Bacteria navigate their way often as individual cells through their chemical and biological environment in aqueous medium or across solid surfaces. They swim when starved or in response to physical and chemical stimuli. Flagella-driven chemotaxis in bacteria has emerged as a paradigm for both signal transduction and cellular decision-making. By altering motility, bacteria swim toward nutrient-rich environments, movement modulated by their chemotaxis systems with the addition of pili for surface movement. The numbers and types of chemoreceptors reflect the bacterial niche and lifestyle, with those adapted to complex environments having diverse metabolic capabilities, encoding far more chemoreceptors in their genomes. The Alpha-proteobacteria typify the latter case, with soil bacteria such as rhizobia, endosymbionts of legume plants, where motility and chemotaxis are essential for competitive symbiosis initiation, among other processes. This review describes the current knowledge of motility and chemotaxis in six model soil bacteria: Sinorhizobium meliloti, Agrobacterium fabacearum, Rhizobium leguminosarum, Azorhizobium caulinodans, Azospirillum brasilense, and Bradyrhizobium diazoefficiens. Although motility and chemotaxis systems have a conserved core, rhizobia possess several modifications that optimize their movements in soil and root surface environments. The soil provides a unique challenge for microbial mobility, since water pathways through particles are not always continuous, especially in drier conditions. The effectiveness of symbiont inoculants in a field context relies on their mobility and dispersal through the soil, often assisted by water percolation or macroorganism movement or networks. Thus, this review summarizes the factors that make it essential to consider and test rhizobial motility and chemotaxis for any potential inoculant.

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Rhizobial Chemotaxis and Motility Systems at Work in the Soil.

The Escherichia coli chemoreceptor array is a supramolecular assembly that enables cells to respond to extracellular cues dynamically and with great precision and sensitivity. In the array, transmembrane receptors organized as trimers of dimers are connected at their cytoplasmic tips by hexameric rings of alternating subunits of the kinase CheA and the scaffolding protein CheW (CheA-CheW rings). Interactions of CheW molecules with the members of receptor trimers not directly bound to CheA-CheW rings may lead to the formation of hexameric CheW rings in the chemoreceptor array. Here, we detected such CheW rings with a cellular cysteine-directed cross-linking assay and explored the requirements for their formation and their participation in array assembly. We found that CheW ring formation varied with cellular CheW abundance, depended on the presence of receptors capable of a trimer-of-dimers arrangement, and did not require CheA. Cross-linking studies of a CheA~CheW fusion protein incapable of forming homomeric CheW oligomers demonstrated that CheW rings were not essential for the assembly of CheA-containing arrays. Förster resonance energy transfer (FRET)–based kinase assays of arrays containing variable amounts of CheW rings revealed that CheW rings enhanced the cooperativity and the sensitivity of the responses to attractants. We propose that six-membered CheW rings provide the additional interconnectivity required for optimal signaling and gradient tracking performance by chemosensory arrays. Description Optimal signaling through E. coli chemoreceptor arrays requires homohexameric rings of the scaffolding protein CheW. Two rings to bind them Bacteria sense attractants and repellants through extensive ordered arrays of transmembrane chemoreceptors and associated intracellular proteins. The core signaling units consist of two trimers of receptor dimers held together by a homodimer of the kinase CheA and two monomers of the scaffolding protein CheW. One CheA molecule and one CheW molecule from each unit also contribute to heterohexameric CheA-CheW rings that tie core units together. Piñas et al. found that receptor arrays in E. coli also contained homohexameric CheW rings that mediated interactions between core units, filling in the spaces not occupied by CheA-CheW rings. These CheW rings augmented the cooperativity between core signaling units and enhanced the overall sensitivity of the receptor array to chemoattractants (see the Focus by Thompson).

Hexameric rings of the scaffolding protein CheW enhance response sensitivity and cooperativity in Escherichia coli chemoreceptor arrays

Plantaricin is a broad-spectrum bacteriocin produced by Lactiplantibacillus plantarum with significant food industry application potential. It was found that the plantaricin production of L. plantarum RX-8 was enhanced when co-culturing with Bacillus subtilis BS-15. This study, therefore, set out to explore how B. subtilis BS-15 induces biosynthesis of plantaricin. The effect of co-culturing with B. subtilis BS-15 on cell growth, plantaricin production, quorum-sensing (QS) signal molecule PlnA/autoinducer-2 (AI-2) secretion, as well as plantaricin biosynthesis gene cluster and AI-2 synthesis-associated gene expression, was investigated in bacteriocin-producer L. plantarum RX-8. When L. plantarum RX-8 and B. subtilis BS-15 were co-inoculated in Man–Rogosa–Sharp (MRS) for 20 h at an inoculum ratio of 1:1 (106:106 CFU/ml), the greatest plantaricin output (2,048 AU/ml) was obtained, rising by 32-fold compared with the monoculture of L. plantarum RX-8. Additionally, co-culture increased PlnA-inducing activity and AI-2 activity by 8- and 1.14-fold, respectively, over monoculture. RT-qPCR findings generated every 4 h (4–32 h) demonstrated that B. subtilis BS-15 remarkably improved the transcription of plnABCD and plnEF, and increased pfs and luxS transcription, even when using 200 mM D-ribose, a kind of AI-2 inhibitor. Based on the above findings, co-culturing with B. subtilis BS-15 as an environmental stimulus could activate the plantaricin induction via the PlnA-mediated intraspecies QS system and the AI-2-mediated interspecies QS system. Moreover, the inducing effect of PlnA and AI-2 in co-culture was independent. Differential proteomics analysis of B. subtilis BS-15 in co-culture indicated that bacteriocin-inducing regulatory mechanism may be related to flagellar assembly, peptidoglycan biosynthesis, anaerobic respiration, glycine cleavage system, or thiamin pyrophosphate biosynthesis.

