Three PilZ Domain Proteins, PlpA, PixA, and PixB, Have Distinct Functions in Regulation of Motility and Development in Myxococcus xanthus.

In bacteria, the nucleotide-based second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pilus-dependent motility and the starvation-induced developmental program that results in formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP effectors. We confirm that the stand-alone PilZ domain protein PlpA is important for regulation of motility independently of the Frz chemosensory system and that Pkn1, which is composed of a Ser/Thr kinase domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pilus-dependent and gliding motility in a Frz-dependent manner as well as development. The acetyltransferase domain is required and sufficient for function during growth, while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility independently of the Frz system, likely by setting up the polarity of the two motility systems. Our results support a model whereby PlpA, PixA, and PixB act in independent pathways and have distinct functions in regulation of motility. IMPORTANCE c-di-GMP signaling controls bacterial motility in many bacterial species by binding to downstream effector proteins. Here, we identify two PilZ domain-containing proteins in Myxococcus xanthus that bind c-di-GMP. We show that PixB, which contains two PilZ domains and an acetyltransferase domain, acts in a manner that depends on the Frz chemosensory system to regulate motility via the acetyltransferase domain, while the intact protein and c-di-GMP binding are essential for PixB to support development. In contrast, PixA acts in a Frz-independent manner to regulate motility. Taking our results together with previous observations, we conclude that PilZ domain proteins and c-di-GMP act in multiple independent pathways to regulate motility and development in M. xanthus.

Structural insights into the mechanism of c-di-GMP-bound YcgR regulating flagellar motility in Escherichia coli.

The motile-sessile transition is critical for bacterial survival and growth. Cyclic-di-GMP (c-di-GMP) plays a central role in controlling this transition and regulating biofilm formation via various effectors. As an effector of c-di-GMP in Escherichia coli and related species, the PilZ domain-containing protein YcgR responds to elevated c-di-GMP concentrations and acts on the flagellar motor to suppress bacterial motility in a brakelike fashion, which promotes bacterial surface attachment. To date, several target proteins within the motor, MotA, FliG, and FliM, along with different regulatory mechanisms have been reported. However, how YcgR acts on these components remains unclear. Here, we report that activated YcgR stably binds to MotA at the MotA-FliG interface and thereby regulates bacterial swimming. Biochemical and structural analyses revealed that c-di-GMP rearranges the PilZ domain configuration, resulting in the formation of a MotA-binding patch consisting of an RXXXR motif and the C-tail helix α3. Moreover, we noted that a conserved region in the YcgR-N domain, which is independent of MotA interaction, is necessary for motility regulation. On the basis of these findings, we infer that the YcgR-N domain is required for activity on other motor proteins. We propose that activated YcgR appends to MotA via its PilZ domain and thereby interrupts the MotA-FliG interaction and simultaneously interacts with other motor proteins via its YcgR-N domain to inhibit flagellar motility. Our findings suggest that the mode of interaction between YcgR and motor proteins may be shared by other PilZ family proteins.

Identification of the Regulatory Components Mediated by the Cyclic di-GMP Receptor Filp and Its Interactor PilZX3 and Functioning in Virulence of Xanthomonas oryzae pv. oryzae.

The degenerate GGDEF/EAL domain protein Filp was previously shown to function as a cyclic di-GMP (c-di-GMP) signal receptor through its specific interaction with an atypical PilZ domain protein PilZX3 (formerly PXO_02715) and that this interaction is involved in regulating virulence in Xanthomonas oryzae pv. oryzae. As a step toward understanding the regulatory role of Filp/PilZX3-mediated c-di-GMP signaling in the virulence of X. oryzae pv. oryzae, differentially expressed proteins (DEPs) downstream of Filp/PilZX3 were identified by isobaric tagging for relative and absolute quantitation (iTRAQ). A total of 2,346 proteins were identified, of which 157 displayed significant differential expression in different strains. Western blot and quantitative reverse transcription-PCR analyses showed that the expression of HrrP (histidine kinase-response regulator hybrid protein), PhrP (PhoPQ-regulated protein), ProP (prophage Lp2 protein 6) were increased in the ∆filp, ∆pilZX3, and ∆filp∆pilZX3 mutant strains, while expression of CheW1 (chemotaxis protein CheW1), EdpX2 (the second EAL domain protein identified in X. oryzae pv. oryzae), HGdpX2 (the second HD-GYP domain protein identified in X. oryzae pv. oryzae) was decreased in all mutant strains compared with that in the wild type, which was consistent with the iTRAQ data. Deletion of the hrrP and proP genes resulted in significant increases in virulence, whereas deletion of the cheW1, hGdpX2, or tdrX2 genes resulted in decreased virulence. Enzyme assays indicated that EdpX2 and HGdpX2 were active phosphodiesterases (PDEs). This study provides a proteomic description of putative regulatory pathway of Filp and PilZX3 and characterized novel factors that contributed to the virulence of X. oryzae pv. oryzae regulated by c-di-GMP signaling.

