ZomB is essential for flagellar motor reversals in Shewanella putrefaciens and Vibrio parahaemolyticus.

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.

The role of FlhF and HubP as polar landmark proteins in Shewanella putrefaciens†CN-32.

Spatiotemporal 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 pili-mediated 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.

Binding of the RNA chaperone Hfq to the type IV pilus base is crucial for its function in Synechocystis sp. PCC 6803.

The bacterial RNA-binding protein Hfq functions in post-transcriptional regulation of gene expression. There is evidence in a range of bacteria for specific subcellular localization of Hfq; however, the mechanism and role of Hfq localization remain unclear. Cyanobacteria harbour a subfamily of Hfq that is structurally conserved but exhibits divergent RNA binding sites. Mutational analysis in the cyanobacterium Synechocystis sp. PCC 6803 revealed that several conserved amino acids on the proximal side of the Hfq hexamer are crucial not only for Hfq-dependent RNA accumulation but also for phototaxis, the latter of which depends on type IV pili. Co-immunoprecipitation and yeast two-hybrid analysis show that the secretion ATPase PilB1 (a component of the type IV pilus base) is an interaction partner of Hfq. Fluorescence microscopy revealed that Hfq is localized to the cytoplasmic membrane in a PilB1-dependent manner. Concomitantly, Hfq-dependent RNA accumulation is abrogated in a ΔpilB1 mutant, indicating that localization to the pilus base via interaction with PilB1 is essential for Hfq function in cyanobacteria.

Light-induced alteration of c-di-GMP level controls motility of Synechocystis sp. PCC 6803.

Cph2 from the cyanobacterium Synechocystis sp. PCC 6803 is a hybrid photoreceptor that comprises an N-terminal module for red/far-red light reception and a C-terminal module switching between a blue- and a green-receptive state. This unusual photoreceptor exerts complex, light quality-dependent control of the motility of Synechocystis sp. PCC 6803 cells by inhibiting phototaxis towards blue light. Cph2 perceives blue light by its third GAF domain that bears all characteristics of a cyanobacteriochrome (CBCR) including photoconversion between green- and blue-absorbing states as well as formation of a bilin species simultaneously tethered to two cysteines, C994 and C1022. Upon blue light illumination the CBCR domain activates the subsequent C-terminal GGDEF domain, which catalyses formation of the second messenger c-di-GMP. Accordingly, expression of the CBCR-GGDEF module in Δcph2 mutant cells restores the blue light-dependent inhibition of motility. Additional expression of the N-terminal Cph2 fragment harbouring a red/far-red interconverting phytochrome fused to a c-di-GMP degrading EAL domain restores the complex behaviour of the intact Cph2 photosensor. c-di-GMP was shown to regulate flagellar and pili-based motility in several bacteria. Here we provide the first evidence that this universal bacterial second messenger is directly involved in the light-dependent regulation of cyanobacterial phototaxis.

P signalling in unicellular cyanobacteria: analysis of redox-signals and energy charge.

This communication presents a short outline of the current knowledge on the molecular basis of P(II) signal transduction in unicellular cyanobacteria with respect to the perception of environmental stimuli. First, the general characteristics of the P(II) signalling system in unicellular cyanobacteria are presented, the hallmark of which is modification by serine-phosphorylation, as compared to the paradigmatic P(II) signal transduction system in proteobacteria, which is based on tyrosyl-uridylylation. Then, the focus is turned on the signals controlling P(II) phosphorylation state. Recently, the cellular phosphatase (termed PphA), which specifically dephosphorylates phosphorylated P(II) (P(II)-P) was identified in Synechocystis sp. strain PCC 6803. With the availability of a PphA-deficient mutant and the purified components for in vitro assay of PphA mediated P(II)-P dephosphorylation, novel insights into the signals, to which P(II)-P dephosphorylation responds, can be obtained. Here we present an investigation of the response of P(II)-P dephosphorylation towards treatments that affect the redox-balance of the cells. Furthermore, a possible role of varying ATP/ADP ratios on P(II)-P dephosphorylation was examined. From these studies, together with previous investigations, we conclude that P(II)-P dephosphorylation specifically responds to changes in the levels of central metabolites of carbon metabolism, in particular 2-oxoglutarate.

The novel protein phosphatase PphA from Synechocystis PCC 6803 controls dephosphorylation of the signalling protein PII.

The family of PII signal transduction proteins consists of one of the most highly conserved signalling proteins in nature. The cyanobacterial PII homologue transmits signals on the nitrogen and carbon status of the cells through phosphorylation of a seryl residue. Recently, we identified a protein phosphatase 2C (PP2C) homologue from the cyanobacterium Synechocystis PCC 6803, termed PphA, to be the cellular phospho-PII (PII-P) phosphatase. In this investigation, we characterized the enzymatic properties of PphA and investigated the regulation of its catalytic activity towards PII-P. PphA dephosphorylates phosphocasein and PII-P with similar efficiency in a strictly Mg2+- or Mn2+-dependent reaction. Low-molecular-weight phosphorylated molecules are poor substrates for PphA. Its reactivity towards PII-P, but not towards phosphocasein, is inhibited by various nucleotides, suggesting that this effect is based on specific properties of the PII protein. The inhibitory effect of ATP can be strongly enhanced by the addition of 2-oxoglutarate or oxaloacetate. At low concentrations of 2-oxoglutarate, changes in the ATP levels within the physiological range affect the degree of PII-Pase inhibition, whereas at 2-oxoglutarate levels beyond 0.1 mM, inhibition is almost complete at very low ATP levels. This suggests that PII dephosphorylation is not only sensitive to 2-oxoglutarate and oxaloacetate levels, it also integrates signals from the energy charge of the cells under specific cellular conditions.

The Synechococcus elongatus P signal transduction protein controls arginine synthesis by complex formation with N-acetyl-L-glutamate kinase.

This communication identifies, for the first time, a receptor protein for signal perception from the P(II) signal transduction protein in the cyanobacterium Synechococcus elongatus. P(II), a phosphoprotein that signals the carbon/nitrogen status of the cells, forms a tight complex with the key enzyme of the arginine biosynthetic pathway, N-acetylglutamate (NAG) kinase. In complex with P(II), the catalytic activity of NAG kinase is strongly enhanced. Complex formation does not require the effector molecules of P(II), 2-oxoglutarate and ATP, but it is highly susceptible to modifications at the phosphorylation site of P(II), Ser-49. Stable complexes were only formed with the non-phosphorylated form of P(II) but not with Ser-49 mutants. In accordance with these data, NAG kinase activity in S. elongatus extracts correlated with the phosphorylation state of P(II), with high NAG kinase activities corresponding to non-phosphorylated P(II) (nitrogen-excess conditions) and low activities to increased levels of P(II) phosphorylation (nitrogen-poor conditions), thus subjecting the key enzyme of arginine biosynthesis to global nitrogen control.