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Bacillus subtilis BS-15 Effectively Improves Plantaricin Production and the Regulatory Biosynthesis in Lactiplantibacillus plantarum RX-8

Magnetospirillum gryphiswaldense employs iron-rich nanoparticles for magnetic navigation within environmental redox gradients. This behavior termed magneto-aerotaxis was previously shown to rely on the sensory pathway CheOp1, but the precise localization of CheOp1-related chemoreceptor arrays during the cell cycle and its possible interconnection with three other chemotaxis pathways have remained unstudied. Here, we analyzed the localization of chemoreceptor-associated adaptor protein CheW1 and histidine kinase CheA1 by superresolution microscopy in a spatiotemporal manner. CheW1 localized in dynamic clusters that undergo occasional segregation and fusion events at lateral sites of both cell poles. Newly formed smaller clusters originating at midcell before completion of cytokinesis were found to grow in size during the cell cycle. Bipolar CheA1 localization and formation of aerotactic swim halos were affected depending on the fluorescent protein tag, indicating that CheA1 localization is important for aerotaxis. Furthermore, polar CheW1 localization was independent of cheOp2 to cheOp4 but lost in the absence of cheOp1 or cheA1 Results were corroborated by the detection of a direct protein interaction between CheA1 and CheW1 and by the observation that cheOp2- and cheOp3-encoded CheW paralogs localized in spatially distinct smaller clusters at the cell boundary. Although the findings of a minor aerotaxis-related CheOp4 phenotype and weak protein interactions between CheOp1 and CheOp4 by two-hybrid analysis implied that CheW1 and CheW4 might be part of the same chemoreceptor array, CheW4 was localized in spatially distinct polar-lateral arrays independent of CheOp1, suggesting that CheOp1 and CheOp4 are also not connected at the molecular level.IMPORTANCE Magnetotactic bacteria (MTB) use the geomagnetic field for navigation in aquatic redox gradients. However, the highly complex signal transduction networks in these environmental microbes are poorly understood. Here, we analyzed the localization of selected chemotaxis proteins to spatially and temporally resolve chemotaxis array localization in Magnetospirillum gryphiswaldense Our findings suggest that bipolar localization of chemotaxis arrays related to the key signaling pathway CheOp1 is important for aerotaxis and that CheOp1 signaling units assemble independent of the three other chemotaxis pathways present in M. gryphiswaldense Overall, our results provide deeper insights into the complex organization of signaling pathways in MTB and add to the general understanding of environmental bacteria possessing multiple chemotaxis pathways.

Spatiotemporal Organization of Chemotaxis Pathways in Magnetospirillum gryphiswaldense.

Chemotaxis and motility are important traits that support bacterial survival in various ecological niches and in pathogenic and symbiotic host interaction Chemotactic stimuli are sensed by chemoreceptors or methyl-accepting chemotaxis proteins (MCPs), which direct the swimming behavior of the bacterial cell In this study, we present evidence that the cellular abundance of chemoreceptors in the plant symbiont Sinorhizobium meliloti can be altered by the addition of several to as few as one amino acid residues and by including common epitope tags such as 3×FLAG and 6×His at their C termini To further dissect this phenomenon and its underlying molecular mechanism, we focused on a detailed analysis of the amino acid sensor McpU Controlled proteolysis is important for the maintenance of an appropriate stoichiometry of chemoreceptors and between chemoreceptors and chemotactic signaling proteins, which is essential for an optimal chemotactic response We hypothesized that enhanced stability is due to interference with protease binding, thus affecting proteolytic efficacy Location of the protease recognition site was defined through McpU stability measurements in a series of deletion and amino acid substitution mutants Deletions in the putative protease recognition site had similar effects on McpU abundance, as did extensions at the C terminus Our results provide evidence that the programmed proteolysis of chemotaxis proteins in S meliloti is cell cycle regulated This posttranslational control, together with regulatory pathways on the transcriptional level, limits the chemotaxis machinery to the early exponential growth phase Our study identified parallels to cell cycle-dependent processes during asymmetric cell division in Caulobacter crescentus IMPORTANCE The symbiotic bacterium Sinorhizobium meliloti contributes greatly to growth of the agriculturally valuable host plant alfalfa by fixing atmospheric nitrogen Chemotaxis of S meliloti cells toward alfalfa roots mediates this symbiosis The present study establishes programmed proteolysis as a factor in the maintenance of the S meliloti chemotaxis system Knowledge about cell cycle-dependent, targeted, and selective proteolysis in S meliloti is important to understand the molecular mechanisms of maintaining a suitable chemotaxis response While the role of regulated protein turnover in the cell cycle progression of Caulobacter crescentus is well understood, these pathways are just beginning to be characterized in S meliloti In addition, our study should alert about the cautionary use of epitope tags for protein quantification

