The number and location of flagella, bacterial organelles of locomotion, are species specific and appear in regular patterns that represent one of the earliest taxonomic criteria in microbiology. However, the mechanisms that reproducibly establish these patterns during each round of cell division are poorly understood. FlhG (previously YlxH) is a major determinant for a variety of flagellation patterns. Here, we show that FlhG is a structural homolog of the ATPase MinD, which serves in cell-division site determination. Like MinD, FlhG forms homodimers that are dependent on ATP and lipids. It interacts with a complex of the flagellar C-ring proteins FliM and FliY (also FliN) in the Gram-positive, peritrichousflagellated Bacillus subtilis and the Gram-negative, polar-flagellated Shewanella putrefaciens. FlhG interacts with FliM/FliY in a nucleotide-independent manner and activates FliM/FliY to assemble with the C-ring protein FliG in vitro. FlhG-driven assembly of the FliM/FliY/FliG complex is strongly enhanced by ATP and lipids. The protein shows a highly dynamic subcellular distribution between cytoplasm and flagellar basal bodies, suggesting that FlhG effects flagellar location and number during assembly of the C-ring. We describe the molecular evolution of a MinD-like ATPase into a flagellation pattern effector and suggest that the underappreciated structural diversity of the C-ring proteins might contribute to the formation of different flagellation patterns.flagellum | FlhG | C-ring | Bacillus | Shewanella

SummarySpatiotemporal regulation of cell polarity plays a role in many fundamental processes in bacteria and often relies on 'landmark' proteins which recruit the corresponding clients to their designated position. Here, we explored the localization of two multi-protein complexes, the polar flagellar motor and the chemotaxis array, in Shewanella putrefaciens CN-32. We demonstrate that polar positioning of the flagellar system, but not of the chemotaxis system, depends on the GTPase FlhF. In contrast, the chemotaxis array is recruited by a transmembrane protein which we identified as the functional ortholog of Vibrio cholerae HubP. Mediated by its periplasmic N-terminal LysM domain, SpHubP exhibits an FlhF-independent localization pattern during cell cycle similar to its Vibrio counterpart and also has a role in proper chromosome segregation. In addition, while not affecting flagellar positioning, SpHubP is crucial for normal flagellar function and is involved in type IV pilimediated twitching motility. We hypothesize that a group of HubP/FimV homologs, characterized by a rather conserved N-terminal periplasmic section required for polar targeting and a highly variable acidic cytoplasmic part, primarily mediating recruitment of client proteins, serves as polar markers in various bacterial species with respect to different cellular functions.

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The coordination of biological activities into daily cycles provides an important advantage for the fitness of diverse organisms. Most eukaryotes possess an internal clock ticking with a periodicity of about one day to anticipate sunrise and sunset. The 24-hour period of the free-running rhythm is highly robust against many changes in the natural environment. Among prokaryotes, only Cyanobacteria are known to harbor such a circadian clock. Its core oscillator consists of just three proteins, KaiA, KaiB, and KaiC that produce 24-hour oscillations of KaiC phosphorylation, even in vitro. This unique three-protein oscillator is well documented for the freshwater cyanobacterium Synechococcus elongatus PCC 7942. Several physiological studies demonstrate a circadian clock also for other Cyanobacteria including marine species. Genes for the core clock components are present in nearly all marine cyanobacterial species, though there are large differences in the specific composition of these genes. In the first section of this review we summarize data on the model circadian clock from S. elongatus PCC 7942 and compare it to the reduced clock system of the marine cyanobacterium Prochlorococcus marinus MED4. In the second part we discuss the diversity of timing mechanisms in other marine Cyanobacteria with regard to the presence or absence of different components of the clock.

In contrast to Synechococcus elongatus PCC 7942, few data exist on the timing mechanism of the widely used cyanobacterium Synechocystis sp. PCC 6803. The standard kaiAB1C1 operon present in this organism was shown to encode a functional KaiC protein that interacted with KaiA, similar to the S. elongatus PCC 7942 clock. Inactivation of this operon in Synechocystis sp. PCC 6803 resulted in a mutant with a strong growth defect when grown under light–dark cycles, which was even more pronounced when glucose was added to the growth medium. In addition, mutants showed a bleaching phenotype. No effects were detected in mutant cells grown under constant light. Microarray experiments performed with cells grown for 1 day under a light–dark cycle revealed many differentially regulated genes with known functions in the ΔkaiABC mutant in comparison with the WT. We identified the genes encoding the cyanobacterial phytochrome Cph1 and the light-repressed protein LrtA as well as several hypothetical ORFs with a complete inverse behaviour in the light cycle. These transcripts showed a stronger accumulation in the light but a weaker accumulation in the dark in ΔkaiABC cells in comparison with the WT. In general, we found a considerable overlap with microarray data obtained for hik31 and sigE mutants. These genes are known to be important regulators of cell metabolism in the dark. Strikingly, deletion of the ΔkaiABC operon led to a much stronger phenotype under light–dark cycles in Synechocystis sp. PCC 6803 than in Synechococcus sp. PCC 7942.

