Pho Regulon Research Articles

Novel Fosfomycin Resistance Mechanism in Pseudomonas entomophila Due to Atypical Pho Regulon Control of GlpT

Background/Objectives: Pseudomonas entomophila is a ubiquitous bacterium capable of killing insects of different orders and has become a model for host–pathogen studies and a promising tool for biological pest control. In the human pathogen Pseudomonas aeruginosa, spontaneous resistance to fosfomycin arises almost exclusively from mutations in the glycerol-3-phosphate transporter (GlpT), the drug’s sole entry route in this species. Here, we investigated whether this specificity is conserved in P. entomophila, as it could provide a valuable marker system for studying mutation rates and spectra and for selection in genetic engineering. Methods: We isolated 16 independent spontaneous fosfomycin-resistant mutants in P. entomophila, and studied the genetic basis of the resistance using a combination of sequencing, phenotyping and computational approaches. Results: We only found two mutants without alterations in glpT or any of its known regulatory elements. Whole-genome sequencing revealed unique inactivating mutations in phoU, a key regulator of the phosphate starvation (Pho) regulon. Computational analyses identified a PhoB binding site in the glpT promoter, and experiments showed that phoU inactivation reduced glpT expression nearly 20-fold. While placing a sugar-phosphate transporter under the Pho regulon may seem advantageous, bioinformatic analysis shows this configuration is atypical among pseudomonads. Conclusions: this atypical Pho regulon control of GlpT probably reflects the peculiarities of P. entomophila’s habitat and lifestyle; highlighting how readily regulatory evolution can lead to the rapid divergence of resistance mechanisms, even among closely related species.

Suppression of inositol pyrophosphate toxicosis and hyper-repression of the fission yeast PHO regulon by loss-of-function mutations in chromatin remodelers Snf22 and Sol1.

Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising phosphate acquisition genes pho1, pho84, and tgp1) is repressed under phosphate-replete conditions by lncRNA-mediated transcriptional interference, mutations of inositol pyrophosphatases that increase IP8 levels derepress the PHO regulon by eliciting precocious termination of lncRNA transcription. Asp1 pyrophosphatase mutations resulting in too much IP8 are cytotoxic in YES medium owing to overexpression of glycerophosphodiester transporter Tgp1. IP8 toxicosis is ameliorated by mutations in cleavage/polyadenylation and termination factors, perturbations of the Pol2 CTD code, and mutations in SPX domain proteins that act as inositol pyrophosphate sensors. Here, we show that IP8 toxicity is alleviated by deletion of snf22+, the gene encoding the ATPase subunit of the SWI/SNF chromatin remodeling complex, by an ATPase-inactivating snf22-(D996A-E997A) allele, and by deletion of the gene encoding SWI/SNF subunit Sol1. Deletion of snf22+ hyper-repressed pho1 expression in phosphate-replete cells; suppressed the pho1 derepression elicited by mutations in Pol2 CTD, termination factor Seb1, Asp1 pyrophosphatase, and 14-3-3 protein Rad24 (that favor precocious prt lncRNA termination); and delayed pho1 induction during phosphate starvation. RNA analysis and lack of mutational synergies suggest that Snf22 is not impacting 3'-processing/termination. Using reporter assays, we find that Snf22 is important for the activity of the tgp1 and pho1 promoters, but not for the promoters that drive the synthesis of the PHO-repressive lncRNAs. Transcription profiling of snf22∆ and snf22-(D996A-E997A) cells identified an additional set of 66 protein-coding genes that were downregulated in both mutants.IMPORTANCERepression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to inositol pyrophosphate dynamics. Cytotoxic asp1-STF alleles derepress the PHO genes via the action of IP8 as an agonist of precocious lncRNA 3'-processing/termination. IP8 toxicosis is alleviated by mutations of the Pol2 CTD and the 3'-processing/termination machinery that dampen the impact of toxic IP8 levels on termination. In this study, a forward genetic screen revealed that IP8 toxicity is suppressed by mutations of the Snf22 and Sol1 subunits of the SWI/SNF chromatin remodeling complex. Genetic and biochemical evidence indicates that the SWI/SNF is not affecting 3'-processing/termination or lncRNA promoter activity. Rather, SWI/SNF is critical for firing the PHO mRNA promoters. Our results implicate the ATP-dependent nucleosome remodeling activity of SWI/SNF as necessary to ensure full access of PHO-activating transcription factor Pho7 to its binding sites in the PHO mRNA promoters.

