Polyphosphate Kinase Research Articles

TNP Analogues Inhibit the Virulence Promoting IP3-4 Kinase Arg1 in the Fungal Pathogen Cryptococcus neoformans.

New antifungals with unique modes of action are urgently needed to treat the increasing global burden of invasive fungal infections. The fungal inositol polyphosphate kinase (IPK) pathway, comprised of IPKs that convert IP3 to IP8, provides a promising new target due to its impact on multiple, critical cellular functions and, unlike in mammalian cells, its lack of redundancy. Nearly all IPKs in the fungal pathway are essential for virulence, with IP3-4 kinase (IP3-4K) the most critical. The dibenzylaminopurine compound, N2-(m-trifluorobenzylamino)-N6-(p-nitrobenzylamino)purine (TNP), is a commercially available inhibitor of mammalian IPKs. The ability of TNP to be adapted as an inhibitor of fungal IP3-4K has not been investigated. We purified IP3-4K from the human pathogens, Cryptococcus neoformans and Candida albicans, and optimised enzyme and surface plasmon resonance (SPR) assays to determine the half inhibitory concentration (IC50) and binding affinity (KD), respectively, of TNP and 38 analogues. A novel chemical route was developed to efficiently prepare TNP analogues. TNP and its analogues demonstrated inhibition of recombinant IP3-4K from C. neoformans (CnArg1) at low µM IC50s, but not IP3-4K from C. albicans (CaIpk2) and many analogues exhibited selectivity for CnArg1 over the human equivalent, HsIPMK. Our results provide a foundation for improving potency and selectivity of the TNP series for fungal IP3-4K.

Make or break: the thermodynamic equilibrium of polyphosphate kinase-catalysed reactions.

Polyphosphate kinases (PPKs) have become popular biocatalysts for nucleotide 5'-triphosphate (NTP) synthesis and regeneration. Two unrelated families are described: PPK1 and PPK2. They are structurally unrelated and use different catalytic mechanisms. PPK1 enzymes prefer the usage of adenosine 5'-triphosphate (ATP) for polyphosphate (polyP) synthesis while PPK2 enzymes favour the reverse reaction. With the emerging use of PPK enzymes in biosynthesis, a deeper understanding of the enzymes and their thermodynamic reaction course is of need, especially in comparison to other kinases. Here, we tested four PPKs from different organisms under the same conditions without any coupling reactions. In comparison to other kinases using phosphate donors with comparably higher phosphate transfer potentials that are characterised by reaction yields close to full conversion, the PPK-catalysed reaction reaches an equilibrium in which about 30% ADP is left. These results were obtained for PPK1 and PPK2 enzymes, and are supported by theoretical data on the basic reaction. At high concentrations of substrate, the different kinetic preferences of PPK1 and PPK2 can be observed. The implications of these results for the application of PPKs in chemical synthesis and as enzymes for ATP regeneration systems are discussed.

Dictyostelium discoideum cells retain nutrients when the cells are about to outgrow their food source.

Dictyostelium discoideum is a unicellular eukaryote that eats bacteria, and eventually outgrows the bacteria. D. discoideum cells accumulate extracellular polyphosphate (polyP), and the polyP concentration increases as the local cell density increases. At high cell densities, the correspondingly high extracellular polyP concentrations allow cells to sense that they are about to outgrow their food supply and starve, causing the D. discoideum cells to inhibit their proliferation. In this report, we show that high extracellular polyP inhibits exocytosis of undigested or partially digested nutrients. PolyP decreases plasma membrane recycling and apparent cell membrane fluidity, and this requires the G protein-coupled polyP receptor GrlD, the polyphosphate kinase Ppk1 and the inositol hexakisphosphate kinase I6kA. PolyP alters protein contents in detergent-insoluble crude cytoskeletons, but does not significantly affect random cell motility, cell speed or F-actin levels. Together, these data suggest that D. discoideum cells use polyP as a signal to sense their local cell density and reduce cell membrane fluidity and membrane recycling, perhaps as a mechanism to retain ingested food when the cells are about to starve. This article has an associated First Person interview with the first author of the paper.

