis protein in A. caulinodans locates at the cell poles with the help of CheA (Jiang et al., 2016). However, it has been reported that CheZ proteins in alpha-and delta-Proteobacteria lack the sequences responsible for CheAs binding (Wuichet et al., 2007), and the localization of CheZ in alpha-Proteobacteria remains unknown.
In the present study, we reported the localization pattern of CheZ in A. caulinodans ORS571 and mapped regions of CheZ AC that are sufficient for polar localization by constructing various truncated mutants of CheZ AC . Furthermore, a novel motif in CheZ AC , which is conserved among Proteobacteria, was found to be involved in the regulation of monopolar CheZ localization.

Bioinformatics Analysis Shows that CheZ AC Lacks Canonical Sites Involved in CheZ EC Polar Localization
The structure of CheZ EC consists of four regions, including an N-terminal helix (residues 1-34) of unknown functions, a fourhelix bundle formed from a dimer of two hairpin structures (residues 35-168), a linker (residues , and a C-terminal helix (residues 199-214) (Zhao et al., 2002;Silversmith, 2005). Because the amino acid sites involved in the CheZ EC polar localization were well studied, we first aligned the CheZ amino acid sequences from E. coli (CheZ EC ) and A. caulinodans (CheZ AC ) using an EMBOSS Needle program (Madeira et al., 2019). Then, we modeled the structure of CheZ AC using online server SWISS-MODEL (Waterhouse et al., 2018) and Jpred4 (Drozdetskiy et al., 2015).
An alignment of CheZ AC and CheZ EC proteins showed significant similarity (33.9%) between them and both of them have conserved phosphatase active sites (Asp 165 and Gln 169 in CheZ AC ) ( Figure 1A), which is consistent with our previous report . Structure modeling results (Figures 1A,B) suggested CheZ AC also has the N-terminal helix (residues 1-86 in CheZ AC ), four-helix bundle hairpin (residues 87-195 in CheZ AC ), the linker (residues 196-225 in CheZ AC ), and the C-terminal helix (residues 225-236 in CheZ AC ). Interestingly, the N-terminal helix in CheZ AC is substantially longer (∼50 residues) than CheZ EC , while the four-helix bundle hairpin (∼25 residues) in CheZ AC is substantially shorter than CheZ EC (Figure 1). And, the gaps in the alignment are similar in size on either side of the hairpin turn (residues ∼140), which is consistent with a shorter bundle (Figures 1A,B). Remaining of residues from 70 to 133 including the tip of hairpin (residues ∼100) is sufficient for polar localization of CheZ EC (Cantwell and Manson, 2009). The similarity of the hairpin tip between CheZ AC and CheZ EC indicates that the hairpin tip might be also employed by CheZ AC to bind potential localization partner proteins. However, a conserved motif D(D/E)WF (residues 95-98) (Cantwell et al., 2003), which is important for CheZ EC polar localization, was not found in CheZ AC ( Figure 1A).
In E. coli, the polar localization of CheZ is achieved by binding with a short form of CheA (CheA EC ), which begins at Met-98 of full length CheA EC (Cantwell et al., 2003;Hao et al., 2009). There is only one CheA protein encoded in A. caulinodans genome, termed as CheA AC . When we made a pairwise sequence alignment of CheA EC and CheA AC , the absence of cognate CheA EC Met-98 in CheA AC suggests that CheA AC does not have a short form of CheA (Supplementary Figure S1). These results The alignment of CheZ EC and CheZ AC was generated using the default settings of EMBOSS Needle software from EMBL. The N-terminal helix, four-helix bundle, linker, and C-terminus of CheZs were marked with blue, yellow, purple, and red color, respectively. The region containing hairpin turn, which is sufficient for localization of CheZ EC , was marked with black frame. The secondary structure was predicted with Jpred4 program. (B) Structure overview of CheZ EC (1KMI) and CheZ AC . The N-terminal helix, four-helix bundle, linker, and C-terminus of CheZs were recolored as above. CheY (marked with pink) was shown in the structure of CheZ EC and the linker region was not shown in CheZ EC . The structure model of CheZ AC was produced by SWISS-MODEL program, and the first 50 amino acids (involved in its N-terminal helix) were not shown.
suggest that CheZ AC may not locate to cell poles or locate to cell poles with a different mechanism from CheZ EC .