Molecular Mechanisms of the Cyanobacterial Response to Different Phosphorus Sources

There are different phosphorus (P) sources of varied concentrations in aquatic ecosystems. The sensing of P by cyanobacteria in the environment is predominantly regulated by two-component signal transduction systems in which the phosphate (Pho) regulon plays a crucial role in maintaining phosphate homeostasis. It responds rapidly and connects to metabolic processes through cross-talk mechanisms. However, the physiological and biochemical mechanisms of the cyanobacterial response to different P sources remain unclear. This review article aims to integrate the physiological and molecular information on the regulatory mechanisms of the cyanobacterial response to different P sources in terms of hydrolysis, transport, and inorganic P (DIP) utilization strategies. Topics covered include enzymatic utilization of DOP (C-O-P, C-P), phosphate transport systems, and exploring the potential P metabolic pathways that might occur in cyanobacteria. This is of great significance for mitigating eutrophication and maintaining the sustainable development of aquatic systems.

PhoB-regulated phosphate assimilation of Ralstonia solanacearum is cross-activated by VsrB in Pi-abundant rich medium

Ralstonia solanacearum is a devastating phytopathogen infecting a broad range of economically important crops. Phosphate (Pi) homeostasis and assimilation play a critical role in the environmental adaptation and pathogenicity of many bacteria. However, the Pi assimilation regulatory mechanism of R. solanacearum remains unknown. This study revealed that R. solanacearum pstSCAB-phoU-phoBR operon expression is sensitive to extracellular Pi concentration, with higher expression under Pi-limiting conditions. The PhoB-PhoR fine-tunes the Pi-responsive expression of the Pho regulon genes, demonstrating its pivotal role in Pi assimilation. By contrast, neither PhoB, PhoR, PhoU, nor PstS was found to be essential for virulence on tomato plants. Surprisingly, the PhoB regulon is activated in a Pi-abundant rich medium. Results showed that histidine kinase VsrB, which is known for the exopolysaccharide production regulation, partially mediates PhoB activation in the Pi-abundant rich medium. The 271 histidine of VsrB is vital for this activation. This cross-activation mechanism between the VsrB and PhoB-PhoR systems suggests the carbohydrate–Pi metabolism coordination in R. solanacearum. Overall, this research provides new insights into the complex regulatory interplay between Pi metabolism and growth in R. solanacearum.

Whole Genome Analysis of Streptomyces spp. Strains Isolated from the Rhizosphere of Vitis vinifera L. Reveals Their Role in Nitrogen and Phosphorus Metabolism

The rhizospheric microorganisms of agricultural crops play a crucial role in plant growth and nutrient cycling. In this study, we isolated two Streptomyces strains, Streptomyces sp. LM32 and Streptomyces sp. LM65, from the rhizosphere of Vitis vinifera L. We then conducted genomic analysis by assembling, annotating, and inferring phylogenomic information from the whole genome sequences. Streptomyces sp. strain LM32 had a genome size of 8.1 Mb and a GC content of 72.14%, while Streptomyces sp. strain LM65 had a genome size of 7.3 Mb and a GC content of 71%. Through ANI results, as well as phylogenomic, pan-, and core-genome analysis, we found that strain LM32 was closely related to the species S. coelicoflavus, while strain LM65 was closely related to the species S. achromogenes subsp. achromogenes. We annotated the functional categories of genes encoded in both strains, which revealed genes involved in nitrogen and phosphorus metabolism. This suggests that these strains have the potential to enhance nutrient availability in the soil, promoting agricultural sustainability. Additionally, we identified gene clusters associated with nitrate and nitrite ammonification, nitrosative stress, allantoin utilization, ammonia assimilation, denitrifying reductase gene clusters, high-affinity phosphate transporter and control of PHO regulon, polyphosphate, and phosphate metabolism. These findings highlight the ecological roles of these strains in sustainable agriculture, particularly in grapevine and other agricultural crop systems.