Polyphosphate Kinases Phosphorylate Thiamine Phosphates

Polyphosphate kinases (PPKs) catalyze the reversible transfer of the γ-phosphate moiety of ATP (or of another nucleoside triphosphate) to a growing chain of polyphosphate (polyP). In this study, we describe that PPKs of various sources are additionally able to phosphorylate thiamine diphosphate (ThP2) to produce thiamine triphosphate (ThP3) and even thiamine tetraphosphate in vitro using polyP as phosphate donor. Furthermore, all tested PPK2s, but not PPK1s, were able to phosphorylate thiamine monophosphate (ThP1) to ThP2 and ThP3 although at low efficiency. The predicted masses and identities of the mono- and oligo-phosphorylated thiamine metabolites were identified by high-performance liquid chromatography tandem mass spectrometry. Moreover, the biological activity of ThP2, that was synthesized by phosphorylation of ThP1 with polyP and PPK, as a cofactor of ThP2-dependent enzymes (here transketolase TktA from Escherichia coli) was confirmed in a coupled enzyme assay. Our study shows that PPKs are promiscuous enzymes in vitro that could be involved in the formation of a variety of phosphorylated metabolites in vivo.

Enzymatic stoichiometry reveals phosphorus limitation-induced changes in the soil bacterial communities and element cycling: Evidence from a long-term field experiment

Soil microbial growth and activity are generally limited by availability of resources such carbon (C), nitrogen (N), or phosphorus (P) in terrestrial ecosystems. However, how soil microbial response to resource limitation in intensive agricultural ecosystems is unclear. Four treatments, namely no fertiliser (CK), chemical fertiliser (NPK), only N fertiliser (N), and organic fertiliser with chemical fertiliser (MNPK), were selected to investigate the effects of different fertiliser practices on the pattern and degree of the limitation of soil microorganisms by elemental availability. The enzyme stoichiometry results indicated that under CK, NPK, and N fertiliser treatments, the soil microbial community as a whole was limited by the availability of C and P. N fertiliser application alone (N) considerably increased the limitation degree of P availability by soil microorganisms, which was caused by the increase in soil N/P. The increased microbial P limitation significantly increased the relative abundance of copiotrophic taxa (Actinobacteria and Sphingomonas) that use labile carbonaceous compounds (such as starch), whereas it significantly decreased the relative abundance of oligotrophic taxa (Acidobacteria and RB41) that use recalcitrant carbonaceous compounds (such as lignin, pectin, and hemicellulose) and important ratios of the microbial community structure (the ratio of fungi/bacteria and the ratio of gram-positive/negative bacteria). Changes in the microbial community structure may be unfavourable for the increase in soil organic matter originating from microorganisms (such as fungi). As per the partial least squares path modelling, N and P cycling was directly affected by microbial P limitation. Linear regression analysis further indicated that with an increase in P limitation, the abundance of functional genes related to N cycling (N fixation, nitrification, and denitrification), P capture (phosphatase and 3-phytase), and P retention (polyphosphate kinase) increased significantly. These results revealed that microorganisms in a P-limited environment adjust their abundance of functional genes to absorb and retain insufficient element (P) while accelerating the turnover of the excess element (N). This study revealed the status and extent of the resource limitation for soil microbial utilization under different long-term fertilisation regimes and indicated that soil bacterial communities respond to resource limitation by adjusting their community structure and element cycling.

Structural insight into substrate binding of Acinetobacter baumannii polyphosphate-AMP phosphotransferase (PPK2), a novel drug target

Acinetobacter baumannii is an opportunistic pathogen known for high morbidity and mortality. It causes life-threatening infections, such as ventilator-associated pneumonia (VAP), bacteremia, meningitis, wound and urinary tract infections (UTI). Increase in carbapenem resistance exhibited by A. baumannii has accentuated the need for novel targets for effective treatment. Despite the pronounced relevance of PPK2 as a pathogenicity determinant in several pathogens, it has not been explored as a drug target in A. baumannii. The present study was piloted to investigate the substrate binding by A. baumannii PPK2 (AbPPK2), a two-domain Class II polyphosphate kinase 2. A homology model of AbPPK2 was developed and validated for molecular docking of ATP and ADP in the predicted binding pocket. Further analysis of AbPPK2 revealed a set of common residues in the catalytic cleft interacting with ATP and ADP which would be useful for the screening of inhibitors against A. baumannii.