Characteristics of CheZ Localization in A. caulinodans ORS571
To study the subcellular localization of CheZ in A. caulinodans cells, we designed a C-terminal GFP fusion to CheZ AC . To avoid artifacts related overexpression, the expression of the fusion gene was controlled by the native promoter of cheZ . When the CheZ AC fusion was expressed in the cheZ mutant strain, the chemotactic behavior of the cheZ null mutant was partially complemented to wild-type levels that contains a control vector pBBR2GFP, indicating the CheZ-GFP retains function (Figure 2A). Another evidence showing that CheZ-GFP functions is that the presence of CheZ-GFP restores cheZ flagella rotating to wild-type level. Wild type and cheZ complemental strains both rotate their flagella between clockwise and counter-clockwise, while cheZ with or without pBBR2GFP always shows counter-clockwise rotation (Unpublished data).
We next determined the spatial distribution of CheZ-GFP by fluorescence microscopy. The CheZ-GFP fusion showed three unique localization patterns ( Figure 2B). We manually determined and quantified these localization patterns using ImageJ, comparing the brightness of polar foci with that of cell body (see Materials and Methods). About 60% of cells demonstrated diffuse CheZ-GFP localization, 30% of cells CheZ-GFP localized to both cell poles, and 10% of cells showed monopolar localization ( Figure 3B). The percent of cells with polar localized CheZ in A. caulinodans are significantly lower than that in E. coli, in which CheZ EC shows polar localization in 85% of the cells (Blat and Eisenbach, 1996). Except polar and diffuse localized pattern, there were also many lateral clusters of CheZ-GFP ( Figure 2C). When analyzing the distance from each lateral cluster to the pole of cell, the position of each lateral cluster distributed along the cell body with a period corresponding to the 1/2 or 1/4 of the cell length ( Figure 2C).

The Polar Localization of CheZ AC Is Independent of Chemotaxis and Flagellar Proteins
Numerous studies have shown that chemotaxis protein can form a polar cluster to better adapt to environmental conditions. To study whether the polar localization of CheZ AC is dependent on CheA or other chemotaxis proteins, we examined the localization patterns of CheZ AC in different backgrounds lacking different chemotaxis proteins. Consistent with our bioinformatics analysis, deletion of cheA does not alter the cellular localization of CheZ AC (Figure 3). We then tested the localization pattern of CheZ AC in the following chemotaxis mutants, cheY1, cheY2, or cheA-R clusters (including cheA, cheY2, cheW, cheB, and cheR) Liu et al., 2020). Interestingly, CheZ AC maintains polar localization in both the cheY1, cheY2, and cheA-R mutants backgrounds (Figure 3).
Next, we tested if flagellar proteins affect CheZ AC polar localization. FliM and FliN are two flagellar motor components and interact with CheY either directly or indirectly (Delalez et al., 2014). Deletion of either one makes A. caulinodans nonflagellated and non-motile , however, neither of them abolished the polar localization of CheZ AC (Figure 3).  These results indicate that CheZ AC is localized to the cell poles independent of chemotaxis or flagellar proteins.