Micronutrients modulate the structure and function of soil bacterial communities

Soil micronutrients are increasingly recognized as critical regulators of biogeochemical cycling and terrestrial ecosystem processes. Despite substantial efforts establishing how belowground microbial communities respond to macronutrients such as N and P, responses to micronutrients remain poorly understood. This is of particular interest in tropical soils, where micronutrients are heterogeneously distributed and often deficient. Using nearly 300 soil samples that span broad gradients in soil micronutrients, we aimed to determine how differences in micronutrients (Ca, Mg, S) and secondary macronutrients (Mn, Fe, Zn) shape soil bacterial communities and their metabolic capabilities. Along with key soil parameters including pH and climatic predictors, we found that Ca, Mg, and Mn were important in shaping the composition and function of soil microbiomes. We also identified lineages that were consistently responsive to specific micronutrients, with those taxa found in low micronutrient conditions often associated with oligotrophic life history strategies. We detected a subset of metabolic attributes related to nutrient cycling that were strongly associated with specific micronutrients. Notably, Mn was important in predicting the abundance of gene systems related to P metabolism including control of Pho regulon and alkylphosphonate utilization, suggesting that it may be important mediators of these responses. Taken together, our results begin to establish a broader understanding of the extent to which belowground systems are shaped by micronutrients and the strategies that soil microbes employ in variable micronutrient environments.

Glycerophosphocholine provision rescues Candida albicans growth and signaling phenotypes associated with phosphate limitation.

Candida albicans is the most commonly isolated species from patients suffering from invasive fungal disease. C. albicans is most commonly a commensal organism colonizing a variety of niches in the human host. The fungus must compete for resources with the host flora to acquire essential nutrients such as phosphate. Phosphate acquisition and homeostasis have been shown to play a key role in C. albicans virulence, with several genes involved in these processes being required for normal virulence and several being upregulated during infection. In addition to inorganic phosphate (Pi), C. albicans can utilize the lipid-derived metabolite glycerophosphocholine (GPC) as a phosphate source. As GPC is available within the human host, we examined the role of GPC in phosphate homeostasis in C. albicans. We find that GPC can substitute for Pi by many though not all criteria and is likely a relevant physiological phosphate source for C. albicans.

Stringent Response of Cyanobacteria and Other Bacterioplankton during Different Stages of a Harmful Cyanobacterial Bloom.

We conducted a field study to investigate the role of stringent response in cyanobacteria and coexisting bacterioplankton during nutrient-deprived periods at various stages of bloom in a freshwater lake (Utah Lake) for the first time. Using metagenomics and metatranscriptomics analyses, we examined the cyanobacterial ecology and expression of important functional genes related to stringent response, N and P metabolism, and regulation. Our findings mark a significant advancement in understanding the mechanisms by which toxic cyanobacteria survive and proliferate during nitrogen (N) and phosphorus (P) limitations. We successfully identified and analyzed the metagenome-assembled genomes (MAGs) of the dominant bloom-forming cyanobacteria, namely, Dolichospermum circinale, Aphanizomenon flos-aquae UKL13-PB, Planktothrix agardhii, and Microcystis aeruginosa. By mapping RNA-seq data to the coding sequences of the MAGs, we observed that these four prevalent cyanobacteria species activated multiple functions to adapt to the depletion of inorganic nutrients. During and after the blooms, the four dominant cyanobacteria species expressed high levels of transcripts related to toxin production, such as microcystins (mcy), anatoxins (ana), and cylindrospermopsins (cyr). Additionally, genes associated with polyphosphate (poly-P) storage and the stringent response alarmone (p)ppGpp synthesis/hydrolysis, including ppk, relA, and spoT, were highly activated in both cyanobacteria and bacterioplankton. Under N deficiency, the main N pathways shifted from denitrification and dissimilatory nitrate reduction in bacterioplankton toward N2-fixing and assimilatory nitrate reduction in certain cyanobacteria with a corresponding shift in the community composition. P deprivation triggered a stringent response mediated by spoT-dependent (p)ppGpp accumulation and activation of the Pho regulon in both cyanobacteria and bacterioplankton, facilitating inorganic and organic P uptake. The dominant cyanobacterial MAGs exhibited the presence of multiple alkaline phosphatase (APase) transcripts (e.g., phoA in Dolichospermum, phoX in Planktothrix, and Microcystis), suggesting their ability to synthesize and release APase enzymes to convert ambient organic P into bioavailable forms. Conversely, transcripts associated with bacterioplankton-dominated pathways like denitrification were low and did not align with the occurrence of intense cyanoHABs. The strong correlations observed among N, P, stringent response metabolisms and the succession of blooms caused by dominant cyanobacterial species provide evidence that the stringent response, induced by nutrient limitation, may activate unique N and P functions in toxin-producing cyanobacteria, thereby sustaining cyanoHABs.

