Ppk Gene Research Articles

Unveiling the potential role of virus-encoded polyphosphate kinases in enhancing phosphorus removal in activated sludge systems

While microbial phosphate removal in activated sludge (AS) systems has been extensively studied, the role of viruses in this process remains largely unexplored. In this study, we identified 149 viral auxiliary metabolic genes associated with phosphorus cycling from 2,510 viral contigs (VCs) derived from AS systems. Notably, polyphosphate kinase 1 (ppk1) and polyphosphate kinase 2 (ppk2) genes, which are primarily responsible for phosphate removal, were found in five unclassified VCs. These genes exhibited conserved protein structures and active catalytic sites, indicating a pivotal role of viruses in enhancing phosphorus removal. Phylogenetic analysis demonstrated a close relationship between viral ppk genes and their bacterial counterparts, suggesting the occurrence of horizontal gene transfer. Furthermore, experimental assays validated that viral ppk genes enhanced host phosphate removal capabilities. VCs carrying ppk genes were observed across diverse ecological and geographical contexts, suggesting their potential to bolster host functions in varied environmental and nutrient settings, spanning natural and engineered systems. These findings uncover a previously underappreciated mechanism by which viruses enhance phosphate removal in wastewater treatment plants. Overall, our study highlights the potential for leveraging virus-encoded genes to improve the efficiency of biological phosphorus removal processes, offering new insights into the microbial ecology of AS systems and the role of viruses in biogeochemical cycling.

Combined effect of polyphosphate kinase and lon protease in Streptomyces coelicolor A3(2) antibiotic production.

The bacterial stringent response is a global regulatory process in which polyphosphate kinase (Ppk) and lon protease are important players. Previous studies have shown that overexpression of the lon gene and deletion of the ppk gene significantly increased actinorhodin production in Streptomyces coelicolor (SCO). In this study, a recombinant SCOΔppk-lon cell, expressing the extra lon gene in Δppk cells, was simulated using a modified in silico (computational) model, ecSco-GEM, and the negative effect of Ppk on actinorhodin production was confirmed. In addition, we identified key enzymes that play a positive role in actinorhodin production. Of these, NADH dehydrogenase/complex-I, beta-ketoacyl-[acyl-carrier-protein] synthase III, glycine cleavage system, and superoxide dismutase were identified as the most significant. By confirming these results with experiments, we have shown that GEMs can be a reliable starting point for in vitro (lab-based) studies of Streptomyces..

Orthophosphate uptake onto woodchips in denitrifying bioreactors is enhanced by anoxic-(anaerobic-)oxic cycling

Woodchips are widely used as a low-cost and renewable organic carbon source for denitrifying biofilms in passive nutrient removal systems. One limitation of wood-based biofiltration systems is their relatively poor removal of phosphorus (P) from subsurface drainage and stormwaters, necessitating the use of additional filter media when co-treatment of nitrogen (N) and P is required. Here, we show that anoxic-oxic cycling of woodchip media, which enhances nitrate (NO3−) removal by increasing the mobilization of organic carbon from wood, also improves orthophosphate (Pi) uptake onto woodchips. Orthophosphate removal rates in flow-through woodchip columns ranged from 0 to 34.9 μg PO43− L−1 h−1 under continuously-saturated (anoxic) conditions, and increased to 17.5 to 71.9 μg PO43− L−1 h−1 in columns undergoing drying-rewetting (oxic-anoxic) cycles. The highest Pi removal efficiencies were observed in the first 20 h after reactors were re-flooded, and were concurrent with maxima in polyphosphate kinase (ppk) gene expression by the polyphosphate accumulating organisms (PAOs) Accumulibacter spp. and Pseudomonas spp. Batch experiments confirmed that anoxic-anaerobic-oxic pre-incubation conditions led to orthophosphate uptake onto woodchips as high as 74.9 ± 0.8 mg PO43−/kg woodchip, and batch tests with autoclaved woodchips demonstrated that Pi uptake was due to biological processes and not adsorption. NO3− removal in batch tests was also greatest under oxic incubation conditions, attributed to greater carbon availability in hypoxic to anoxic zones in woodchip biofilms. While further research is needed to elucidate the mechanisms controlling enhanced Pi uptake by woodchip biofilms under anoxic-(anaerobic-)oxic cycling, these results suggest a role for enhanced Pi uptake by PAOs in a nature-based system for treatment of nonpoint source nutrients.

