Pseudomonas aeruginosa is an ubiquitous pathogen able to infect humans, animals and plants. Chemotaxis was found to be associated with the virulence of this and other pathogens. Although established as a model for chemotaxis research, the majority of the 26 P. aeruginosa chemoreceptors remain functionally un-annotated. We report here the identification of PA5072 (named McpK) as chemoreceptor for -ketoglutarate (KG). High-throughput thermal shift assays and isothermal titration calorimetry studies (ITC) of the recombinant McpK ligand binding domain (LBD) show that it recognizes exclusively -ketoglutarate. The ITC analysis indicated that the ligand bound with positive cooperativity (Kd1=301 µM, Kd2=81 µM). McpK is predicted to possess a helical bimodular (HBM) type of LBD and this and other studies suggest that this domain type may be associated with the recognition of organic acids. Analytical ultracentrifugation (AUC) studies revealed that McpK-LBD is present in a monomer-dimer equilibrium. Alpha-KG binding stabilized the dimer and dimer self-dissociation constants of 55 µM and 5.9 µM were derived for ligand-free and KG-bound forms of McpK-LBD, respectively. Ligand-induced LBD dimer stabilization has been observed for other HBM domain containing receptors and may correspond to a general mechanism of this protein family. Quantitative capillary chemotaxis assays demonstrated that P. aeruginosa showed chemotaxis to a broad range of KG concentrations with maximal responses at 500 µM. Deletion of the mcpK gene reduced chemotaxis over the entire concentration range to close to background levels and wild type like chemotaxis was recovered following complementation. Real-time PCR studies indicated that the presence of KG does not modulate mcpK expression. Since KG is present in plant root exudates it was investigated whether the deletion of mcpK altered maize root colonization. However, no significant changes with respect to the wild type strain were observed. The existence of a chemoreceptor specific for KG may be due to its central metabolic role as well as to its function as signaling molecule. This work expands the range of known chemoreceptor types and underlines the important physiological role of chemotaxis towards tricarboxylic acid (TCA) cycle intermediates.

Plant root colonization by rhizobacteria can protect plants against pathogens and promote plant growth, and chemotaxis to root exudates was shown to be an essential prerequisite for efficient root colonization. Since many chemoattractants control the transcript levels of their cognate chemoreceptor genes, we have studied here the transcript levels of the 27 Pseudomonas putida KT2440 chemoreceptor genes in the presence of different maize root exudate (MRE) concentrations. Transcript levels were increased for 10 chemoreceptor genes at low MRE concentrations, whereas almost all receptor genes showed lower transcript levels at high MRE concentrations. The exposure of KT2440 to different MRE concentrations did not alter c-di-GMP levels, indicating that changes in chemoreceptor transcripts are not mediated by this second messenger. Data suggest that rhizosphere colonization unfolds in a temporal fashion. Whereas at a distance to the root, exudates enhance chemoreceptor gene transcript levels promoting in turn chemotaxis, this process is reversed in root vicinity, where the necessity of chemotaxis toward the root may be less important. Insight into KT2440 signaling processes were obtained by analyzing mutants defective in the three cheA paralogous genes. Whereas a mutant in cheA1 showed reduced c-di-GMP levels and impaired biofilm formation, a cheA2 mutant was entirely deficient in MRE chemotaxis, indicating the existence of homologs of the P. aeruginosa wsp and che (chemotaxis) pathways. Signaling through both pathways was important for efficient maize root colonization. Future studies will show whether the MRE concentration dependent effect on chemoreceptor gene transcript levels is a feature shared by other species.