Serratia nevei 9rpt1, a Potential Microorganism for Phosphorus Recovery

Aims: Phosphorus (P) is an essential element for life, finite and irreplaceable. Its constant exploration on a global scale has motivated frequent alerts regarding an eventual crisis due to the shortage of this nutrient. However, it is possible to recycle it and reintroduce it into the ecological cycle. One viable alternative is the microbial recovery of phosphate.
 Study design: This study is based on systematic bioprospection of bacteria in phosphate-deficient Amazon regions.
 Place and Duration of Study: Bacteria were isolated from black water samples, collected in the Rio Pretinho, located at Serra do Aracá, Barcelos, Amazonas, Brazil, from January to July 2019.
 Methodology: Microbial isolation was performed in Luria Bertani agar medium. For the genomic study, the isolate with the best performance in the phosphate uptake test was chosen. The WGS was carried out in a Illumina HiSeq 2500 System. The assembly of the draft genome was carried out with the SPAdes and the annotation by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).
 Results: Serratia nevei 9rpt1 recovers 90% of the phosphate available in the culture medium. Its draft genome comprises 5.4 MB, the GC content is 59.52% and 4,922 coding sequences were identified, among these, two pst operons: one complete, containing the five pst genes and one missing pstS, pstC and phoU genes.
 Conclusion: Serratia nevei 9rpt1, isolated from an Amazonian environment poor in phosphate, is very efficient to uptake this nutrient in a Pi starvation condition. The genomic findings revealed this strain has an additional high affinity Pi uptake pst system containing the ATP-binding protein PstB, the canonical permease PstA, a putative permease other than PstC, upstream of the PstA and two essential enzymes in the polyphosphate metabolism: polyphosphate kinase 1 and exopolyphosphatase.

Ddp1 Cooperates with Ppx1 to Counter a Stress Response Initiated by Nonvacuolar Polyphosphate.

ABSTRACTIn diverse cells from bacterial to mammalian species, inorganic phosphate is stored in long chains called polyphosphate (polyP). These nearly universal polymers, ranging from three to thousands of phosphate moieties in length, are associated with molecular functions, including energy homeostasis, protein folding, and cell signaling. In many cell types, polyphosphate is concentrated in subcellular compartments or organelles. In the budding yeast Saccharomyces cerevisiae, polyP synthesis by the membrane-bound vacuolar transporter chaperone (VTC) complex is coupled to its translocation into the lumen of the vacuole, a lysosome-like organelle, where it is stored at high concentrations. In contrast, the ectopic expression of the bacterial polyphosphate kinase (PPK) results in the toxic accumulation of polyP outside the vacuole. In this study, we used label-free mass spectrometry to investigate the mechanisms underlying this toxicity. We find that PPK expression results in the activation of a stress response mediated in part by the Hog1 and Yak1 kinases and the Msn2/Msn4 transcription factors as well as by changes in protein kinase A (PKA) activity. This response is countered by the combined action of the Ddp1 and Ppx1 polyphosphatases that function together to counter polyP accumulation and downstream toxicity. In contrast, the ectopic expression of previously proposed mammalian polyphosphatases did not impact PPK-mediated toxicity in this model, suggesting either that these enzymes do not function directly as polyphosphatases in vivo or that they require cofactors unique to higher eukaryotes. Our work provides insight into why polyP accumulation outside lysosome-like organelles is toxic. Furthermore, it serves as a resource for exploring how polyP may impact conserved biological processes at a molecular level.

Polyphosphate Kinase Is Required for the Processes of Virulence and Persistence in Acinetobacter baumannii.