Emerging paradigms for PilZ domain-mediated C-di-GMP signaling.

PilZ domain-containing proteins constitute a large family of bacterial signaling proteins. As a widely distributed protein domain for the binding of the second messenger c-di-GMP, the canonical PilZ domain contains a set of motifs that define the binding site for c-di-GMP and an allosteric switch for propagating local conformational changes. Here, we summarize some new insights gathered from recent studies on the commonly occurring single-domain PilZ proteins, YcgR-like proteins and PilZ domain-containing cellulose synthases. The studies collectively illuminate how PilZ domains function as cis- or trans-regulatory domains that enable c-di-GMP to control the activity of its cellular targets. Overall, the review highlights the diverse protein structure, biological function and regulatory mechanism of PilZ domain-containing proteins, as well as the challenge of deciphering the function and mechanism of orphan PilZ proteins.

The FilZ Protein Contains a Single PilZ Domain and Facilitates the Swarming Motility of Pseudoalteromonas sp. SM9913.

Swarming regulation is complicated in flagellated bacteria, especially those possessing dual flagellar systems. It remains unclear whether and how the movement of the constitutive polar flagellum is regulated during swarming motility of these bacteria. Here, we report the downregulation of polar flagellar motility by the c-di-GMP effector FilZ in the marine sedimentary bacterium Pseudoalteromonas sp. SM9913. Strain SM9913 possesses two flagellar systems, and filZ is located in the lateral flagellar gene cluster. The function of FilZ is negatively controlled by intracellular c-di-GMP. Swarming in strain SM9913 consists of three periods. Deletion and overexpression of filZ revealed that, during the period when strain SM9913 expands quickly, FilZ facilitates swarming. In vitro pull-down and bacterial two-hybrid assays suggested that, in the absence of c-di-GMP, FilZ interacts with the CheW homolog A2230, which may be involved in the chemotactic signal transduction pathway to the polar flagellar motor protein FliMp, to interfere with polar flagellar motility. When bound to c-di-GMP, FilZ loses its ability to interact with A2230. Bioinformatic investigation indicated that filZ-like genes are present in many bacteria with dual flagellar systems. Our findings demonstrate a novel mode of regulation of bacterial swarming motility.

Cyclic di-GMP modulates sessile-motile phenotypes and virulence in Dickeya oryzae via two PilZ domain receptors.

Dickeya oryzae is a bacterial pathogen causing the severe rice stem rot disease in China and other rice-growing countries. We showed recently that the universal bacterial second messenger c-di-GMP plays an important role in modulation of bacterial motility and pathogenicity, but the mechanism of regulation remains unknown. In this study, bioinformatics analysis of the D. oryzae EC1 genome led to the identification of two proteins, YcgR and BcsA, both of which contain a conserved c-di-GMP receptor domain, known as the PilZ-domain. By deleting all the genes encoding c-di-GMP-degrading enzymes in D. oryzae EC1, the resultant mutant 7ΔPDE with high c-di-GMP levels became nonmotile, formed hyperbiofilm, and lost the ability to colonize and invade rice seeds. These phenotypes were partially reversed by deletion of ycgR in the mutant 7ΔPDE, whereas deletion of bcsA only reversed the hyperbiofilm phenotype of mutant 7ΔPDE. Significantly, double deletion of ycgR and bcsA in mutant 7ΔPDE rescued its motility, biofilm formation, and virulence to levels of wild-type EC1. In vitro biochemical experiments and in vivo phenotypic assays further validated that YcgR and BcsA proteins are the receptors for c-di-GMP, which together play a critical role in regulating the c-di-GMP-associated functionality. The findings from this study fill a gap in our understanding of how c-di-GMP modulates bacterial motility and biofilm formation, and provide useful clues for further elucidation of sophisticated virulence regulatory mechanisms in this important plant pathogen.

Corrigendum: The Stand-Alone PilZ-Domain Protein MotL Specifically Regulates the Activity of the Secondary Lateral Flagellar System in Shewanella putrefaciens.

[This corrects the article DOI: 10.3389/fmicb.2021.668892.].

The Stand-Alone PilZ-Domain Protein MotL Specifically Regulates the Activity of the Secondary Lateral Flagellar System in Shewanella putrefaciens.

A number of bacterial species control the function of the flagellar motor in response to the levels of the secondary messenger c-di-GMP, which is often mediated by c-di-GMP-binding proteins that act as molecular brakes or clutches to slow the motor rotation. The gammaproteobacterium Shewanella putrefaciens possesses two distinct flagellar systems, the primary single polar flagellum and a secondary system with one to five lateral flagellar filaments. Here, we identified a protein, MotL, which specifically regulates the activity of the lateral, but not the polar, flagellar motors in response to the c-di-GMP levels. MotL only consists of a single PilZ domain binding c-di-GMP, which is crucial for its function. Deletion and overproduction analyses revealed that MotL slows down the lateral flagella at elevated levels of c-di-GMP, and may speed up the lateral flagellar-mediated movement at low c-di-GMP concentrations. In vitro interaction studies hint at an interaction of MotL with the C-ring of the lateral flagellar motors. This study shows a differential c-di-GMP-dependent regulation of the two flagellar systems in a single species, and implicates that PilZ domain-only proteins can also act as molecular regulators to control the flagella-mediated motility in bacteria.