N-terminal Helical Regions Are Sufficient for CheZ AC Polar Localization
Although there is low conservation between E. coli and A. caulinodans CheZ, the C-terminal sequences including CheY-P binding region and phosphatase active sites are conserved (Blat and Eisenbach, 1996;Zhao et al., 2002;Wuichet et al., 2007;Silversmith, 2010;. The N-terminal helix, whose function remains unknown, and the middle four-helix bundle hairpin of CheZ, which is required for localization in E. coli, are variable (Lertsethtakarn and Ottemann, 2010). To map the region sufficient for polar localization, various portions of the N-terminal helix (residues 1-86), and middle four-helix bundle regions (residues 87-195) of CheZ AC were fused to GFP ( Figure 4A). CheZ 51-236, containing a portion of N-terminal helix of CheZ AC , failed to localize to the cell poles (Figures 4B,C). Surprisingly, CheZ 71-236, containing nearly all regions of the N-terminal helix can locate to cellular poles, though the number of cells with bipolar localized CheZ AC decreased from 30 to 5% compared to full-length CheZ AC (Figures 4B,C). Because the four-helix bundle, especially its hairpin tip, is essential for the polar localized pattern of CheZ EC (Hao et al., 2009), we made a longer truncated mutant containing a portion of the four-helix bundle CheZ 140-236. Intriguingly, CheZ 140-236 can localize to cell poles, however, the cell ratio showed polar localized pattern decreased no more than 5%. When the remaining residues extended from 1-139 to 1-169, including almost all the region of N-terminal helix and four-helix bundle, the CheZ 170-236 mutant can locate to mono-or bi-polar poles in cells above 70% ( Figure 4). These results suggest that the role of N-terminal helix and four-helix bundle on the polar localization of CheZ AC is different from that of CheZ EC . The N-terminal helical region is sufficient for polar localization of CheZ AC , and the four-helix bundle is involved in the regulation of CheZ polar localization.
To further determine the regions of CheZ that are responsible for the polar localization in A. caulinodans, CheZ AC proteins with various deletions at the N-terminal helix and four-helix bundle were fused to GFP ( Figure 4A). All the fusion proteins were introduced into the cheZ mutant and expressed with the native promoter. When part of the CheZ AC N-terminal helix was deleted, including residues from 2 to 31 or from 2 to 50 (termed as CheZ 2-31 and CheZ 2-50), the truncated mutant maintained polar localization, though the polar localized CheZ AC decreased from 40 to 25, and 10%, respectively (Figures 4B,C). Unexpectedly, deletions of N-terminal helical regions from residues 2-70 (CheZ 2-70) or 51-70 (CheZ 51-70) did not abolish the polar localization of CheZ AC (Figures 4B,C). These results suggest that the region from residues 2-70 might not be the sole region sufficient for polar localization of CheZ AC . We further tested the polar localized pattern of mutants lacking part of the four-helix bundle hairpin, CheZ 71-100, CheZ 97-137, and CheZ 138-169, and we found that all of them remained the polar location of CheZ AC . These results suggest that CheZ AC might be anchored to cell poles via multiple motifs. Interestingly, CheZ 138-169 not only maintains polar localization, but also shows 100% monopolar localized pattern (Figure 4).

Mining for a Novel Motif Involved in the Regulation of CheZ AC Localization
We next sought to identify what protein or residues are responsible for the unique monopolar localization pattern of CheZ 138-169. When CheZ 138-169 was introduced into various chemotaxis and flagella mutants, it always maintained nearly 100% monopolar localization (Supplementary Figure S2). These results suggest that the role of residues from 138 to 169 on the localization of CheZ AC is not affected by other chemotaxis or flagellar proteins.
To find potential conserved sites required for the monopolar localized pattern of CheZ, we analyzed twenty-five amino acid sequences of CheZ proteins from alpha-Proteobacteria which are closely related to A. caulinodans ( Figure 5A). Within residues 138-169, two conserved features were found. One is the conserved phosphatase motif (DXXXQ) (Lertsethtakarn and Ottemann, 2010), and the other is an uncharacterized conserved motif (ACNFQ), which is close to the phosphatase motif (Figures 5B,C). Interestingly, deletion of the ACNFQ motif (CheZ 158-164) was sufficient to cause the monopolar localized pattern of CheZ AC (Figure 5D). To further investigate whether the monopolar localization resulted from the deletion of ACNFQ, three point-directed mutants, A160R, C161A, and F163L, were constructed successfully. The localization pattern of CheZ_C161A and CheZ_F163L were similar with that of wild-type CheZ AC , indicating these residues are not required. CheZ_A160R showed different localization that was nearly 100% bipolar ( Figure 5E). These results suggest that the novel motif ACNFQ is involved in the monopolar localization of CheZ AC and conserved site A160 within the motif might contribute more to the function.