Isolation, Identification and Survival Strategy of <i>Dietzia maris</i> MX2 Halotolerant Strain from the Yakshinskoe Mineral Salts Deposit

Halophilic and halotolerant microorganisms have a high biotechnological potential. They are producers of biologically active substances, stress-protective agents, hydrolytic enzymes, and are used for environmental bioremediation. At the same time, the characterization of novel halotolerant bacteria and the disclosure of their salt tolerance strategy are topical fundamental problems. In the present work, a new strain MX2 was isolated from the salt well brine of the Yakshinskoe potassium-magnesium salt deposit. The isolate is represented by aerobic gram-positive non-motile bacteria that do not produce spores. The cell morphology varies from cocci to short rods that are capable of producing V-shaped forms. Colonies on the surface of agar nutrient medium were circular with an entire edge and raised center, glistening and orange. Bacteria of strain MX2 are halotolerant microorganisms capable of growing at NaCl concentrations up to 9%. Strain MX2 was sequenced. Its size was estimated at 3747717 b. p., the number of protein-coding genes — 3562. Strain MX2 was identified as belonging to the species Dietzia maris based on analysis of 16S rRNA, gyrB, rpoB, recA, ppk gene sequences and using time-of-flight mass spectrometry (MALDI-TOF-MS). D. maris MX2 has complete metabolic pathways for the synthesis of ectoine, hydroxyectoine, and trehalose, as well as transport systems for ectoine, hydroxyectoine, trehalose, glycerol, glycerol-3-phosphate, L-proline, and glycine-betaine. Thus, to ensure the osmotic balance, D. maris MX2 uses the strategy of accumulating compatible organic solutes.

Macrogenomics reveal the effects of inter-cropping perilla on kiwifruit: impact on inter-root soil microbiota and gene expression of carbon, nitrogen, and phosphorus cycles in kiwifruit.

Intercropping systems can improve soil fertility and health, however, soil microbial communities and functional genes related to carbon, nitrogen and phosphorus cycling under the intercropping system of mesquite and perilla have not been studied. Therefore, in the present study, different planting densities and varieties of Perilla frutescens (L.) Britt and kiwifruit were used for intercropping, and changes in soil microbial communities and carbon, nitrogen, and phosphorus cycling genes in kiwifruit inter-roots under inter-cropping conditions were investigated by macro-genome sequencing technology. The results showed that intercropping with Perill caused a decrease in most soil nutrients, soil enzyme activities, and had a significant impact on the microbial (bacteria and fungi) diversity. Inter-cropping increased the relative abundance of the dominant bacterial phylum "Proteobacteria" and "Actinobacteria" by 47 and 57%, respectively, but decreased the relative abundance of the dominant fungal phylum "Chordata" and "Streptophyta" by 11 and 20%, respectively, in the inter-root soil of kiwifruit, and had a significant impact on the microbial (bacteria and fungi) diversity. In addition, inter-cropping could greatly increase the inter-root soil carbon sequestration (PccA, korA/B/C/D, fhs, and rbcl/s), carbon degradation (abfD), organic nitrogen mineralization (GDH2), denitrification (napA/B, nirB, norB), organic phosphorus mineralization (phop, phn), and inorganic phosphorus solubilization (gcd, ppk) gene abundance. The gene co-occurrence network indicated that soil korB, nirB, and gnd key functional genes for carbon, nitrogen, and phosphorus cycling in kiwifruit inter-root soils and their expression was up-regulated in the inter-cropping group. Structural equation (SEM) further showed that soil total nitrogen, organic matter, total carbon and acid phosphatase had significant effects on microbial diversity (p < 0.05) and soil carbon cycling gene korB and phosphorus cycling gene purH (p < 0.001), while korB and purH had positive effects on kiwifruit quality. In conclusion, intercropping perilla in kiwifruit orchards changed the structure of bacterial and fungal communities in the inter-root soil of kiwifruit, but I believe that intercropping perilla stimulates carbon degradation, leading to carbon emission and serious loss of soil nutrients, and that prolonged intercropping may adversely affect the quality of kiwifruit, and thus its limitations should be noted in future studies.