ABSTRACTAcinetobacter baumannii, one of the most successful bacteria causing severe nosocomial infection, was identified as a top-priority pathogen by the WHO. Thus, genetic manipulations to clarify the potential targets for fighting A. baumannii resistance and virulence are vital. Polyphosphate (polyP) kinase (PPK) is conserved in nearly all bacteria and is responsible for polyP formation, which is associated with bacterial pathogenicity and antibiotic resistance. In this study, ppk1-deficient (Δppk1::Apr), ppk1-complemented (Δppk1::Apr/PJL02-ppk1), and wild-type strains of A. baumannii ATCC 17978 were used to determine the influence of PPK1 on A. baumannii virulence and persistence mainly by polyP quantification, surface motility, biofilm formation, and bacterial persistence assays. Our work found that PPK1 is indispensable for polyP formation in vivo and that the motility of the PPK1-deficient strain was significantly impaired due to the lack of a pilus-like structure typically present compared with the complemented and wild-type strains. The deficiency of PPK1 also inhibited the biofilm formation of A. baumannii and decreased bacterial persistence under stimuli of high-concentration ampicillin (Amp) treatment, H2O2 stress, heat shock, and starvation stress. Furthermore, ppk1-deficient bacterium-infected mice showed a significantly reduced bacterial load and a decreased inflammatory response. However, complementation with PPK1 effectively rescued the impaired virulence and persistence of ppk1-deficient A. baumannii. In addition, metabonomic analysis revealed that PPK1 was associated with glycerophospholipid metabolism and fatty acid biosynthesis. Taken together, our results suggest that targeting PPK1 to control A. baumannii pathogenicity and persistence is a feasible strategy to fight this pathogen.IMPORTANCE A. baumannii was identified as a top-priority pathogen by the WHO due to its antibiotic resistance. Meanwhile, the pathogenicity of A. baumannii mediated by several vital virulence factors also cannot be ignored. Here, the role of PPK1 in A. baumannii was also explored. We found that the motility ability and biofilm formation of a PPK1-deficient strain were significantly impaired. Furthermore, PPK1 was essential for its persistence maintenance to resist stimuli of high-concentration Amp treatment, H2O2 stress, heat shock, and starvation stress. Metabonomic analysis revealed that PPK1 was associated with glycerophospholipid metabolism and fatty acid biosynthesis. In addition, ppk1-deficient bacterium-infected mice showed significantly reduced bacterial loads and a decreased inflammatory responses in vivo. Together, our results suggest that PPK1 is vital for A. baumannii pathogenicity and persistence.

Metagenomics Unveils Microbial Diversity and Their Biogeochemical Roles in Water and Sediment of Thermokarst Lakes in the Yellow River Source Area.

Thermokarst lakes have long been recognized as biogeochemical hotspots, especially as sources of greenhouse gases. On the Qinghai-Tibet Plateau, thermokarst lakes are experiencing extensive changes due to faster warming. For a deep understanding of internal lake biogeochemical processes, we applied metagenomic analyses to investigate the microbial diversity and their biogeochemical roles in sediment and water of thermokarst lakes in the Yellow River Source Area (YRSA). Sediment microbial communities (SMCs) had lower species and gene richness than water microbial communities (WMCs). Bacteria were the most abundant component in both SMCs and WMCs with significantly different abundant genera. The functional analyses showed that both SMCs and WMCs had low potential in methanogenesis but strong in aerobic respiration, nitrogen assimilation, exopolyphosphatase, glycerophosphodiester phosphodiesterases, and polyphosphate kinase. Moreover, SMCs were enriched in genes involved in anaerobic carbon fixation, aerobic carbon fixation, fermentation, most nitrogen metabolism pathways, dissimilatory sulfate reduction, sulfide oxidation, polysulfide reduction, 2-phosphonopropionate transporter, and phosphate regulation. WMCs were enriched in genes involved in assimilatory sulfate reduction, sulfur mineralization, phosphonoacetate hydrolase, and phosphonate transport. Functional potentials suggest the differences of greenhouse gas emission, nutrient cycling, and living strategies between SMCs and WMCs. This study provides insight into the main biogeochemical processes and their properties in thermokarst lakes in YRSA, improving our understanding of the roles and fates of these lakes in a warming world.