The AXXFQ Motif Is Conserved in Proteobacteria
CheZ distributes broadly among alpha-, beta-, gamma-, delta-, and epsilon-Proteobacteria (Wuichet et al., 2007). Although the degree of identity and similarity between these CheZ proteins are low, the catalytic residues in phosphatase active motif are highly FIGURE 5 | Identification of a novel motif and the effect of some conserved sites of CheZ on polar localization. The phylogenetic tree constructed by twenty-five amino acid sequences of CheZ from species that are closely related to A. caulinodans in alpha-Proteobacteria (A). Multiple alignment of CheZ protein sequences (B). The position of "ACNFQ" and "DXXXQ" motifs are marked with red and blue boxes, respectively. The construction of a motif (ACNFQ) deleted CheZ mutant (C). The letter "D" and "Q" indicate the position of conserved phosphatase motif DXXXQ. The localization pattern of the novel-motif-deleted mutant (D). The roles of other conserved sites on CheZ sublocalization (E). The "-" means strain containing cognate CheZ derivatives cannot restore chemotaxis.
conserved among them (Wuichet et al., 2007). To determine the distribution of the novel motif ACNFQ in Proteobacteria, the representative CheZ sequences from each class (alpha-, beta-, gamma-, delta-, and epsilon-Proteobacteria) were selected for alignment. All these CheZ proteins have the novel ACNFQ motif close to phosphatase sites (Figure 6A), although the second and third amino acids in the motif are variable among Proteobacteria, which was renamed as AXXFQ motif. We then used more than 200 representative sequences from each class to align and build a WebLogo of the conserved region consensus sequences (Crooks et al., 2004). The glutamine residue (Q164 in A. caulinodans) near phosphatase sites DXXXQ is the most conserved site among different Proteobacteria (Figure 6B). Alanine and phenylalanine residues (A160 and F163 in A. caulinodans) are the second conserved sites (Figure 6B). In epsilon-Proteobacteria, there is a tyrosine residue instead of phenylalanine ( Figure 6B). Considering both tyrosine and phenylalanine have a benzene ring, this might be a conservative substitution. These results showed that the novel motif AXXF(Y)Q is widely distributed and conserved among Proteobacteria.

Phosphatase Active Sites Affect CheZ AC Location
The proximity between these two motifs (AXXFQ and DXXXQ) led us to assess if the localization pattern could be affected by phosphatase active sites. To investigate the role of phosphatase activity on the subcellular localization of CheZ AC , sitedirected mutants of D165A and Q169A, both critical for the phosphatase activity of CheZ AC (Zhao et al., 2002), were fused to GFP. CheZ_Q169A showed a small increase in diffuse localization, and D165A caused an obvious decrease of polar localization (Figures 6C,D), suggesting the subcellular localization of CheZ AC might be affected by phosphatase active sites. Because the localization of CheZ AC is affected by phosphatase active sites, in turn, the role of different regions