Programmed Proteolysis of Chemotaxis Proteins in Sinorhizobium meliloti: Features in the C-Terminal Region Control McpU Degradation.

Convergent evolution in the supercoiling of prokaryotic flagellar filaments

Chemotaxis systems enable microbes to sense their immediate environment, moving toward beneficial stimuli and away from those that are harmful. In an effort to better understand the chemotaxis system of Sinorhizobium meliloti, a symbiont of the legume alfalfa, the cellular stoichiometries of all ten chemotaxis proteins in S. meliloti were determined. A combination of quantitative immunoblot and mass spectrometry revealed that the protein stoichiometries in S. meliloti varied greatly from those in Escherichia coli and Bacillus subtilis To compare protein ratios to other systems, values were normalized to the central kinase CheA. All S. meliloti chemotaxis proteins exhibited increased ratios to various degrees. The 10-fold higher molar ratio of adaptor proteins CheW1 and CheW2 to CheA might result in the formation of rings in the chemotaxis array that consist of only CheW instead of CheA and CheW in a 1:1 ratio. We hypothesize that the higher ratio of CheA to the main response regulator CheY2 is a consequence of the speed-variable motor in S. meliloti, instead of a switch-type motor. Similarly, proteins involved in signal termination are far more abundant in S. meliloti, which utilizes a phosphate sink mechanism based on CheA retrophosphorylation to inactivate the motor response regulator versus CheZ-catalyzed dephosphorylation as in E. coli and B. subtilis Finally, the abundance of CheB and CheR, which regulate chemoreceptor methylation, was increased compared to CheA, indicative of variations in the adaptation system of S. meliloti Collectively, these results mark significant differences in the composition of bacterial chemotaxis systems.IMPORTANCE The symbiotic soil bacterium Sinorhizobium meliloti contributes greatly to host-plant growth by fixing atmospheric nitrogen. The provision of nitrogen as ammonium by S. meliloti leads to increased biomass production of its legume host alfalfa and diminishes the use of environmentally harmful chemical fertilizers. To better understand the role of chemotaxis in host-microbe interaction, a comprehensive catalogue of the bacterial chemotaxis system is vital, including its composition, function, and regulation. The stoichiometry of chemotaxis proteins in S. meliloti has very few similarities to the systems in Escherichia coli and Bacillus subtilis In addition, total amounts of proteins are significantly lower. S. meliloti exhibits a chemotaxis system distinct from known models by incorporating new proteins as exemplified by the phosphate sink mechanism.

Cellular stoichiometry of chemotaxis proteins in Sinorhizobium meliloti


 The final phase of the reionization process can be probed by rest–frame UV absorption spectra of quasars at z ≳ 6, shedding light on the properties of the diffuse intergalactic medium within the first Gyr of the Universe. The ESO Large Programme ‘XQR-30: the ultimate XSHOOTER legacy survey of quasars at z ≃ 5.8–6.6’ dedicated ∼250 hours of observations at the VLT to create a homogeneous and high-quality sample of spectra of 30 luminous quasars at z ∼ 6, covering the rest wavelength range from the Lyman limit to beyond the Mg ii emission. Twelve quasar spectra of similar quality from the XSHOOTER archive were added to form the enlarged XQR-30 sample, corresponding to a total of ∼350 hours of on-source exposure time. The median effective resolving power of the 42 spectra is R ≃ 11400 and 9800 in the VIS and NIR arm, respectively. The signal-to-noise ratio per 10 km s−1 pixel ranges from ∼11 to 114 at λ ≃ 1285 Å rest frame, with a median value of ∼29. We describe the observations, data reduction and analysis of the spectra, together with some first results based on the E-XQR-30 sample. New photometry in the H and K bands are provided for the XQR-30 quasars, together with composite spectra whose characteristics reflect the large absolute magnitudes of the sample. The composite and the reduced spectra are released to the community through a public repository, and will enable a range of studies addressing outstanding questions regarding the first Gyr of the Universe.

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Amanda L. Sebastian

Papers

Convergent evolution in the supercoiling of prokaryotic flagellar filaments

Cellular stoichiometry of chemotaxis proteins in Sinorhizobium meliloti

XQR-30: The ultimate XSHOOTER quasar sample at the reionization epoch