Interaction of calcium responsive proteins and transcriptional factors with the PHO regulon in yeasts and fungi.

Phosphate and calcium ions are nutrients that play key roles in growth, differentiation and the production of bioactive secondary metabolites in filamentous fungi. Phosphate concentration regulates the biosynthesis of hundreds of fungal metabolites. The central mechanisms of phosphate transport and regulation, mediated by the master Pho4 transcriptional factor are known, but many aspects of the control of gene expression need further research. High ATP concentration in the cells leads to inositol pyrophosphate molecules formation, such as IP3 and IP7, that act as phosphorylation status reporters. Calcium ions are intracellular messengers in eukaryotic organisms and calcium homeostasis follows elaborated patterns in response to different nutritional and environmental factors, including cross-talking with phosphate concentrations. A large part of the intracellular calcium is stored in vacuoles and other organelles forming complexes with polyphosphate. The free cytosolic calcium concentration is maintained by transport from the external medium or by release from the store organelles through calcium permeable transient receptor potential (TRP) ion channels. Calcium ions, particularly the free cytosolic calcium levels, control the biosynthesis of fungal metabolites by two mechanisms, 1) direct interaction of calcium-bound calmodulin with antibiotic synthesizing enzymes, and 2) by the calmodulin-calcineurin signaling cascade. Control of very different secondary metabolites, including pathogenicity determinants, are mediated by calcium through the Crz1 factor. Several interactions between calcium homeostasis and phosphate have been demonstrated in the last decade: 1) The inositol pyrophosphate IP3 triggers the release of calcium ions from internal stores into the cytosol, 2) Expression of the high affinity phosphate transporter Pho89, a Na+/phosphate symporter, is controlled by Crz1. Also, mutants defective in the calcium permeable TRPCa7-like of Saccharomyces cerevisiae shown impaired expression of Pho89. This information suggests that CrzA and Pho89 play key roles in the interaction of phosphate and calcium regulatory pathways, 3) Finally, acidocalcisomes organelles have been found in mycorrhiza and in some melanin producing fungi that show similar characteristics as protozoa calcisomes. In these organelles there is a close interaction between orthophosphate, pyrophosphate and polyphosphate and calcium ions that are absorbed in the polyanionic polyphosphate matrix. These advances open new perspectives for the control of fungal metabolism.

Nucleosome Remodeling at the Yeast PHO8 and PHO84 Promoters without the Putatively Essential SWI/SNF Remodeler.

Chromatin remodeling by ATP-dependent remodeling enzymes is crucial for all genomic processes, like transcription or replication. Eukaryotes harbor many remodeler types, and it is unclear why a given chromatin transition requires more or less stringently one or several remodelers. As a classical example, removal of budding yeast PHO8 and PHO84 promoter nucleosomes upon physiological gene induction by phosphate starvation essentially requires the SWI/SNF remodeling complex. This dependency on SWI/SNF may indicate specificity in remodeler recruitment, in recognition of nucleosomes as remodeling substrate or in remodeling outcome. By in vivo chromatin analyses of wild type and mutant yeast under various PHO regulon induction conditions, we found that overexpression of the remodeler-recruiting transactivator Pho4 allowed removal of PHO8 promoter nucleosomes without SWI/SNF. For PHO84 promoter nucleosome removal in the absence of SWI/SNF, an intranucleosomal Pho4 site, which likely altered the remodeling outcome via factor binding competition, was required in addition to such overexpression. Therefore, an essential remodeler requirement under physiological conditions need not reflect substrate specificity, but may reflect specific recruitment and/or remodeling outcomes.