DISCUSSION
The location of CheZ to cell poles can improve the sensitivity to chemotactic stimuli. In E. coli, CheZ_F98S, a CheZ EC variant that abolished localization, showed a decreased chemotactic response to external signals (Cantwell et al., 2003;Vaknin and Berg, 2004). Furthermore, the spatial distribution of chemotactic proteins, including CheZ, provides a region for specialized functions which are similar as the membrane-bound organelles in eukaryotic cells (Maddock and Shapiro, 1993). Three localization patterns of CheZ were found in A. caulinodans, diffuse, bipolar, and monopolar, indicating the localization pattern of CheZ in A. caulinodans is more complex than that in E. coli (Sourjik and Berg, 2000;Cantwell et al., 2003). The ratio of cells that demonstrated polar localization of CheZ AC is much lower than that of CheZ EC , indicating the role of CheZ AC localization may be different between them. Similar to CheZ in E. coli, the localization of CheZ at lateral body of A. caulinodans cells showed a typical periodic distribution, and this phenomenon may be interpreted by a "stochastic nucleation model" (Greenfield et al., 2009;Saaki et al., 2018).
For many bacteria, CheZ locates near cell poles where CheY-P is generated, and the phosphatase activity of CheZ is 5-to 10-folds higher at the position (Vaknin and Berg, 2004). The enhanced phosphatase activity of CheZ ensures that peritrichously located flagellar motors experience a uniform concentration of CheY-P, which is critical for the coordinated regulation of flagellar motility (Cluzel et al., 2000;Lipkow et al., 2005;Ringgaard et al., 2011). We determined CheZ AC can still locate to cellular poles despite lacking a CheA binding site (Wuichet et al., 2007) or other chemotaxis or flagellar proteins. The localization of a remote CheZ ortholog in Helicobacter pylori has been studied (Lertsethtakarn and Ottemann, 2010;Lertsethtakarn et al., 2015). And, in contrast to E. coli CheZ, H. pylori CheZ localization is  pBBRCheZQ169A-GFP pBBR-1-MCS-2 with cheZ with a C161A substitution fused with egfp; Km R This study a Amp R , ampicillin resistance; Gm R , gentamicin resistance; Km R , kanamycin resistance; Nal R , nalidixic acid; Tc R , tetracycline.
independent of CheA or other typical chemotaxis proteins, but dependent on ChePep, a novel chemotaxis protein distributed among epsilon-Proteobacteria (Howitt et al., 2011;Lertsethtakarn et al., 2015). Because the genes encoding homologs of ChePep were not found in A. caulinodans genome, we suspect that there might be other partner proteins that contribute CheZ to localization clusters. The four-helix bundle of CheZ EC , especially the tip of the hairpin, is responsible for polar localization in E. coli. In this work, we found that the N-terminal helix is sufficient for the polar localization of CheZ AC . The sequence conservation of N-terminus of CheZ between E. coli and A. caulinodans is low, and interestingly, deletion of the N-terminal helix, CheZ AC still remained the polar location, indicating more than one region is sufficient for its polar localization. However, the reason why CheZ AC has two independent regions that are sufficient for polar localization is unknown.
CheZ 138-169 results in monopolar localization. In this study, a conserved motif AXXF(Y)Q which is close to the phosphatase active motif DXXXQ was found to be responsible for the unique monopolar localized pattern of CheZ AC . Although AXXF(Y)Q is conserved among Proteobacteria, its role in localization and chemotaxis had not been studied. We speculate the high level of polar localization of CheZ in E. coli and H. pylori under common conditions may mask the observation of the role of AXXF(Y)Q on localization changes (Sourjik and Berg, 2000;Cantwell et al., 2003;Lertsethtakarn et al., 2015). The biological significance of the polar localization is that each daughter cell can inherit a CheZ after cell division (Jones and Armitage, 2015;Mauriello et al., 2018). For example, the location of chemotactic proteins transfers from monopolar to bipolar clusters in Vibrio cholerae before cell division (Ringgaard et al., 2011). The unipolar localization of CheZ 138-169 or CheZ 158-164 indicates that one daughter cell cannot inherit CheZ AC , and the residues 138-169 might be involved in the dissociation between CheZ AC and its binding partners for localization. In E. coli, the polar localization of CheZ EC can be improved by the interaction with CheA at the chemotaxis signaling complex (Wang and Matsumura, 1996; Vaknin and Berg, 2004). These results further suggest that there may be other proteins that recruit CheZ to the clusters and/or affect the catalysis activity of CheZ, as seen in H. pylori (Lertsethtakarn and Ottemann, 2010;Howitt et al., 2011;Lertsethtakarn et al., 2015).
In this study, we mapped the critical regions sufficient for CheZ AC localization and assessed the role of regions in the N-terminal helix and four-helix bundle of CheZ AC on both localization changes and chemotaxis. Furthermore, a novel and widespread motif affecting monopolar localization of CheZ AC was identified, which might be also important for the modulation of CheZ polar localization in other Proterobacteria.