Phytic acid contributes to the phosphate-zinc signaling crosstalk in Arabidopsis

Inorganic phosphate (Pi) and zinc (Zn) are two essential nutrients for plant growth. Crosstalk between these two elements to control their uptake and homeostasis in plants has been previously demonstrated. However, the signaling molecule(s) required for the mechanisms underlying this interaction remain unknown.Phytic acid (PA), the main P storage form in plants, serves also as a signalling molecule in processes controlling plant growth and development as well as responses to different stimuli. In this study, we investigated the involvement of PA in the control of Zn-Pi homeostasis interaction in Arabidopsis. For this purpose, we used two classes of low phytic acid (lpa) lines: the inositol polyphosphate kinase 1 gene (ipk1-1) mutant and two transgenic lines expressing the bacterial phytase PHY-US417. The transgenic lines exhibit an enhanced root growth under Zn-deficiency compared to wild type (WT) and ipk1-1. In addition, higher Pi and Zn contents were detected in the lpa lines under standard and also deficient conditions (-Pi and -Zn). However, the activation of shoot Pi accumulation which occurs in WT in response to Zn depletion was not observed in the lpa lines. Finally, we noticed that the changes in Pi and Zn accumulation seem to be correlated with a tight regulation of Pi and Zn transporters in the lpa lines. All these findings underline a regulatory role of PA in the control of the Zn-Pi crosstalk but also open the door to possible involvement of additional unknown signaling molecules in this process.

Tagging Polyphosphate Kinase Alters Enzymatic Activity and Affect Stress Recovery in E. coli

Many bacterial species, including Escherichia coli (E. coli) utilize the enzyme polyphosphate kinase (PPK) to synthesize polyphosphate (polyP) in response to biological stress. Multiple studies have shown that impairing PPK activity impairs bacterial pathogenicity and survival, which has made it a target for therapeutic development. Unfortunately, PPK regulation is poorly understood. To address this, we previously described a series of mutations in E. coli ppk, termed ppk*, that result in high polyP accumulation in vivo. However, the specific activity of PPK* enzyme in vitro, when purified using a C‐term 6X His‐tag (PPK*‐HT), was comparable to purified WT enzyme (PPK‐HT).PPK activity is tied to its oligomeric state, with the dimeric form synthesizing polyP. To determine if the ppk* mutation altered oligomerization, we tested PPK‐HT and PPK*‐HT enzymes in an analytical ultracentrifuge. We found that the PPK‐HT enzyme was mostly dimeric but had a small monomeric population. Conversely, the PPK*‐HT enzyme was exclusively dimeric. This suggested that the ppk* mutation may affect oligomerization, but the His‐tag masked any effect on activity.We next hypothesized that the C‐term His tag alters the in vitro activity. To test this, we compared the specific activities of PPK‐HT and PPK*‐HT to that of PPK purified using a smaller C‐term C tag (PPK‐CT) and PPK enzyme purified with no tags (PPK‐NT). PPK*‐HT and PPK‐HT had comparable activities in vitro but were significantly more active than the untagged enzyme. PPK‐CT and PPK‐NT had comparable activities. This suggests that the C‐tag does not alter PPK activity while the His‐tag does.We next tested the purified enzymes’ sensitivity to substrate by testing PPK activity in 6mM and 20mM ATP. We found that the His‐tag increased the enzyme’s activity in the higher ATP concentrations. Comparing the 20mM and 6mM samples, the His‐tag enzymes showed an increase in activity of ~3.5 fold compared to their 6mM conditions. Conversely, the PPK‐CT and PPK‐UT only showed a 1.4‐ and 1.9‐ fold increase in activity, respectively.PolyP synthesis impairs bacterial growth and replication. We hypothesized that a more active PPK would impair recovery following a prolonged stationary phase. We developed strains carrying one of four expression vectors: an empty vector control (EV), PPK‐HT, PPK‐CT, or native PPK. Using these four strains, we developed a bacterial growth recovery curve assay. We found that while the PPK‐CT and untagged PPK strains had comparable recovery and growth rates, the PPK‐HT strain had a slower recovery rate resulting in a later shift into log phase.Taken together, our results have significant implications for polyP biology. We have shown that the standard purification method produces an enzyme with altered substrate sensitivities, making it a poorer candidate for drug development studies. C‐tag enzyme is comparable to untagged enzyme, making it a reliable candidate for future studies. Finally, the in vitro differences produce an in vivo phenotype, making it easy to test the effect of the tags in living systems.

