Algae represent a unique opportunity to develop a sustainable production platform for many valuable products. A key hindrance to exploiting algae's potential as a bio-product manufacturing platform is our limited understanding of nuclear genetic regulatory systems. To begin to address this deficiency we identified prospective transcription factor-promoter pairs that have the potential for use in a controllable synthetic transcription system. We constructed a library of 92 recombinant nuclear transcription factors (TFs) from the model alga Chlamydomonas reinhardtii. Using a yeast one-hybrid assay, we identified TFs with the ability to activate transcription and chose TF64 for further analysis. We generated two independent C. reinhardtii strains that constitutively expressed the gene encoding TF64 and used RNA-sequencing on these strains to identify transcripts that were altered by expression of the TF's gene. Many mRNAs were regulated by TF64, and we confirmed activation of transcription of nine genes from the LHCBM family by RT-qPCR. Overall, we were able to identify numerous potential TF-cognate promoter binding partners in C. reinhardtii, thus laying the groundwork to develop a controllable synthetic nuclear transcription system for microalgae, an essential tool for realizing their full biotechnological potential.

A new class of protein phosphatases has emerged in the study of bacterial/archaeal chemotaxis, the CheC-type phosphatases. These proteins are distinct and unrelated to the well-known CheY-P phosphatase CheZ, though they have convergently evolved to dephosphorylate the same target. The family contains a common consensus sequence D/S-X(3)-E-X(2)-N-X(22)-P that defines the phosphatase active site, of which there are often two per protein. Three distinct subgroups make up the family: CheC, FliY and CheX. Further, the CheC subgroup can be divided into three classes. Bacillus subtilis CheC typifies the first class and might function as a regulator of CheD. Class II CheCs likely function as phosphatases in systems other than chemotaxis. Class III CheCs are found in the archaeal class Halobacteria and might function as class I CheCs. FliY is the main phosphatase in the B. subtilis chemotaxis system. CheX is quite divergent from the rest of the family, forms a dimer and some may function outside chemotaxis. A model for the evolution of the family is discussed.

Much study of two-component systems deals with the excitation of the histidine kinase, activation of the response regulator, and the ultimate target of the signal. Removal of the message is of great importance to these signaling systems. Many methods have evolved in two-component systems to this end. These include autodephosphorylation of the response regulator, hydrolysis of the phosphoryl group by the kinase, or a dedicated phosphatase protein. It has long been known that CheZ is the phosphatase in the chemotaxis system of Escherichia coli and related bacteria. Most bacteria and archaea, however, do not have a cheZ gene, but instead rely on the CheC, CheX, and FliY family of CheY-P phosphatases. Here, we describe assays to test these chemotactic phosphatases, applicable to many other response regulator phosphatases.

ABSTRACT Bacterial chemotaxis involves the regulation of motility by a modified two-component signal transduction system. In Escherichia coli, CheZ is the phosphatase of the response regulator CheY but many other bacteria, including Bacillus subtilis, use members of the CheC-FliY-CheX family for this purpose. While Bacillus subtilis has only CheC and FliY, many systems also have CheX. The effect of this three-phosphatase system on chemotaxis has not been studied previously. CheX was shown to be a stronger CheY-P phosphatase than either CheC or FliY. In Bacillus subtilis, a cheC mutant strain was nearly complemented by heterologous cheX expression. CheX was shown to overcome the ΔcheC adaptational defect but also generally lowered the counterclockwise flagellar rotational bias. The effect on rotational bias suggests that CheX reduced the overall levels of CheY-P in the cell and did not truly replicate the adaptational effects of CheC. Thus, CheX is not functionally redundant to CheC and, as outlined in the discussion, may be more analogous to CheZ.

The bacterial chemotaxis system is one of the most extensively studied signal transduction systems in biology. The response regulator CheY controls flagellar rotation and is phosphorylated by the CheA histidine kinase to its active form. CheC is a CheY-P phosphatase, and this activity is enhanced in a CheC-CheD heterodimer. CheC is also critical for chemotactic adaptation, the return to the prestimulus system state despite persistent attractant concentrations. Here, CheC point mutants were examined in Bacillus subtilis for in vivo complementation and in vitro activity. The mutants were identified separating the three known abilities of CheC: CheD binding, CheY-P binding, and CheY-P phosphatase activity. Remarkably, the phosphatase ability was not as critical to the in vivo function of CheC as the ability to bind both CheY-P and CheD. Additionally, it was confirmed that CheY-P increases the affinity of CheC for CheD, the later of which is known to be necessary for receptor activation of CheA. These data suggest a model of CheC as a CheY-P-induced regulator of CheD. Here, CheY-P would cause CheC to sequester CheD from the chemoreceptors, inducing adaptation of the chemotaxis system. This model represents the first plausible means for feedback from the output of the system, CheY-P, to the receptors.

Signal transduction underlying bacterial chemotaxis involves excitatory phosphorylation and feedback control through deamidation and methylation of sensory receptors. The structure of a complex between the signal-terminating phosphatase, CheC, and the receptor-modifying deamidase, CheD, reveals how CheC mimics receptor substrates to inhibit CheD and how CheD stimulates CheC phosphatase activity. CheD resembles other cysteine deamidases from bacterial pathogens that inactivate host Rho-GTPases. CheD not only deamidates receptor glutamine residues contained within a conserved structural motif but also hydrolyzes glutamyl-methyl-esters at select regulatory positions. Substituting Gln into the receptor motif of CheC turns the inhibitor into a CheD substrate. Phospho-CheY, the intracellular signal and CheC target, stabilizes the CheC:CheD complex and reduces availability of CheD. A point mutation that dissociates CheC from CheD impairs chemotaxis in vivo. Thus, CheC incorporates an element of an upstream receptor to influence both its own effect on receptor output and that of its binding partner, CheD.

Rapid restoration of prestimulus levels of the chemotactic response regulator, CheY- P, is important for preparing bacteria and archaea to respond sensitively to new stimuli. In an extension of previous work ( Szurmant, H., Bunn, M. W., Cannistraro, V. J., and Ordal, G. W. ( 2003) J. Biol. Chem. 278, 48611 - 48616), we describe a new family of CheY- P phosphatases, the CYX family, that is widespread among the bacteria and archaea. These proteins provide another pathway, in addition to the ones involving CheZ of the gamma- and beta- proteobacteria ( e. g. Escherichia coli) or the alternative CheY that serves as a " phosphate sink" among the alpha- proteobacteria ( e. g. Sinorhizobium meliloti), for dephosphorylating CheY- P. In particular, we identify CheC, known previously to be involved in adaptation to stimuli in Bacillus subtilis, as a CheY- P phosphatase. Using an in vitro assay used previously to demonstrate that the switch protein FliY is a CheY- P phosphatase, we have shown that increasing amounts of CheC accelerate the hydrolysis of CheY- P. In vivo, a double mutant lacking cheC and the region of fliY that encodes the CheY- P binding domain is almost completely smooth swimming, implying that these cells contain very high levels of CheY- P. CheC appears to be primarily involved in restoring normal CheY- P levels following the addition of attractant, whereas FliY seems to act on CheY- P constitutively. The activity of CheC is relatively low compared to that of FliY, but we have shown that the chemotaxis protein CheD enhances the activity of CheC 5- fold. We suggest a model for how FliY, CheC, and CheD work together to regulate CheY- P levels in the bacterium.