Bacterial Strains and Growth Conditions
Azorhizobium caulinodans ORS571, its derivatives, and E. coli strains are listed in Table 1. A. caulinodans strains were grown in TY media at 37 • C. E. coli strains were cultured in Luria broth (Luria et al., 1960) at 37 • C.

Generation of CheZ Variants
To construct CheZ variants, a fragment including cheZ gene and its native promoter was amplified by polymerase chain reaction (PCR). Then an egfp gene encoding enhanced GFP was amplified from pEGFP-N1. The two fragments were linked by overlap extension, as previously described (Ho et al., 1989). Next, the resulting construct CheZ-GFP fusion was cloned into a broadhost-range plasmid pBBR1MCS-2 (Kovach et al., 1995), and the pBBR1-CheZ-GFP was used as a temple to construct other CheZ variants. Both the truncated mutants such as CheZ 2-31-GFP and site-directed mutants such as CheZC161A-GFP were constructed by overlap extension PCR as described by Ho et al. (1989). All the CheZ variants were introduced into the cheZ mutant strain by triparental conjugation using a helper plasmid pRK2013 (Ditta et al., 1980). Primer pairs used in the construction are listed in Table 2.

Microscopy and Data Analysis
After growing in TY solid medium for overnight with shaking, cells with GFP fusion were used for observation. Agarose pads were used to immobile bacteria as described by Meier and Scharf (2009). Images were taken by an Olympus DP73 camera on an Olympus BX53 system fluorescence microscope with a 100 × objective and controlled by a cellSens Dimension 1.7 imaging software (Olympus Inc.,). A space between 505 to 550 nm filter was used to detect fluorescence signals. The images analyzing spatial distribution of CheZ were processed by ImageJ 1 as described by Thiem et al. (2007). Distribution of CheZ AC was manually enumerated and classified into three types (diffuse, bipolar, and monopolar localization). CheZ AC cells with monopolar or bipolar localization showed obvious bright spots at one end or both ends of cell. When the brightness in the whole cell distribute evenly, the localization of CheZ AC in these cells is counted as diffuse. ImageJ was used to quantify the brightness at different regions of cells. Experiments were repeated at least three times, and for each sample at least 100 cells were counted.

Soft Agar Plate Assay
The chemotactic behavior of cheZ mutant derivative strains was assessed using soft agar plate assay, as previously described (Miller et al., 2009). Overnight bacterial cultures were washed with chemotaxis buffer at least two times and then adjusted to OD 600 of 0.6. Five microliter of cells was dropped in the center of 0.3% soft agar plate. After culturing for 3 days at 37 • C, the chemotactic rings on soft agar plate were counted. Ten mM sodium lactate was used as sole carbon source. Experiments were repeated at least three times.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
XL and ZX conceived and designed the experiments, analyzed the data, prepared the figures and tables, and wrote the manuscript. XL, YL, and XD carried out the experiments. KJ helped with the improvement and revision of the manuscript. All authors approved the submission for publication.

FUNDING
This work was financed by the NSFC-Shandong Joint Fund Key Projects (U1806206) and the National Natural Science Foundation of China (31870020).

ACKNOWLEDGMENTS
We thank Robert B. Bourret, Karen M. Ottemann, Shuai Hu, and John S. Parkinson for helpful and insightful comments on the manuscript.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2020.585140/full#supplementary-material Supplementary Figure 1 | The alignment of CheA EC and CheA AC was generated using the default settings of EMBOSS Needle software from EMBL. The beginning Met of the short form of CheA EC was marked with black frame.
Supplementary Figure 2 | The localization pattern of CheZ 138-169 in cheA, cheA-R, cheY1, cheY2, fliM, and fliN mutant. The mutein CheZ 138-169 was fused to GFP, and CheZ 138-169GFP including its own promoter were inserted into pBBR2 and then were transformed into each mutant strain.
Supplementary Figure 3 | The representative images of chemotactic rings formed by cheZ mutant containing different CheZ derivatives fused to GFP. Ten mM sodium lactate was used as sole carbon source. The cognate results of each strain also shown in Figures 4A and 5A.