Bacterial communities in surface and basal ice of a glacier terminus in the headwaters of Yangtze River on the Qinghai\u2013Tibet Plateau

BackgroundOn the front lines of climate change, glacier termini play crucial roles in linking glaciers and downstream ecosystems during glacier retreat. However, we lack a clear understanding of biological processes that occur in surface and basal ice at glacier termini.MethodsHere, we studied the bacterial communities in surface ice and basal ice (the bottom layer) of a glacier terminus in the Yangtze River Source, Qinghai–Tibet Plateau.ResultsSurface and basal ice harbored distinct bacterial communities but shared some core taxa. Surface ice communities had a higher α-diversity than those in basal ice and were dominated by Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Cyanobacteria while basal ice was dominated by Firmicutes and Proteobacteria. The bacterial communities were also substantially different in functional potential. Genes associated with functional categories of cellular processes and metabolism were significantly enriched in surface ice, while genes connected to environmental information processing were enriched in basal ice. In terms of biogeochemical cycles of carbon, nitrogen, phosphorus, and sulfur, bacterial communities in surface ice were enriched for genes connected to aerobic carbon fixation, aerobic respiration, denitrification, nitrogen assimilation, nitrogen mineralization, sulfur mineralization, alkaline phosphatase, and polyphosphate kinase. In contrast, bacterial communities in basal ice were enriched for genes involved in anaerobic carbon fixation, fermentation, nitrate reduction, 2-aminoethylphosphonic acid pathway, G3P transporter, glycerophosphodiester phosphodiesterase, and exopolyphosphatase. Structural equation modeling showed that total nitrogen and environmental carbon:phosphorus were positively while environmental nitrogen:phosphorus was negatively associated with taxonomic β-diversity which itself was strongly associated with functional β-diversity of bacterial communities.ConclusionsThis study furthers our understanding of biogeochemical cycling of the mountain cryosphere by revealing the genetic potential of the bacterial communities in surface and basal ice at the glacier terminus, providing new insights into glacial ecology as well as the influences of glacier retreat on downstream systems.

Functional Genetic Diversity and Plant Growth Promoting Potential of Polyphosphate Accumulating Bacteria in Soil.

ABSTRACTPolyphosphate (polyP) accumulation is an important trait of microorganisms. Implication of polyP accumulating bacteria (PAB) in enhanced biological phosphate removal, heavy metal sequestration, and dissolution of dental enamel is well studied. Phosphorous (P) accumulated within microbial biomass also regulates labile P in soil; however, abundance and diversity of the PAB in soil is still unexplored. Present study investigated the genetic and functional diversity of PAB in rhizosphere soil. Here, we report the abundance of Pseudomonas spp. as high PAB in soil, suggesting their contribution to global P cycling. Additional subset analysis of functional genes i.e., polyphosphate kinase (ppk) and exopolyphosphatase (ppx) in all PAB, indicates their significance in bacterial growth and metabolism. Distribution of functional genes in phylogenetic tree represent a more biologically realistic discrimination for the two genes. Distribution of ppx gene disclosed its phylogenetic conservation at species level, however, clustering of ppk gene of similar species in different clades illustrated its environmental condition mediated modifications. Selected PAB showed tolerance to abiotic stress and strong correlation with plant growth promotary (PGP) traits viz. phosphate solubilization, auxin and siderophore production. Interaction of PAB with A. thaliana enhanced the growth and phosphate status of the plant under salinity stress, suggestive of their importance in P cycling and stress alleviation.IMPORTANCE Study discovered the abundance of Pseudomonas genera as a high phosphate accumulator in soil. The presence of functional genes (polyphosphate kinase [ppk] and exopolyphosphatase [ppx]) in all PAB depicts their importance in polyphosphate metabolism in bacteria. Genetic and functional diversity reveals conservation of the ppx gene at species level. Furthermore, we found a positive correlation between PAB and plant growth promotary traits, stress tolerance, and salinity stress alleviation in A. thaliana.

