\u3ci\u3eAzospirillum brasilense\u3c/i\u3e Signaling via PilZ-containing Chemotaxis Receptors and Chemotaxis Protein Paralogs

Chemotaxis is the biased movement of bacteria in response to environmental stimuli. Chemotaxis is initiated with dedicated receptors that sense environmental signals, and these signals are transduced through a phosphorylation cascade that can influence flagellar rotation and swimming direction. Most knowledge of chemotaxis signal transduction comes from studies of Escherichia coli, which possesses a simple system consisting of five chemotaxis receptors and a single chemotactic signaling pathway. Unfortunately, E. coli is not representative of the diversity of chemotaxis signal transduction systems revealed by the analysis of completely sequenced bacterial genomes. Most sequenced bacterial genomes to date suggest most motile bacteria able of chemotaxis rely on at least two distinct chemotaxis signaling systems and a large number (\u3e20) chemotaxis receptors. This dissertation focuses on the plant growth promoting alphaproteobacterium Azospirillum brasilense as a representative model for chemotaxis. The genome of A. brasilense encodes four chemotaxis operons and fifty-one chemotaxis receptors. Here we show that chemotaxis paralogs encoded in two of these chemotaxis systems, Che1 and Che4, interact to produce two functional chemotaxis signaling arrays segregated by chemotaxis receptor lengths. One of these arrays contains a subclass of receptors that possess an additional Cterminal domain and comprise the PilZ-containing chemotaxis receptors. In bacteria, the Cterminal PilZ domain was shown to bind the secondary messenger cyclic diguanylate monophosphate which affects enzyme activity, gene expression and protein-protein interactions. Here we develop a novel real time tool (slide-in-chamber) and assay (root-in-pool) for monitoring the role of A. brasilense chemotaxis and PilZ-containing chemotaxis receptors (Tlp1 and Aer) in bacterial behavior and colonization of wheat rhizospheres. We found that chemotaxis is essential for A. brasilense to sense and subsequently colonize specific zones of wheat roots. A. brasilense exhibits an attractant response to the root hair and elongation zones of wheat and is repelled from the wheat root tip, and this response is dependent upon functional PilZ receptors Tlp1 and Aer. Furthermore, cyclic diguanylate monophosphate binding to Tlp1 and Aer is essential for bacterial response to roots. Taken together, these data reveal a new role for cyclic diguanylate monophosphate in modulating sensitivity to complex gradients

Modeling aerotaxis band formation in Azospirillum brasilense

Background Bacterial chemotaxis, the ability of motile bacteria to navigate gradients of chemicals, plays key roles in the establishment of various plant-microbe associations, including those that benefit plant growth and crop productivity. The motile soil bacterium Azospirillum brasilense colonizes the rhizosphere and promotes the growth of diverse plants across a range of environments. Aerotaxis, or the ability to navigate oxygen gradients, is a widespread behavior in bacteria. It is one of the strongest behavioral responses in A. brasilense and it is essential for successful colonization of the root surface. Oxygen is one of the limiting nutrients in the rhizosphere where density and activity of organisms are greatest. The aerotaxis response of A. brasilense is also characterized by high precision with motile cells able to detect narrow regions in a gradient where the oxygen concentration is low enough to support their microaerobic lifestyle and metabolism. Results Here, we present a mathematical model for aerotaxis band formation that captures most critical features of aerotaxis in A. brasilense. Remarkably, this model recapitulates experimental observations of the formation of a stable aerotactic band within 2 minutes of exposure to the air gradient that were not captured in previous modeling efforts. Using experimentally determined parameters, the mathematical model reproduced an aerotactic band at a distance from the meniscus and with a width that matched the experimental observation. Conclusion Including experimentally determined parameter values allowed us to validate a mathematical model for aerotactic band formation in spatial gradients that recapitulates the spatiotemporal stability of the band and its position in the gradient as well as its overall width. This validated model also allowed us to capture the range of oxygen concentrations the bacteria prefer during aerotaxis, and to estimate the effect of parameter values (e.g. oxygen consumption rate), both of which are difficult to obtain in experiments

A PilZ-Containing Chemotaxis Receptor Mediates Oxygen and Wheat Root Sensing in Azospirillum brasilense

Chemotactic bacteria sense environmental changes via dedicated receptors that bind to extra- or intracellular cues and relay this signal to ultimately alter direction of movement toward beneficial cues and away from harmful environments. In complex environments, such as the rhizosphere, bacteria must be able to sense and integrate diverse cues. Azospirillum brasilense is a microaerophilic motile bacterium that promotes growth of cereals and grains. Root surface colonization is a prerequisite for the beneficial effects on plant growth but how motile A. brasilense navigates the rhizosphere is poorly studied. Previously only 2 out of 51 A. brasilense chemotaxis receptors have been characterized, AerC and Tlp1, and only Tlp1 was found to be essential for wheat root colonization. Here we describe another chemotaxis receptor, named Aer, that is homologous to the Escherichia coli Aer receptor, likely possesses an FAD cofactor and is involved in aerotaxis (taxis in an air gradient). We also found that the A. brasilense Aer contributes to sensing chemical gradients originating from wheat roots. In addition to A. brasilense Aer having a putative N-terminal FAD-binding PAS domain, it possesses a C-terminal PilZ domain that contains all the conserved residues for binding c-di-GMP. Mutants lacking the PilZ domain of Aer are altered in aerotaxis and are completely null in wheat root colonization and they also fail to sense gradients originating from wheat roots. The PilZ domain of Aer is also vital in integrating Aer signaling with signaling from other chemotaxis receptors to sense gradients from wheat root surfaces and colonizing wheat root surfaces

Clostridium amazonense sp. nov. an obliqately anaerobic bacterium isolated from a remote Amazonian community in Peru

A strictly anaerobic Gram-stain positive, spore-forming, rod-shaped bacterium designated NE08VT, was isolated from a fecal sample of an individual residing in a remote Amazonian community in Peru. Phylogenetic analysis based on the 16S rRNA gene sequence showed the organism belonged to the genus Clostridium and is most closely related to Clostridium vulturis (97.4% sequence similarity) and was further characterized using biochemical and chemotaxonomic methods. The major cellular fatty acids were anteiso C13:0 and C16:0 with a genomic DNA G + C content of 31.6 mol%. Fermentation products during growth with PYG were acetate and butyrate. Based on phylogenetic, phenotypic and chemotaxonomic information, strain NE08V was identified as representing a novel species of the genus Clostridium, for which the name Clostridium amazonense sp. nov. is proposed. The type strain is NE08VT (DSM 23598T = CCUG 59712T)