Effects of phosphorus sources on the transformation of phosphorus forms, microbial community, and functional genes in up-flow anaerobic sludge bed reactor

Considering the removal of pollutants and the resource recovery in wastewater, it is crucial to address the conflict between phosphorus pollution and phosphorus resources during wastewater treatment. This study investigated the transformation law of phosphorus in an up-flow anaerobic sludge bed reactor using different phosphorus source substrates. In addition, metagenomics sequencing was used to analyze microbial community succession and changes in the key functional genes in the reactor. Results showed that when sodium hypophosphite and sodium phosphite were used as the phosphorus sources, the average removal efficiency of total phosphorus was 6.43 % and 5.69 %, respectively. The resulting average phosphine concentrations were 109.76 mg/m3 and 72.77 mg/m3, respectively. Sodium hypophosphite was the more effective phosphorus source for Proteobacteria growth. The ppa, ppk, ppx and gcd genes were all up-regulated when exposed to the two phosphorus sources. On day 90 of the experiment, the relative abundances of these four genes in the reactor with sodium hypophosphite as phosphorus source were 0.0046 %, 0.0025 %, 0.0036 %, and 0.0014 %, respectively. In contrast, in the reactor with sodium phosphite as a phosphorus source, the relative abundances of the ppa, ppk, ppx and gcd genes were 0.0009 %, 0.0005 %, 0.0014 %, and 0.0001 %, respectively. Sodium hypophosphite was more effective than sodium phosphite in promoting the dissolution of inorganic phosphorus in microbial cells.

Refinement of the "Candidatus Accumulibacter" genus based on metagenomic analysis of biological nutrient removal (BNR) pilot-scale plants operated with reduced aeration.

Members of the "Candidatus Accumulibacter" genus are widely studied as key polyphosphate-accumulating organisms (PAOs) in biological nutrient removal (BNR) facilities performing enhanced biological phosphorus removal (EBPR). This diverse lineage includes 18 "Ca. Accumulibacter" species, which have been proposed based on the phylogenetic divergence of the polyphosphate kinase 1 (ppk1) gene and genome-scale comparisons of metagenome-assembled genomes (MAGs). Phylogenetic classification based on the 16S rRNA genetic marker has been difficult to attain because most "Ca. Accumulibacter" MAGs are incomplete and often do not include the rRNA operon. Here, we investigate the "Ca. Accumulibacter" diversity in pilot-scale treatment trains performing BNR under low dissolved oxygen (DO) conditions using genome-resolved metagenomics. Using long-read sequencing, we recovered medium- and high-quality MAGs for 5 of the 18 "Ca. Accumulibacter" species, all with rRNA operons assembled, which allowed a reassessment of the 16S rRNA-based phylogeny of this genus and an analysis of phylogeny based on the 23S rRNA gene. In addition, we recovered a cluster of MAGs that based on 16S rRNA, 23S rRNA, ppk1, and genome-scale phylogenetic analyses do not belong to any of the currently recognized "Ca. Accumulibacter" species for which we propose the new species designation "Ca. Accumulibacter jenkinsii" sp. nov. Relative abundance evaluations of the genus across all pilot plant operations revealed that regardless of the operational mode, "Ca. A. necessarius" and "Ca. A. propinquus" accounted for more than 40% of the "Ca. Accumulibacter" community, whereas the newly proposed "Ca. A. jenkinsii" accounted for about 5% of the "Ca. Accumulibacter" community.IMPORTANCEOne of the main drivers of energy use and operational costs in activated sludge processes is the amount of oxygen provided to enable biological phosphorus and nitrogen removal. Wastewater treatment facilities are increasingly considering reduced aeration to decrease energy consumption, and whereas successful BNR has been demonstrated in systems with minimal aeration, an adequate understanding of the microbial communities that facilitate nutrient removal under these conditions is still lacking. In this study, we used genome-resolved metagenomics to evaluate the diversity of the "Candidatus Accumulibacter" genus in pilot-scale plants operating with minimal aeration. We identified the "Ca. Accumulibacter" species enriched under these conditions, including one novel species for which we propose "Ca. Accumulibacter jenkinsii" sp. nov. as its designation. Furthermore, the MAGs obtained for five additional "Ca. Accumulibacter" species further refine the phylogeny of the "Ca. Accumulibacter" genus and provide new insight into its diversity within unconventional biological nutrient removal systems.