Perfect adaptation achieved by transport limitations governs the inorganic phosphate response in S. cerevisiae

Cells cope with and adapt to ever-changing environmental conditions. Sophisticated regulatory networks allow cells to adjust to these fluctuating environments. One such archetypal system is the Saccharomyces cerevisiae Pho regulon. When external inorganic phosphate (Pi) concentration is low, the Pho regulon activates, expressing genes that scavenge external and internal Pi. However, the precise mechanism controlling this regulon remains elusive. We conducted a systems analysis of the Pho regulon on the single-cell level under well-controlled environmental conditions. This analysis identified a robust, perfectly adapted Pho regulon state in intermediate Pi conditions, and we identified an intermediate nuclear localization state of the transcriptional master regulator Pho4p. The existence of an intermediate nuclear Pho4p state unifies and resolves outstanding incongruities associated with the Pho regulon, explains the observed programmatic states of the Pho regulon, and improves our general understanding of how nature evolves and controls sophisticated gene regulatory networks. We further propose that robustness and perfect adaptation are not achieved through complex network-centric control but by simple transport biophysics. The ubiquity of multitransporter systems suggests that similar mechanisms could govern the function of other regulatory networks as well.

Differential expression and cross-correlation between global regulator and pho regulon genes involved in decision-making under phosphate stress.

The differential gene expression under phosphate stress conditions leads to cross-talk between the global regulator, pho regulon, and metabolic genes. Promoter activity analysis of the selected 23 genes reveals the dynamic nature of real-time gene expression under different phosphate conditions. The expression profiles of the global regulator (rpoD, soxR, soxS, arcB, and fur), pho regulon (phoH, phoR, phoB, and ugpB), and metabolic genes (sdh, pfkA, ldh) varied significantly on phosphate level variation. Under stress conditions, soxR switches expression partners and co-expresses with rpoS instead of soxS. The partner-switching behavior of the genes under a challenging environment represents the intelligence of functional execution and ensures cell survival. The dynamic expression profile of the selected genes applies a time-lagged correlation to provide insight into the differential gene interaction between time-shifted expression profiles. Under different phosphate conditions, the minimum spanning tree graph revealed a different clustering pattern of selected genes depending on the computed distance and its proximity to other promoters.

A Dual Regulatory Role of the PhoU Protein in Salmonella Typhimurium.

ABSTRACTBacteria utilize two-component regulatory systems to sense and respond to their surroundings. Unlike other two-component systems that directly sense through a sensory domain in the histidine kinase (HK), the PhoB/PhoR two-component system requires additional proteins, including the PstSCAB phosphate transporter and the PhoU protein, to sense phosphate levels. Although PhoU is involved in phosphate signaling by connecting the PstSCAB transporter and PhoR histidine kinase, the mechanism by which PhoU controls expression of pho regulon genes has not yet been clearly understood. Here, we identified PhoU residues required for interacting with PhoR histidine kinase from the intracellular pathogen Salmonella enterica serovar Typhimurium. The PhoU Ala147 residue interacts with the PhoR PAS domain and is involved in repressing pho expression in high phosphate. Unexpectedly, the PhoU Arg184 residue interacts with the PhoR histidine kinase domain and is required for activating pho expression in low Mg2+ by increasing PhoR autophosphorylation, revealing its new function. The substitution of the Arg184 to Gly codon decreased Salmonella virulence both in macrophages and in mice, suggesting that PhoU’s role in promoting PhoR autophosphorylation is required during Salmonella infection.

