Distinct Domains of CheA Confer Unique Functions in Chemotaxis and Cell Length in Azospirillum brasilense Sp7

Abstract

Chemotaxis is the movement of cells in response to gradients of diverse chemical cues. Motile bacteria utilize a conserved chemotaxis signal transduction system to bias their motility and navigate through a gradient. A central regulator of chemotaxis is the histidine kinase CheA. This cytoplasmic protein interacts with membrane-bound receptors, which assemble into large polar arrays, to propagate the signal. In the alphaproteobacterium Azospirillum brasilense, Che1 controls transient increases in swimming speed during chemotaxis, but it also biases the cell length at division. However, the exact underlying molecular mechanisms for Che1-dependent control of multiple cellular behaviors are not known. Here, we identify specific domains of the CheA1 histidine kinase implicated in modulating each of these functions. We show that CheA1 is produced in two isoforms: a membraneanchored isoform produced as a fusion with a conserved seven-transmembrane domain of unknown function (TMX) at the N terminus and a soluble isoform similar to prototypical CheA. Site-directed and deletion mutagenesis combined with behavioral assays confirm the role of CheA1 in chemotaxis and implicate the TMX domain in mediating changes in cell length. Fluorescence microscopy further reveals that the membrane-anchored isoform is distributed around the cell surface while the soluble isoform localizes at the cell poles. Together, the data provide a mechanism for the role of Che1 in controlling multiple unrelated cellular behaviors via acquisition of a new domain in CheA1 and production of distinct functional isoforms.IMPORTANCE Chemotaxis provides a significant competitive advantage to bacteria in the environment, and this function has been transferred laterally multiple times, with evidence of functional divergence in different genomic contexts. The molecular principles that underlie functional diversification of chemotaxis in various genomic contexts are unknown. Here, we provide a molecular mechanism by which a single CheA protein controls two unrelated functions: chemotaxis and cell length. Acquisition of this multifunctionality is seemingly a recent evolutionary event. The findings illustrate a mechanism by which chemotaxis function may be co-opted to regulate additional cellular functions.

Distinct Chemotaxis Protein Paralogs Assemble into Chemoreceptor Signaling Arrays To Coordinate Signaling Output

“…We analyzed the dependence of chemoreceptor cluster localization on Che1 and Che4 proteins to localize using yellow fluorescent protein (YFP) fusions of Che1 and Che4 chemotaxis protein paralogs (CheA, CheW, and CheY) and imaged each in wild-type and Δ che1 , Δ che4 , and Δ che1 Δ che4 mutant backgrounds. The functionality of all tagged proteins was verified through chemotaxis soft agar assays before imaging (29). A. brasilense Sp7 contains two isoforms of CheA1: full-length CheA1 (which is membrane anchored, localizes at the cell surface, and is dispensable for chemotaxis) and CheA1ΔTMX (which lacks the transmembrane domains and is vital to chemotaxis) (29).…”

“…The functionality of all tagged proteins was verified through chemotaxis soft agar assays before imaging (29). A. brasilense Sp7 contains two isoforms of CheA1: full-length CheA1 (which is membrane anchored, localizes at the cell surface, and is dispensable for chemotaxis) and CheA1ΔTMX (which lacks the transmembrane domains and is vital to chemotaxis) (29). We used CheA1ΔTMX-YFP to assess chemotaxis-dependent localization.…”

“…To test whether Che1 and Che4 proteins physically interact, which would be required to form stable chemoreceptor arrays, we used a bacterial adenylate cyclase two-hybrid (BACTH) assay. Given that YFP fusions to the C terminus of CheA1ΔTMX, CheA4, CheW1, CheW4, CheY1, and CheY4 are functional (26, 29), we tested all interactions with the catalytic domains fused at the C terminus of the proteins expressed from both BACTH vectors. We first determined that proteins encoded by the same operon are able to interact.…”

Multiple CheY Proteins Control Surface-Associated Lifestyles of Azospirillum brasilense

“…A lower swimming speed for this strain would increase its residence time in proximity to surfaces, which could promote enhanced adhesion for this mutant relative to the other strains. Several lines of experimental evidence indicate that chemotaxis signaling regulates non-chemotaxis functions in A. brasilense, though the mechanism(s) are not known (Bible et al, 2008;Gullett et al, 2017;Ganusova et al, 2021). It is thus also possible that the cheY mutants have different cell surface properties which would modulate their ability to adhere to surfaces.…”

Draft Genome Sequence of Bacillus subtilis 2C-9B, a Strain with Biocontrol Potential against Chili Pepper Root Pathogens and Tolerance to Pb and Zn

“…Also, a cadmium-translocating P-type ATPase, a copper-translocating P-type ATPase, a copper-binding protein, and the copper transporter CopZ were found. Furthermore, CheA and CheC are present in the genome of this bacterium; these proteins are usually involved in chemotaxis and adaptation ( 14 , 15 ). An endoglucanase and an N -acetylglucosamine-6-phosphate deacetylase, which are enzymes involved mainly in the consumption of carbon sources, were also found.…”