A cocktail of protein engineering strategies: Breaking the enzyme bottleneck one by one for high UTP production in vitro.

The pyrimidine metabolic pathway is tightly regulated in microorganisms, allowing limited success in metabolic engineering for the production of pathway-related substances. Here, we constructed a four-enzyme coupled system for the in vitro production of uridine triphosphate (UTP). The enzymes used include nucleoside kinase, uridylate kinase, nucleoside diphosphate kinase, and polyphosphate kinase for energy regeneration. All these enzymes are derived from extremophiles. To increase the total and unit time yield of the product, three enzymes other than polyphosphate kinase were modified separately by multiple protein engineering strategies. A nucleoside kinase variant with increased specific activity from 2.7 to 36.5 U/mg, a uridylate kinase variant (specific activity of 37.1 U/mg) with a 5.2-fold increase in thermostability, and a nucleoside diphosphate kinase variant with a 2-fold increase in a specific activity to over 900 U/mg were obtained, respectively. The reaction conditions of the coupled system were further optimized, and a two-stage method was taken to avoid the problem of enzymatic pH adaptation mismatch. Under optimal conditions, this system can produce more than 65 mM UTP (31.5 g/L) in 3.0 h. The substrate conversion rate exceeded 98% and the maximum UTP productivity reached 40 mM/h.

Integration of Microbial Transformation Mechanism of Polyphosphate Accumulation and Sulfur Cycle in Subtropical Marine Mangrove Ecosystems with Spartina alterniflora Invasion.

Excessive phosphorus can lead to eutrophication in marine and coastal ecosystems. Sulfur metabolism-associated microorganisms stimulate biological phosphorous removal. However, the integrating co-biotransformation mechanism of phosphorus and sulfur in subtropical marine mangrove ecosystems with Spartina alterniflora invasion is poorly understood. In this study, an ecological model of the coupling biotransformation of sulfur and phosphorus is constructed using metagenomic analysis and quantitative polymerase chain reaction strategies. Phylogenetic analysis profiling, a distinctive microbiome with high frequencies of Gammaproteobacteria and Deltaproteobacteria, appears to be an adaptive characteristic of microbial structures in subtropical mangrove ecosystems. Functional analysis reveals that the levels of sulfate reduction, sulfur oxidation, and poly-phosphate (Poly-P) aggregation decrease with increasing depth. However, at depths of 25-50cm in the mangrove ecosystems with S. alterniflora invasion, the abundance of sulfate reduction genes, sulfur oxidation genes, and polyphosphate kinase (ppk) significantly increased. A strong positive correlation was found among ppk, sulfate reduction, sulfur oxidation, and sulfur metabolizing microorganisms, and the content of sulfide was significantly and positively correlated with the abundance of ppk. Further microbial identification suggested that Desulfobacterales, Anaerolineales, and Chromatiales potentially drove the coupling biotransformation of phosphorus and sulfur cycling. In particular, Desulfobacterales exhibited dominance in the microbial community structure. Our findings provided insights into the simultaneous co-biotransformation of phosphorus and sulfur bioconversions in subtropical marine mangrove ecosystems with S. alterniflora invasion.

Rational Design to Enhance Enzyme Activity for the Establishment of an Enzyme–Inorganic Hybrid Nanoflower Co-Immobilization System for Efficient Nucleotide Production

Increasing yields while reducing costs is one of the ultimate pursuits of industrial production. To achieve this goal in the enzymatic production of multiple nucleotides, in this study, a co-immobilized polyphosphate kinase-nucleoside kinase hybridized nanoflower system (PPK@NK) was constructed. To improve the productivity, the nucleoside kinase (NK) used was rationally designed, and a variant with significantly increased activity compared to the wild type was obtained. The polyphosphate kinase (PPK) and NK could be sequentially adsorbed on the surface of hybrid nanoflowers at room temperature (25 °C) through the interaction of Cu2+ and proteins without any other chemical pretreatment. The optimal preparation conditions and reaction parameters of PPK@NK hybrid nanoflowers were investigated. Under optimal reaction conditions, the newly prepared co-immobilization system could catalyze the conversion of 100 mM uridine, cytidine, and inosine to the corresponding nucleotides completely within 4 h and could be reused at least six times. The storage stability of the co-immobilized system was more than 2-fold higher than that of the free enzyme, and there was no significant difference in thermostability. PPK@NK hybridized nanoflowers have properties such as easy preparation and storage and low cost, indicating their suitability for the efficient production of nucleotides.