Two Pathways Diverge: Glycerophosphocholine Impacts Both Phosphate and Lipid Metabolism in Candida albicans

Candida albicans (C. albicans)is a leading source of fungal infections in humans, resulting in over 250,000 deaths annually. C. albicanscolonizes multiple host niches and causes invasive disease, requiring its adaptation to wide variations in nutrient availability. Phosphate is an essential nutrient, and several genes associated with phosphate acquisition are differentially expressed during infection. In addition to inorganic phosphate (Pi), C. albicans utilizes the abundant lipid metabolite, glycerophosphocholine (GPC), as a phosphate source. GPC is transported into the cell via the Git3 and Git4 transporters. The Git transporters are required for full virulence in a mouse model of disseminated candidiasis, but the mechanism for that virulence defect is unknown. Here, we probe the importance of GPC uptake to cellular metabolism by examining two aspects of its metabolic fate: i) its catabolism to liberate Pi), and ii) its potential conversion to phosphatidylcholine (PC) via reacylation. To query its role in phosphate homeostasis, we employed a knockout strain of the high affinity phosphate transporter, Pho84. Cells lacking Pho84 display sensitivity to several host‐related stressors and decreased virulence. Our results show that provision of cells with GPC as phosphate source alleviates hypersensitivity of pho84∆/∆ mutants to osmotic, oxidative, and cell wall stress. The rate at which GPC and Pi are transported into the cell varies depending upon the total level of phosphate provided, with Pitransport exceeding GPC transport by a maximum of two‐fold at 500 µM total phosphate. The effect of exogenous GPC on induction of the PHO regulon, the cellular system of phosphate homeostasis, is complex and concentration dependent. Beyond its role in phosphate metabolism, GPC has other physiologically relevant metabolic fates, namely its reacylation to form a phosphatidylcholine (PC) molecule. In Saccharomyces cerevisiae, the first step of this novel reacylation pathway is catalyzed by the acyltransferase, Gpc1, which converts GPC to lyso‐PC. Initial in vivo labeling studies under phosphate replete conditions indicate that the C. albicans homolog of Gpc1 is a GPC acyltransferase and that the reacylation pathway is functional in C. albicans. In vitro assays in strains lacking or containing Gpc1 are underway. Taken together, our results indicate that C. albicans can utilize the abundant lipid metabolite, GPC, via at least two metabolic pathways to promote its growth and survival in the human host.

Soil Microbial Community Succession Based on PhoD and Gcd Genes along a Chronosequence of Sand-Fixation Forest

Revegetation by planting shrubs on moving sand dunes is widely used to control desertification in arid/semi-arid areas. The soil including microbial community can gradually be improved along with plantation development. The purposes of this study were (1) to investigate the responses of microbial communities involved in the mineralization of soil organic phosphorus (OP) and dissolution of inorganic P (IOP) in the development of sand-fixating plantation and (2) to discuss the interactions between P turnover microbial communities and soil properties. We assessed the compositions of soil phoD gene (one of the Pho regulons encoding alkaline phosphomonoesterases) and gcd gene (encoding glucose dehydrogenase) in microbial community by using high-throughput Illumina MiSeq sequencing in a chronosequence of Caragana microphylla plantations (0-, 10-, 20-, and 37-year plantations and a native C. microphylla shrub forest) in Horqin Sandy Land, Northeast China. Soil properties including soil nutrients, enzymatic activity, and P fractions were also determined. The abundance of phoD and gcd genes linearly increased with the plantation age. However, the diversity of soil phoD microbes was more abundant than that of gcd. The phoD gene abundance and the fractions of total OP and IOP were positively correlated with the activity of phosphomonoesterase. Actinobacteria and Streptomycetaceae were the dominant phoD taxa, while Proteobacteria and Rhizobiaceae were the dominant gcd taxa. Plantation development facilitated the progressive successions of soil phoD and gcd communities resulting from the increase in the abundance of dominant taxa. Total soil N, NH4-N, and available K were the main factors affecting the structures of phoD and gcd communities, while pH was not significantly influencing factor in such arid and nutrient-poor sandy soil. Many phoD or gcd OTUs were classified into Rhizobium and Bradyrhizobium, suggesting the coupling relationship between soil P turnover and N fixation.