Bloodstream form trypanosomes avoid the host immune response by switching the expression of their surface proteins between Variant Surface Glycoproteins (VSG), only one of which is expressed at any given time. Monoallelic transcription of the telomeric VSG Expression Site (ES) by RNA polymerase I (RNA pol I) localizes to a unique nuclear body named the ESB. Most work has focused on silencing mechanisms of inactive VSG-ESs, but the mechanisms involved in transcriptional activation of a single VSG-ES remain largely unknown. Here, we identify a highly SUMOylated focus (HSF) in the nucleus of the bloodstream form that partially colocalizes with the ESB and the active VSG-ES locus. SUMOylation of chromatin-associated proteins was enriched along the active VSG-ES transcriptional unit, in contrast to silent VSG-ES or rDNA, suggesting that it is a distinct feature of VSG-ES monoallelic expression. In addition, sequences upstream of the active VSG-ES promoter were highly enriched in SUMOylated proteins. We identified TbSIZ1/PIAS1 as the SUMO E3 ligase responsible for SUMOylation in the active VSG-ES chromatin. Reduction of SUMO-conjugated proteins by TbSIZ1 knockdown decreased the recruitment of RNA pol I to the VSG-ES and the VSG-ES-derived transcripts. Furthermore, cells depleted of SUMO conjugated proteins by TbUBC9 and TbSUMO knockdown confirmed the positive function of SUMO for VSG-ES expression. In addition, the largest subunit of RNA pol I TbRPA1 was SUMOylated in a TbSIZ-dependent manner. Our results show a positive mechanism associated with active VSG-ES expression via post-translational modification, and indicate that chromatin SUMOylation plays an important role in the regulation of VSG-ES. Thus, protein SUMOylation is linked to active gene expression in this protozoan parasite that diverged early in evolution. African trypanosomes have evolved one of the most complex strategies of immune evasion by routinely switching the expression of surface proteins called Variant Surface Glycoproteins (VSG), only one of which is expressed at any given time. Previous work has suggested that the recruitment of a single VSG telomeric locus to a discrete nuclear body (ESB) underlies the mechanism responsible for VSG monoallelic expression. Our findings establish unexpected roles for SUMOylation as a specific post-translational modification that marks the ESB and the VSG-ES chromatin. We describe a highly SUMOylated focus (HSF) as a novel nuclear structure that partially colocalizes with the VSG-ES locus and the nuclear body ESB. Furthermore, chromatin SUMOylation is a distinct feature of the active VSG-ES locus, in contrast to other loci investigated. SUMOylation of chromatin-associated proteins is required for efficient recruitment of the polymerase to the VSG-ES promoter and for VSG-ES expression. Altogether, these data suggest the presence of a large number of SUMOylated proteins associated with monoallelic expression as Protein Group SUMOylation. In contrast to the wealth of literature focused on VSG regulation by silencing, our results indicate a positive mechanism via SUMOylation to regulate VSG expression in the infectious form of this protozoan parasite.

Protein-coding genes in trypanosomes occur in polycistronic transcription units (PTUs). How RNA polymerase II (Pol II) initiates transcription of PTUs has not been resolved; the current model favors chromatin modifications inducing transcription rather than sequence-specific promoters. Here, we uncover core promoters by functional characterization of Pol II peaks identified by chromatin immunoprecipitation sequencing (ChIP-seq). Two distinct promoters are located between divergent PTUs, each driving unidirectional transcription. Detailed analysis identifies a 75-bp promoter that is necessary and sufficient to drive full reporter expression and contains functional motifs. Analysis of further promoters suggests transcription initiation is regulated and promoters are either focused or dispersed. In contrast to the previous model of unregulated and promoter-independent transcription initiation, we find that sequence-specific promoters determine the initiation of Pol II transcription of protein-coding genes PTUs. These findings in Trypanosoma brucei suggest that in addition of chromatin modifications, promoter motifs-based regulation of gene expression is deeply conserved among eukaryotes.

Malaria, caused by Plasmodium parasites, is still one of the biggest global health challenges. P. falciparum is the deadliest species to humans. In this review, we discuss how this parasite develops and adapts to the complex and heterogenous environments of its two hosts thanks to varied chromatin-associated and epigenetic mechanisms. First, one small family of transcription factors, the ApiAP2 proteins, functions as master regulators of spatio-temporal patterns of gene expression through the parasite life cycle. In addition, chromatin plasticity determines variable parasite cell phenotypes that link to parasite growth, virulence and transmission, enabling parasite adaptation within host conditions. In recent years, epitranscriptomics is emerging as a new regulatory layer of gene expression. We present evidence of the variety of tRNA and mRNA modifications that are being characterized in Plasmodium spp., and the dynamic changes in their abundance during parasite development and cell fate. We end up outlining that new biological systems, like the mosquito model, to decipher the unknowns about epigenetic mechanisms in vivo; and novel methodologies, to study the function of RNA modifications; are needed to discover the Achilles heel of the parasite. With this new knowledge, future strategies manipulating the epigenetics and epitranscriptomic machinery of the parasite have the potential of providing new weapons against malaria.