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How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli. We first identify ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum, characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we trace the evolution of the system through γ-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost.

Nearly all motile bacterial cells use a highly sensitive and adaptable sensory system to detect changes in nutrient concentrations in the environment and guide their movements toward attractants and away from repellents. The best-studied bacterial chemoreceptor arrays are membrane-bound. Many motile bacteria contain one or more additional, sometimes purely cytoplasmic, chemoreceptor systems. Vibrio cholerae contains three chemotaxis clusters (I, II, and III). Here, using electron cryotomography, we explore V. cholerae's cytoplasmic chemoreceptor array and establish that it is formed by proteins from cluster I. We further identify a chemoreceptor with an unusual domain architecture, DosM, which is essential for formation of the cytoplasmic arrays. DosM contains two signaling domains and spans the two-layered cytoplasmic arrays. Finally, we present evidence suggesting that this type of receptor is important for the structural stability of the cytoplasmic array.chemotaxis | chemoreceptor arrays | Vibrio cholerae | electron cryotomography | microscopy M ost motile bacteria move toward favorable environments through a process called chemotaxis. The molecular basis of this behavior is best understood in Escherichia coli, where transmembrane methyl-accepting chemotaxis proteins (MCPs, or chemoreceptors) form large arrays at the cell pole. The chemoreceptors bind attractants or repellents in the periplasm (1-3) and relay signals to histidine kinases (CheA) in the cytoplasm (4). When activated, CheA first autophosphorylates and then transfers the phosphoryl group to the response regulators CheY and CheB. Phosphorylated CheY binds to the flagellar motor, changing the direction of flagellar rotation. This allows the cells to switch from swimming forward smoothly (so-called "runs") to tumbling randomly. Changes in the duration and frequency of run and tumble phases drive a biased random walk that moves the cells toward favorable environments (5). The other response regulator, CheB, is a methylesterase, which, in conjunction with the constitutively active methyltransferase CheR, tunes the sensitivity of the system by changing the methylation state of the chemoreceptors (6-8).Although there is only one chemotaxis system in E. coli, most chemotactic bacterial and archaeal species have multiple systems (9). For instance, Rhodobacter sphaeroides has three chemotaxis systems encoded in three distinct clusters of genes, one of which (CheOp2) encodes two receptors without predicted transmembrane domains, TlpT and TlpC (10). Analysis of fluorescently tagged TlpT and TlpC revealed that they localize in foci around midcell. The foci split before cell division and are segregated to the midcell position within the future daughter cells. The chromosome-associated ParA-like ATPase PpfA controls the localization and segregation of the foci through an interaction with the N terminus of TlpT in a ParB-like manner (11).Using electron cryotomography (ECT), we discovered that these cytoplasmic foci in R. sphaerodes are ordered arrays of chemore...

Suppressive subtractive hybridisation was applied to the analysis of late stage arbuscular mycorrhizal development in pea. 96 cDNA clones were amplified and 81, which carried fragments more than 200 nt in size, were sequence analysed. Among 67 unique fragments, 10 showed no homology and 10 were similar to sequences with unknown function. RNA accumulation of the corresponding 67 genes was analysed by hybridisation of macro-arrays. The cDNAs used as probes were derived from roots of wild type and late mutant pea genotypes, inoculated or not with the AM fungus Glomus mosseae. After calibration, a more than 2.5-fold mycorrhiza-induced RNA accumulation was detected in two independent experiments in the wild type for 25 genes, 22 of which seemed to be induced specifically during late stage AM development. Differential expression for 7 genes was confirmed by RT-PCR using RNA from mycorrhiza and from controls of a different pea cultivar. In order to confirm arbuscule-related expression, the Medicago truncatula EST data base was screened for homologous sequences with putative mycorrhiza-related expression and among a number of sequences with significant similarities, a family of trypsin inhibitor genes could be identified. Mycorrhiza-induced RNA accumulation was verified for five members by real-time PCR and arbuscule-related activation of the promoter could be shown in transgenic roots for one of the genes, MtTi 1.

The ability of most bacterial flagellar motors to reverse the direction of rotation is crucial for efficient chemotaxis. In Escherichia coli, motor reversals are mediated by binding of phosphorylated chemotaxis protein CheY to components of the flagellar rotor, FliM and FliN, which induces a conformational switch of the flagellar C-ring. Here, we show that for Shewanella putrefaciens, Vibrio parahaemolyticus and likely a number of other species an additional transmembrane protein, ZomB, is critically required for motor reversals as mutants lacking ZomB exclusively exhibit straightforward swimming also upon full phosphorylation or overproduction of CheY. ZomB is recruited to the cell poles by and is destabilized in the absence of the polar landmark protein HubP. ZomB also co-localizes to and may thus interact with the flagellar motor. The ΔzomB phenotype was suppressed by mutations in the very C-terminal region of FliM. We propose that the flagellar motors of Shewanella, Vibrio and numerous other species harboring orthologs to ZomB are locked in counterclockwise rotation and may require interaction with ZomB to enable the conformational switch required for motor reversals. Regulation of ZomB activity or abundance may provide these species with an additional means to modulate chemotaxis efficiency.