Phosphate Starvation by Energy Metabolism Disturbance in Candida albicansvip1Δ/Δ Induces Lipid Droplet Accumulation and Cell Membrane Damage.

Phosphorus in the form of phosphate (Pi) is an essential element for metabolic processes, including lipid metabolism. In yeast, the inositol polyphosphate kinase vip1 mediated synthesis of inositol heptakisphosphate (IP7) regulates the phosphate-responsive (PHO) signaling pathway, which plays an important role in response to Pi stress. The role of vip1 in Pi stress and lipid metabolism of Candida albicans has not yet been studied. We found that when vip1Δ/Δ was grown in glucose medium, if Pi was supplemented in the medium or mitochondrial Pi transporter was overexpressed in the strain, the lipid droplet (LD) content was reduced and membrane damage was alleviated. However, further studies showed that neither the addition of Pi nor the overexpression of the Pi transporter affected the energy balance of vip1Δ/Δ. In addition, the LD content of vip1Δ/Δ grown in Pi limitation medium PNMC was lower than that grown in SC, and the metabolic activity of vip1Δ/Δ grown in PNMC was also lower than that grown in SC medium. This suggests that the increase in Pi demand by a high energy metabolic rate is the cause of LD accumulation in vip1Δ/Δ. In addition, in the vip1Δ/Δ strains, the core transcription factor PHO4 in the PHO pathway was transported to the vacuole and degraded, which reduced the pathway activity. However, this does not mean that knocking out vip1 completely blocks the activation of the PHO pathway, because the LD content of vip1Δ/Δ grown in the medium with β-glycerol phosphate as the Pi source was significantly reduced. In summary, the increased Pi demand and the decreased PHO pathway activity in vip1Δ/Δ ultimately lead to LD accumulation and cell membrane damage.

Polyphosphate Kinase 2 (PPK2) Enzymes: Structure, Function, and Roles in Bacterial Physiology and Virulence.

Inorganic polyphosphate (polyP) has been implicated in an astonishing array of biological functions, ranging from phosphorus storage to molecular chaperone activity to bacterial virulence. In bacteria, polyP is synthesized by polyphosphate kinase (PPK) enzymes, which are broadly subdivided into two families: PPK1 and PPK2. While both enzyme families are capable of catalyzing polyP synthesis, PPK1s preferentially synthesize polyP from nucleoside triphosphates, and PPK2s preferentially consume polyP to phosphorylate nucleoside mono- or diphosphates. Importantly, many pathogenic bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii encode at least one of each PPK1 and PPK2, suggesting these enzymes may be attractive targets for antibacterial drugs. Although the majority of bacterial polyP studies to date have focused on PPK1s, PPK2 enzymes have also begun to emerge as important regulators of bacterial physiology and downstream virulence. In this review, we specifically examine the contributions of PPK2s to bacterial polyP homeostasis. Beginning with a survey of the structures and functions of biochemically characterized PPK2s, we summarize the roles of PPK2s in the bacterial cell, with a particular emphasis on virulence phenotypes. Furthermore, we outline recent progress on developing drugs that inhibit PPK2 enzymes and discuss this strategy as a novel means of combatting bacterial infections.

Insoluble Protein Applications: The Use of Bacterial Inclusion Bodies as Biocatalysts.

Biocatalysis and biotransformations have a broad application in industrial synthetic chemistry. In addition to the whole cell catalysis, purified recombinant enzymes are successfully used for biocatalysis of specific chemical reactions. In this contribution, we report characterization, immobilization, and application of several model target enzymes (D-amino acid oxidase, sialic acid aldolase, maltodextrin phosphorylase, polyphosphate kinase, UDP-glucose pyrophosphorylase) physiologically aggregated within inclusion bodies retaining their biological activity as immobilized biocatalysts.