No Light, No Germination: Excitation of the Rhodospirillum centenum Photosynthetic Apparatus Is Necessary and Sufficient for Cyst Germination

ABSTRACT Rhodospirillum centenum is a Gram-negative alphaproteobacterium that is capable of differentiating into dormant cysts that are metabolically inactive and desiccation resistant. Like spores synthesized by many Gram-positive species, dormant R. centenum cysts germinate in response to an environmental signal, indicating that conditions favor survival and proliferation. Factors that induce germination are called germinants and are often both niche and species specific. In this study, we have identified photosynthesis as a niche-specific germinant for R. centenum cyst germination. Specifically, excitation of wild-type cysts suspended in a nutrient-free buffer with far-red light at >750 nm results in rapid germination. This is in stark contrast to mutant strains deficient in photosynthesis that fail to germinate upon exposure to far-red light under all assayed conditions. We also show that photosynthesis-induced germination occurs in a carbon- and nitrogen-free buffer even in strains that are deficient in carbon or nitrogen fixation. These results demonstrate that photosynthesis not only is necessary for germination but is itself sufficient for the germination of R. centenum cysts. IMPORTANCE Environmental cues that signal Gram-positive spores to germinate (termed germinants) have been identified for several Bacillus and Clostridium species. These studies showed that germinants are niche and species specific. For example, Clostridium difficile spores sense bile salts as a germinant as their presence informs these cells of an intestinal environment. Bacillus fastidiosus spores use uric acid as a germinant that is present in soil and poultry litter as this species inhabits poultry litter. It is evident from these studies that dormant cells sample their environment to assess whether conditions are advantageous for the propagation and survival of vegetative cells. To date, a limited number of germinants have been defined for only a few Gram-positive spore-forming species. Beyond that group, there is scant information on what cues signal dormant cells to exit dormancy. In our study, we show that the versatile Gram-negative photosynthetic bacterium Rhodospirillum centenum uses light-driven photosynthesis, and not the availability of nutrients, to trigger the germination of dormant cysts. This use of light-driven photosynthesis as a germinant is surprising as this species is also capable of growing under dark conditions using exogenous carbon sources for energy. Consequently, photosynthetic growth appears to be the preferred growth mechanism by this species.

Adenylate Charge Regulates Sensor Kinase CheS3 To Control Cyst Formation in Rhodospirillum centenum

ABSTRACTRhodospirillum centenumforms metabolically dormant cysts under unfavorable growth conditions such as desiccation or nutrient starvation. The development of cysts is tightly regulated and involves a cyst-repressing chemotaxis-like signal transduction pathway called the Che3signaling cascade. The Che3cascade is comprised of a methyl chemoreceptor (MCP3), receptor-methylating/demethylating proteins CheB3and CheR3, two CheW3linker proteins, a CheA3-CheY hybrid histidine kinase, and a single-domain response regulator, CheY3. In addition to Che-like components, the Che3cascade also contains a second hybrid histidine kinase, CheS3. Recent biochemical and genetic studies show that CheA3does not serve as a phosphor donor for CheY3; instead, CheA3inhibits a CheS3→CheY3two-component system by phosphorylating an inhibitory receiver domain of CheS3. In this study, we show that in addition to phosphorylation by CheA3, the phosphorylation state of CheS3is also regulated by the cellular energy level as quantified by the molar ratio of ATP/(ATP + ADP). A 35% decrease in cellular energy is shown to occurin vivoupon a nutrient downshift that gives rise to cyst formation. When this energy decline is replicatedin vitro, the phosphorylation level of CheS3is reduced by ~75%. Finally, we also show that ADP-mediated reduction of CheS3phosphorylation is a consequence of ADP enhancing autodephosphorylation of CheS3.IMPORTANCEUpon starvation,Rhodospirillum centenumundergoes a developmental process that forms metabolically dormant cysts, which withstand desiccation and nutritional limitation. This study explores the role of the cellular energy state as measured by the ratio of ATP to ADP as an important regulator of cyst formation inRhodospirillum centenum. We show thatR. centenumcells experience a significant reduction in ATP during cyst formation using ATP/(ATP + ADP) as a measurement. When thisin vivolevel of energy starvation is simulatedin vitro, CheS3phosphorylation is reduced by 75%. This profound reduction in CheS3autophosphorylation is contrasted with a much lower 25% decrease in CheA3phosphorylation in response to a similar downward shift in ATP/(ATP + ADP). We argue that even though adenylate energy affects all ATP-dependent enzymes to an extent, the enhanced inhibition of CheS3activity in response to a reduction in the ATP/(ATP + ADP) ratio likely functions as an important input signal to regulate cyst development.

Structure of a Novel Bacterial c-GMP Binding Protein

cAMP is an important secondary messenger molecule widely distributed across all living kingdoms, whereas cGMP is generally considered to be restricted to eukaryotes. Recently, solid evidences for cGMP signaling in Rhodospirillum centenum have been provided, and it is proposed that cGMP could also be adapted to deliver messages to diverse outputs via unknown mechanisms. While the structures and functions of binding between cAMP and its receptor protein CRP have been well studied in the past, currently no structure of prokaryotic cGMP-binding protein complex is known. Here we report the first determination of a cGMP-receptor crystal structure from the plant pathogen Xanthomonas campestris (Xcc) to a resolution of 2.2 Å. The new cGMP receptor Xcc0249 is found to belong to the CRP/FNR family protein containing both a cyclic-Nucleotide Binding Domain (cNBD) and a GGDEF domains, and exhibits strong cGMP binding and diguanylate cyclase activities. Mutations of crucial amino acid residues responsible for cGMP binding to Xcc0249 are found to significantly reduce the biofilm formation and virulence in Xcc. Isothermal calorimetry (ITC) measurements demonstrate that Xcc0249 can bind preferentially to cGMP with a much stronger affinity (KD: 2.93E-7) than cAMP (KD: 1.79E-5). cGMP binding to Xcc0249 is also found to enhance the GGDEF diguanylate cyclase activity, implying a broader functional role of cGMP and a possible linkage between the cGMP and c-di-GMP interaction networks in bacteria. References