Effects of Escherichia coli Alkaline Phosphatase PhoA on the Mineralization of Dissolved Organic Phosphorus

Alkaline phosphatases, which play the key role in the mineralization of organic phosphorus, have been grouped into three distinct families, PhoA, PhoX, and PhoD. PhoA is still an important component of the Pho regulon for many microbes although its distribution is not as wide as that of PhoX and PhoD. However, several questions remain unclear about the effect of PhoA mineralization of dissolved organic phosphorus. In this study, the role of Escherichia coli alkaline phosphatase PhoA (hereinafter referred to as PhoA) in the mineralization of different organic phosphorus including phosphate monoesters, phosphate diesters, and phytic acids was investigated. The influence of the reaction time, organic phosphorus concentration, and L-amino acid on PhoA mineralization was examined. The results show that PhoA specifically hydrolyzes phosphate monoesters except for phytic acid and the optimal reaction time is around 12 h. The PhoA mineralization rate of glucose 6-phosphate disodium (G6P), 5′-adenosine monophosphate (AMP), and sodium glycerophosphate (BGP) significantly decreased by 38.01%, 55.31%, and 57.08%, respectively (p < 0.01), while the concentration of organic phosphorus increased from 0.50 to 5.00 mg/L. Overall, L-amino acids inhibited PhoA mineralization in a concentration-independent manner. The inhibitory effect of neutral amino acids serine (L-Ser) and tyrosine (L-Tyr) was significantly higher than that of basic amino acids arginine (L-Arg), lysine (L-Lys), and histidine (L-His). All the five amino acids can inhibit PhoA mineralization of AMP, with the highest inhibition rate observed for L-Tyr (23.77%), the lowest—for L-Arg (1.54%). Compared with other L-amino acids, L-Tyr has the highest G6P and BGP mineralization inhibition rate, with the average inhibition rates of 12.89% and 11.65%, respectively. This study provides meaningful information to better understand PhoA mineralization.

The role of three‐tandem Pho Boxes in the control of the C‐P lyase operon in a thermophilic cyanobacterium

Cyanobacteria have an inherited advantage in phosphonate phytoremediation. However, studies on phosphonate metabolism in cyanobacteria are rare and mostly focus on physiology and ecology. Here, C-P lyase gene cluster regulation in an undomesticated thermophilic Synechococcus OS-B' was examined in Synechocystis sp. PCC6803, a unicellular cyanobacterial model. Phylogenetic and cluster synteny analysis of C-P lyase genes revealed a closer relationship between Syn OS-B' and Thermus thermophilus, than with other cyanobacteria. Pho boxes were identified in the 5'-end-flanking region of the C-P lyase gene cluster, through which the downstream gene expression was regulated in a phosphate concentration-dependent manner. Unexpectedly, the phosphate concentration that thoroughly inhibited Pho boxes was almost two orders of magnitude higher than that of any natural or anthropogenic wastewater reported so far. The Pho boxes mediated regulation was achieved through the Pho regulon two-component system, and the absence of either SphS or SphR ablated the cell's ability to sense ambient phosphate changes. The three tandems of Pho boxes maintained inequivalent roles, of which the third tandem was not essential; however, it played a role in adjusting Pho boxes response in both positive and negative manner under phosphorus limitation.

Inactivation of phosphate regulator (SphU) in cyanobacterium Synechocystis sp. 6803 directly induced acetyl phosphate pathway leading to enhanced PHB level under nitrogen-sufficient condition

Poly-β-hydroxybutyrate (PHB) is a biodegradable and biocompatible polymer that has potential in the fields of environmental, agricultural, and biomedical sciences. Cyanobacteria are considered an excellent source of PHB by bioconversion of CO2. This study aimed to prolong PHB production under nitrogen-sufficient condition in the model cyanobacterium Synechocystis sp. PCC 6803. Interestingly, the lack of phosphate regulator (SphU) enabled the mutant strain (ΔSphU) to have the ability to accumulate phosphate with higher expression of Pho regulon. When strain ΔSphU was cultured in nitrogen complete medium for 14 days, the PHB granules were more extensively accumulated in the ΔSphU strain than in the wild type. Photosynthesis activity slightly increased in ΔSphU strain, with no significant difference in chlorophyll a content between wild-type and ΔSphU strain in nitrogen-containing medium, indicating that the higher PHB content (14.57% (w/w) cell dry weight) was not influent of chlorosis. The RT-qPCR analysis revealed that genes involved in PHB biosynthesis and acetyl phosphate pathway were more upregulated in ΔSphU strain. Moreover, the level of acetate production in ΔSphU cells was higher than that in the wild type, suggesting that the deletion of the phosphate regulator could directly induce PHB metabolism by activation of the acetyl phosphate pathway. This research provides better understanding of PHB production regulation in cyanobacteria which are a promising hosts for industrial production of biodegradable plastics.