Identification of Cbp1, a c-di-GMP Binding Chemoreceptor in Azorhizobium caulinodans ORS571 Involved in Chemotaxis and Nodulation of the Host Plant.

Cbp1, a chemoreceptor containing a PilZ domain was identified in Azorhizobium caulinodans ORS571, a nitrogen-fixing free-living soil bacterium that induces nodule formation in both the roots and stems of the host legume Sesbania rostrata. Chemoreceptors are responsible for sensing signals in the chemotaxis pathway, which guides motile bacteria to beneficial niches and plays an important role in the establishment of rhizobia-legume symbiosis. PilZ domain proteins are known to bind the second messenger c-di-GMP, an important regulator of motility, biofilm formation and virulence. Cbp1 was shown to bind c-di-GMP through the conserved RxxxR motif of its PilZ domain. A mutant strain carrying a cbp1 deletion was impaired in chemotaxis, a feature that could be restored by genetic complementation. Compared with the wild type strain, the Δcbp1 mutant displayed enhanced aggregation and biofilm formation. The Δcbp1 mutant induced functional nodules when inoculated individually. However, the Δcbp1 mutant was less competitive than the wild type in competitive root colonization and nodulation. These data are in agreement with the hypothesis that the c-di-GMP binding chemoreceptor Cbp1 in A. caulinodans is involved in chemotaxis and nodulation.

Arginine within a specific motif near the N-terminal of FimY is critical for the maximal production of type 1 fimbriae in Salmonella enterica serovar Typhimurium.

An important Salmonella serovar for both human and animals Salmonella Typhimurium possesses 13 gene clusters that have the potential to produce fimbrial structure, among which the type 1 fimbriae with the binding specificity to mannose residue is the most commonly found type. Six structural genes and five regulatory genes comprise the fim gene cluster that is responsible for the production of type 1 fimbriae in S. Typhimurium. The fimY gene encodes a positive regulator for type 1 fimbrial expression since a deletion in fimY abolished the production of fimbriae. The N-terminal portion of FimY contains amino acid residues that exhibit some similarity as those found in the proteins possessing the PilZ domain, which is engaged in cyclic di-GMP binding. A fimY allele that had a change from arginine to alanine at position 7 (R7A) or 7 and 11 (R7/11A) generated by site-directed mutagenesis in a 6 RRERH11 R motif near N-terminal, when cloned in pACYC184 and transformed into a fimY-deleted strain, decreased the expression of fimA and fimZ. The number of type 1 fimbriae in these two transformants was also less than those of the other transformants that contained different fimY alleles in pACYC184 when observed in electron microscopy. However, changing from arginine to alanine at position 11 (R11A) remained the same as the wild-type fimY allele. It is likely that the arginine at the 7th position of FimY is critical for its maximal activating activity upon fimZ. Another motif 83 DI85 SLWIEK91 G motif did not affect the function of FimY. Although FimY has the two aforementioned motifs, which contain some amino acids that are present within those of the PilZ domain proteins, secondary structure prediction analysis did not reveal that FimY has a conformation commonly observed in PilZ-like proteins. Therefore, FimY and PilZ domain proteins are not homologs. Further investigation for a detailed analysis of FimY is thus warranted.

Cellulose production, activated by cyclic di-GMP through BcsA and BcsZ, is a virulence factor and an essential determinant of the three-dimensional architectures of biofilms formed by Erwinia amylovora Ea1189.

Bacterial biofilms are multicellular aggregates encased in an extracellular matrix mainly composed of exopolysaccharides (EPSs), protein and nucleic acids, which determines the architecture of the biofilm. Erwinia amylovora Ea1189 forms a biofilm inside the xylem of its host, which results in vessel plugging and water transport impairment. The production of the EPSs amylovoran and levan is critical for the formation of a mature biofilm. In addition, cyclic dimeric GMP (c-di-GMP) has been reported to positively regulate amylovoran biosynthesis and biofilm formation in E. amylovora Ea1189. In this study, we demonstrate that cellulose is synthesized by E. amylovora Ea1189 and is a major modulator of the three-dimensional characteristics of biofilms formed by this bacterium, and also contributes to virulence during systemic host invasion. In addition, we demonstrate that the activation of cellulose biosynthesis in E. amylovora is a c-di-GMP-dependent process, through allosteric binding to the cellulose catalytic subunit BcsA. We also report that the endoglucanase BcsZ is a key player in c-di-GMP activation of cellulose biosynthesis. Our results provide evidence of the complex composition of the extracellular matrix produced by E. amylovora and the implications of cellulose biosynthesis in shaping the architecture of the biofilm and in the expression of one of the main virulence phenotypes of this pathogen.