Evidence of defined temporal expression patterns that lead a gram-negative cell out of dormancy.

Many bacterial species are capable of forming long-lived dormant cells. The best characterized are heat and desiccation resistant spores produced by many Gram-positive species. Less characterized are dormant cysts produced by several Gram-negative species that are somewhat tolerant to increased temperature and very resistant to desiccation. While there is progress in understanding regulatory circuits that control spore germination, there is scarce information on how Gram-negative organisms emerges from dormancy. In this study, we show that R. centenum cysts germinate by emerging a pair of motile vegetative cells from a thick cyst cell wall coat ~ 6 hrs post induction of germination. Time-lapse transcriptomic analysis reveals that there is a defined temporal pattern of gene expression changes during R. centenum cyst germination. The first observable changes are increases in expression of genes for protein synthesis, an increase in expression of genes involved in the generation of a membrane potential and the use of this potential for ATP synthesis via ATPase expression. These early events are followed by expression changes that affect the cell wall and membrane composition, followed by expression changes that promote chromosome replication. Midway through germination, expression changes occur that promote the flow of carbon through the TCA cycle to generate reducing power and parallel synthesis of electron transfer components involved in oxidative phosphorylation. Finally, late expression changes promote the synthesis of a photosystem as well as flagellar and chemotaxis components for motility.

DNA-binding properties of a cGMP-binding CRP homologue that controls development of metabolically dormant cysts of Rhodospirillum centenum.

Rhodospirillum centenum utilizes 3',5'-cyclic guanosine monophosphate (cGMP) as a messenger to regulate development of desiccation-resistant cysts. In this study, we demonstrated that gcyA, gcyB and gcyC, coding for putative subunits of a guanylyl cyclase, increase expression from 8- to 500-fold when cells transition from vegetative to cyst phases of growth. This induction did not occur in a strain that is defective in cGMP synthesis or in a strain that contains a deletion of cgrA that codes for a cGMP-binding homologue of Escherichia coli catabolite repressor protein (CRP). We also demonstrated that cgrA auto-induces its own expression in the presence of cGMP, indicating that a feed-forward loop is used to ramp up cGMP production as cells undergo encystment. Inspection of an intragenic region upstream of gcyB revealed a sequence that is identical to the CRP consensus sequence from E. coli. DNase I and fluorescence anisotropy analyses demonstrated that CgrA bound to this target sequence at a protein : cGMP ratio of 1 : 2 with Kd ∼61 nM. This was in contrast to CgrA in the presence of cAMP, which exhibited Kd ∼1795 nM. CgrA thus constitutes a novel variant of CRP that utilizes cGMP to regulate production of cGMP synthase for the control of cyst development.

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

UNLABELLED: Rhodospirillum centenum forms 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 Che3 signaling cascade. The Che3 cascade is comprised of a methyl chemoreceptor (MCP3), receptor-methylating/demethylating proteins CheB3 and CheR3, two CheW3 linker proteins, a CheA3-CheY hybrid histidine kinase, and a single-domain response regulator, CheY3. In addition to Che-like components, the Che3 cascade also contains a second hybrid histidine kinase, CheS3. Recent biochemical and genetic studies show that CheA3 does not serve as a phosphor donor for CheY3; instead, CheA3 inhibits a CheS3→CheY3 two-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 CheS3 is 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 occur in vivo upon a nutrient downshift that gives rise to cyst formation. When this energy decline is replicated in vitro, the phosphorylation level of CheS3 is reduced by ~75%. Finally, we also show that ADP-mediated reduction of CheS3 phosphorylation is a consequence of ADP enhancing autodephosphorylation of CheS3. IMPORTANCE: Upon starvation, Rhodospirillum centenum undergoes 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 in Rhodospirillum centenum. We show that R. centenum cells experience a significant reduction in ATP during cyst formation using ATP/(ATP + ADP) as a measurement. When this in vivo level of energy starvation is simulated in vitro, CheS3 phosphorylation is reduced by 75%. This profound reduction in CheS3 autophosphorylation is contrasted with a much lower 25% decrease in CheA3 phosphorylation 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 CheS3 activity in response to a reduction in the ATP/(ATP + ADP) ratio likely functions as an important input signal to regulate cyst development.

Transcriptome analysis of cyst formation in Rhodospirillum centenum reveals large global changes in expression during cyst development.