The Phosin PptA Plays a Negative Role in the Regulation of Antibiotic Production in Streptomyces lividans.

In Streptomyces, antibiotic biosynthesis is triggered in phosphate limitation that is usually correlated with energetic stress. Polyphosphates constitute an important reservoir of phosphate and energy and a better understanding of their role in the regulation of antibiotic biosynthesis is of crucial importance. We previously characterized a gene, SLI_4384/ppk, encoding a polyphosphate kinase, whose disruption greatly enhanced the weak antibiotic production of Streptomyces lividans. In the condition of energetic stress, Ppk utilizes polyP as phosphate and energy donor, to generate ATP from ADP. In this paper, we established that ppk is co-transcribed with its two downstream genes, SLI_4383, encoding a phosin called PptA possessing a CHAD domain constituting a polyphosphate binding module and SLI_4382 encoding a nudix hydrolase. The expression of the ppk/pptA/SLI_4382 operon was shown to be under the positive control of the two-component system PhoR/PhoP and thus mainly expressed in condition of phosphate limitation. However, pptA and SLI_4382 can also be transcribed alone from their own promoter. The deletion of pptA resulted into earlier and stronger actinorhodin production and lower lipid content than the disruption of ppk, whereas the deletion of SLI_4382 had no obvious phenotypical consequences. The disruption of ppk was shown to have a polar effect on the expression of pptA, suggesting that the phenotype of the ppk mutant might be linked, at least in part, to the weak expression of pptA in this strain. Interestingly, the expression of phoR/phoP and that of the genes of the pho regulon involved in phosphate supply or saving were strongly up-regulated in pptA and ppk mutants, revealing that both mutants suffer from phosphate stress. Considering the presence of a polyphosphate binding module in PptA, but absence of similarities between PptA and known exo-polyphosphatases, we proposed that PptA constitutes an accessory factor for exopolyphosphatases or general phosphatases involved in the degradation of polyphosphates into phosphate.

A genetic screen for suppressors of hyper-repression of the fission yeast PHO regulon by Pol2 CTD mutation T4A implicates inositol 1-pyrophosphates as agonists of precocious lncRNA transcription termination.

Fission yeast phosphate homeostasis genes are repressed in phosphate-rich medium by transcription of upstream lncRNAs that interferes with activation of the flanking mRNA promoters. lncRNA control of PHO gene expression is influenced by the Thr4 phospho-site in the RNA polymerase II CTD and the 3′ processing/termination factors CPF and Rhn1, mutations of which result in hyper-repression of the PHO regulon. Here, we performed a forward genetic screen for mutations that de-repress Pho1 acid phosphatase expression in CTD-T4A cells. Sequencing of 18 independent STF (Suppressor of Threonine Four) isolates revealed, in every case, a mutation in the C-terminal pyrophosphatase domain of Asp1, a bifunctional inositol pyrophosphate (IPP) kinase/pyrophosphatase that interconverts 5-IP7 and 1,5-IP8. Focused characterization of two STF strains identified 51 coding genes coordinately upregulated vis-à-vis the parental T4A strain, including all three PHO regulon genes (pho1, pho84, tgp1). Whereas these STF alleles—asp1-386(Stop) and asp1-493(Stop)—were lethal in a wild-type CTD background, they were viable in combination with mutations in CPF and Rhn1, in which context Pho1 was also de-repressed. Our findings implicate Asp1 pyrophosphatase in constraining 1,5-IP8 or 1-IP7 synthesis by Asp1 kinase, without which 1-IPPs can accumulate to toxic levels that elicit precocious termination by CPF/Rhn1.