Bacteria commonly exhibit a high degree of cellular organization and polarity which affect many vital processes such as replication, cell division, and motility. In Shewanella and other bacteria, HubP is a polar marker protein which is involved in proper chromosome segregation, placement of the chemotaxis system, and various aspects of pilus- and flagellum-mediated motility. Here, we show that HubP also recruits a transmembrane multidomain protein, PdeB, to the flagellated cell pole. PdeB is an active phosphodiesterase and degrades the second messenger c-di-GMP. In Shewanella putrefaciens, PdeB affects both the polar and the lateral flagellar systems at the level of function and/or transcription in response to environmental medium conditions. Mutant analysis on fluorescently labeled PdeB indicated that a diguanylate cyclase (GGDEF) domain in PdeB is strictly required for HubP-dependent localization. Bacterial two-hybrid and in vitro interaction studies on purified proteins strongly indicate that this GGDEF domain of PdeB directly interacts with the C-terminal FimV domain of HubP. Polar localization of PdeB occurs late during the cell cycle after cell division and separation and is not dependent on medium conditions. In vitro activity measurements did not reveal a difference in PdeB phosphodiesterase activities in the presence or absence of the HubP FimV domain. We hypothesize that recruitment of PdeB to the flagellated pole by HubP may create an asymmetry of c-di-GMP levels between mother and daughter cells and may assist in organization of c-di-GMP-dependent regulation within the cell. IMPORTANCE c-di-GMP-dependent signaling affects a range of processes in many bacterial species. Most bacteria harbor a plethora of proteins with domains which are potentially involved in synthesis and breakdown of c-di-GMP. A potential mechanism to elicit an appropriate c-di-GMP-dependent response is to organize the corresponding proteins in a spatiotemporal fashion. Here, we show that a major contributor to c-di-GMP levels and flagellum-mediated swimming in Shewanella, PdeB, is recruited to the flagellated cell pole by the polar marker protein HubP. Polar recruitment involves a direct interaction between HubP and a GGDEF domain in PdeB, demonstrating a novel mechanism of polar targeting by the widely conserved HubP/FimV polar marker.

21Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation 22 strategies and differences in the presence of circadian clock proteins. In Synechococcus elongatus 23 PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity 24 of KaiC correlates with the period of the clock and mediates temperature compensation. 25 Synechocystis sp. PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins 26 were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore 27 characterized the enzymatic activity of KaiC3 as a representative of non-standard KaiC homologs 28 in vitro. KaiC3 displayed ATPase activity, which were lower compared to the Synechococcus 29 elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature-dependent. Hence, KaiC3 is 30 missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid 31 analysis showed that KaiC3 interacts with KaiB3, KaiC1 and KaiB1. Further, KaiB3 and KaiB1 32reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in 33 constant darkness is completely abolished after deletion of ∆kaiAB1C1 and reduced in the 34 absence of kaiC3. We therefore suggest a role for adaptation to darkness for KaiC3 as well as a 35 crosstalk between the KaiC1 and KaiC3 based systems. 36 Importance: The circadian clock influences the cyanobacterial metabolism and deeper 37 understanding of its regulation will be important for metabolic optimizations in context of 38 industrial applications. Due to the heterogeneity of cyanobacteria, characterization of clock 39 systems in organisms apart from the circadian model Synechococcus elongatus PCC 7942 is 40 required. Synechocystis PCC 6803 represents a major cyanobacterial model organism and harbors 41 phylogenetically diverged homologs of the clock proteins, which are present in various other non-42 3 cyanobacterial prokaryotes. By our in vitro studies we unravel the interplay of the multiple 43 Synechocystis Kai proteins and characterize enzymatic activities of the non-standard clock 44 homolog KaiC3. We show that the deletion of kaiC3 affects growth in constant darkness 45 suggesting its involvement in the regulation of non-photosynthetic metabolic pathways. 46 Introduction: 47 Cyanobacteria have evolved the circadian clock system to sense, anticipate and respond to 48 predictable environmental changes based on the rotation of Earth and the resulting day-night 49 cycle. Circadian rhythms are defined by three criteria: (i) oscillations with a period of about 24 50 hours without external stimuli, (ii) synchronization of the oscillator with the environment and (iii) 51 compensation of the usual temperature dependence of biochemical reactions, so that the period 52 of the endogenous oscillation does not depend on temperature in a physiological range (2). The 53 cyanobacterial circadian clock system has been studied in much detail in the unicellular model 54 cyanoba...

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