BACKGROUND: Rhodospirillum centenum is a photosynthetic member of the Gram-negative Azospirillum clade members of which exhibit a complex developmental life-cycle featuring morphologically distinct cell types. Under periods of nutrient deprivation, replicative vegetative cells differentiate into metabolically dormant cysts that survive harsh environmental stresses such as desiccation. Encystment involves a multi-stage developmental process that includes the rounding of cells, production of large intracellular storage granules of poly-hydroxybutyrate (PHB) and the excretion of a protective exopolysaccharide coating that envelops dormant cysts. RESULTS: To study the process of cyst development, we performed RNA-seq studies on cells that were induced to undergo cyst development. To assay for temporal changes in gene expression, RNA was extracted at 4, 24, 48, 72, 96 hours during development and subjected to deep sequence analysis. These results show that 812 genes exhibit log2 ≥ 1.5-fold changes in expression over a 96 hour cyst induction period demonstrating large global changes in gene expression during cyst development. CONCLUSIONS: Notable changes in expression occurred in numerous genes involved in cell wall and lipid biosynthesis, metabolic enzymes, and numerous regulatory genes such as histidine kinases and transcription factors. Many genes involved in protein synthesis and DNA replication were also significantly reduced during late stages of cyst development. Genes previously identified by genetic screens as being critical for cyst development also exhibited changes of expression during cyst induction. This study provides the first transcriptome profile of global changes in gene expression that occur during development of cysts in a Gram-negative species.

4-Hydroxy-3-methyl-6-(1-methyl-2-oxoalkyl)pyran-2-one synthesis by a type III polyketide synthase from Rhodospirillum centenum.

The purple photosynthetic bacterium Rhodospirillum centenum has a putative type III polyketide synthase gene (rpsA). Although rpsA was known to be transcribed during the formation of dormant cells, the reaction catalyzed by RpsA was unknown. Thus we examined the RpsA reaction in vitro, using various fatty acyl-CoAs with even numbers of carbons as starter substrates. RpsA produced tetraketide pyranones as major compounds from one C(10-14) fatty acyl-CoA unit, one malonyl-CoA unit and two methylmalonyl-CoA units. We identified these products as 4-hydroxy-3-methyl-6-(1-methyl-2-oxoalkyl)pyran-2-ones by NMR analysis. RpsA is the first bacterial type III PKS that prefers to incorporate two molecules of methylmalonyl-CoA as the extender substrate. In addition, in vitro reactions with (13)C-labeled malonyl-CoA revealed that RpsA produced tetraketide 6-alkyl-4-hydroxy-1,5-dimethyl-2-oxocyclohexa-3,5-diene-1-carboxylic acids from C(14-20) fatty acyl-CoAs. This class of compounds is likely synthesized through aldol condensation induced by methine proton abstraction. No type III polyketide synthase that catalyzes this reaction has been reported so far. These two unusual features of RpsA extend the catalytic functions of the type III polyketide synthase family.

Evolutionary remodeling of ßγ-crystallins for domain stability at cost of Ca2+ binding.

The topologically similar ßγ-crystallins that are prevalent in all kingdoms of life have evolved for high innate domain stability to perform their specialized functions. The evolution of stability and its control in ßγ-crystallins that possess either a canonical (mostly from microorganisms) or degenerate (principally found in vertebrate homologues) Ca2+-binding motif is not known. Using equilibrium unfolding of ßγ-crystallin domains (26 wild-type domains and their mutants) in apo- and holo-forms, we demonstrate the presence of a stability gradient across these members, which is attained by the choice of residues in the (N/D)(N/D)XX(S/T)S Ca2+-binding motif. The occurrence of a polar, hydrophobic, or Ser residue at the 1st, 3rd, or 5th position of the motif is likely linked to a higher domain stability. Partial conversion of a microbe-type domain (with a canonical Ca2+-binding motif) to a vertebrate-type domain (with a degenerate Ca2+-binding motif) by mutating serine to arginine/lysine disables the Ca2+-binding but significantly augments its stability. Conversely, stability is compromised when arginine (in a vertebrate-type disabled domain) is replaced by serine (as a microbe type). Our results suggest that such conversions were acquired as a strategy for desired stability in vertebrate members at the cost of Ca2+-binding. In a physiological context, we demonstrate that a mutation such as an arginine to serine (R77S) mutation in this motif of γ-crystallin (partial conversion to microbe-type), implicated in cataracts, decreases the domain stability. Thus, this motif acts as a "central tuning knob" for innate as well as Ca2+-induced gain in stability, incorporating a stability gradient across ßγ-crystallin members critical for their specialized functions.

Cyclic GMP controls Rhodospirillum centenum cyst development.

Adenylyl cyclases are widely distributed across all kingdoms whereas guanylyl cyclases are generally thought to be restricted to eukaryotes. Here we report that the α-proteobacterium Rhodospirillum centenum secretes cGMP when developing cysts and that a guanylyl cyclase deletion strain fails to synthesize cGMP and is defective in cyst formation. The R. centenum cyclase was purified and shown to effectively synthesize cGMP from GTP in vitro, demonstrating that it is a functional guanylyl cyclase. A homologue of the Escherichia coli cAMP receptor protein (CRP) is linked to the guanylyl cyclase and when deleted is deficient in cyst development. Isothermal calorimetry (ITC) and differential scanning fluorimetry (DSF) analyses demonstrate that the recombinant CRP homologue preferentially binds to, and is stabilized by cGMP, but not cAMP. This study thus provides evidence that cGMP has a crucial role in regulating prokaryotic development. The involvement of cGMP in regulating bacterial development has broader implications as several plant-interacting bacteria contain a similar cyclase coupled by the observation that Azospirillum brasilense also synthesizes cGMP when inducing cysts.

Metabolic flexibility revealed in the genome of the cyst-forming alpha-1 proteobacterium Rhodospirillum centenum.

BACKGROUND: Rhodospirillum centenum is a photosynthetic non-sulfur purple bacterium that favors growth in an anoxygenic, photosynthetic N2-fixing environment. It is emerging as a genetically amenable model organism for molecular genetic analysis of cyst formation, photosynthesis, phototaxis, and cellular development. Here, we present an analysis of the genome of this bacterium. RESULTS: R. centenum contains a singular circular chromosome of 4,355,548 base pairs in size harboring 4,105 genes. It has an intact Calvin cycle with two forms of Rubisco, as well as a gene encoding phosphoenolpyruvate carboxylase (PEPC) for mixotrophic CO2 fixation. This dual carbon-fixation system may be required for regulating internal carbon flux to facilitate bacterial nitrogen assimilation. Enzymatic reactions associated with arsenate and mercuric detoxification are rare or unique compared to other purple bacteria. Among numerous newly identified signal transduction proteins, of particular interest is a putative bacteriophytochrome that is phylogenetically distinct from a previously characterized R. centenum phytochrome, Ppr. Genes encoding proteins involved in chemotaxis as well as a sophisticated dual flagellar system have also been mapped. CONCLUSIONS: Remarkable metabolic versatility and a superior capability for photoautotrophic carbon assimilation is evident in R. centenum.

Regulation of aerobic photosystem synthesis in the purple bacterium Rhodospirillum centenum by CrtJ and AerR.

Genes coding for putative CrtJ and AerR homologs were identified and characterized in the purple photosynthetic bacterium Rhodospirillum centenum (also known as Rhodocista centenaria), an organism that synthesizes photopigments even under highly aerated conditions. Mutational analysis indicated that in Rsp. centenum, gene crtJ codes for a repressor for photosynthesis gene expression as in Rhodobacter capsulatus, which exhibits a high level of oxygen repression of photosystem synthesis. In contrast to Rba. capsulatus, AerR in Rsp. centenum appears to be an aerobic activator; an aerR mutation resulted in significantly reduced levels of photopigment synthesis. Both aerR and crtJ mutants retained essentially normal levels of photosystem synthesis under anaerobic conditions, indicating that their activities are specific for aerobic photosystem synthesis. The readthrough transcript from crtE promoter, which is regulated by AerR and CrtJ, seems to be significant in maintaining the expression levels of the light harvesting I (puf) genes in Rsp. centenum. We suggest that AerR and CrtJ regulate aerobic photosystem synthesis primarily through controlling activity of the transcriptional readthrough.

Involvement of a Che-like signal transduction cascade in regulating cyst cell development in Rhodospirillum centenum.

Homologues of the E. coli chemotaxis (Che) signal transduction pathway are present in nearly all motile bacteria. Although E. coli contains only one Che cascade, many other bacteria are known to possess multiple sets of che genes. The role of multiple che-like gene clusters could potentially code for parallel Che-like signal transduction pathways that have distinctly different input and output functions. In this study, we describe a che-like gene cluster in Rhodospirillum centenum that controls a developmental cycle. In-frame deletion mutants of homologues of CheW (DeltacheW(3a)and DeltacheW(3b)), CheR (DeltacheR(3)), CheA (DeltacheA(3)) and a methyl-accepting chemotaxis protein (Deltamcp(3)) are defective in starvation-induced formation of heat and desiccation resistant cyst cells. In contrast, mutants of homologues of CheY (DeltacheY(3)), CheB (DeltacheB(3)), and a second input kinase designated as CheS (DeltacheS(3)) result in cells that are derepressed in the formation of cysts. A model of signal transduction is presented in which there are three distinct Che-like signal transduction cascades; one that is involved in chemotaxis, one that is involved in flagella biosynthesis and the third that is involved in cyst development.