Possible Role of CHAD Proteins in Copper Resistance.

Conserved Histidine Alpha-helical Domain (CHAD) proteins attached to the surface of polyphosphate (PolyP) have been studied in some bacteria and one archaeon. However, the activity of CHAD proteins is unknown beyond their interaction with PolyP granules. By using bioinformatic analysis, we report that several species of the biomining acidophilic bacteria contain orthologs of CHAD proteins with high sequence identity. Furthermore, the gene coding for the CHAD protein is in the same genetic context of the enzyme polyphosphate kinase (PPK), which is in charge of PolyP synthesis. Particularly, the group of ppk and CHAD genes is highly conserved. Metallosphaera sedula and other acidophilic archaea used in biomining also contain CHAD proteins. These archaea show high levels of identity in genes coding for a cluster having the same organization. Amongst these genes are chad and ppx. In general, both biomining bacteria and archaea contain high PolyP levels and are highly resistant to heavy metals. Therefore, the presence of this conserved genetic organization suggests a high relevance for their metabolism. It has been formerly reported that a crystallized CHAD protein contains a copper-binding site. Based on this previous knowledge, in the present report, it was determined that all analyzed CHAD proteins are very conserved at their structural level. In addition, it was found that the lack of YgiF, an Escherichia coli CHAD-containing protein, decreases copper resistance in this bacterium. This phenotype was not only complemented by transforming E. coli with YgiF but also by expressing CHAD from Acidithiobacillus ferrooxidans in it. Interestingly, the strains in which the possible copper-binding sites were mutated were also more metal sensitive. Based on these results, we propose that CHAD proteins are involved in copper resistance in microorganisms. These findings are very interesting and may eventually improve biomining operations in the future.

Assessing the influence of diverse phosphorus sources on bacterial communities and the abundance of phosphorus cycle genes in acidic paddy soils.

The impact of chemical fertilizers on soil microbial communities is well acknowledged. This study assesses the influence of various phosphorus sources on soil bacterial composition, abundance, and Phosphorus Cycle Gene Abundance. Three phosphorus sources (natural phosphate rock, triple super phosphate (TSP), and chemical fertilizer NPK) were field tested following two rice cultivation cycles. Soil samples were subsequently collected and analyzed for bacterial groups and phosphorus cycle genes. Results indicated that the bacterial community composition remained consistent, comprising five main phyla: Firmicutes, Actinobacteria, Proteobacteria, Halobacterota, and Chloroflexia, regardless of fertilizer type. NPK fertilizer significantly reduced the relative abundance of Chloroflexia by 19% and Firmicutes by 16.4%, while increasing Actinobacteria and Proteobacteria by 27.5 and 58.8%, respectively. TSP fertilizer increased Actinobacteria by 27.1% and Halobacterota by 24.8%, but reduced Chloroflexia by 8.6%, Firmicutes by 12.6%, and Proteobacteria by 0.6%. Phosphate rock application resulted in reductions of Chloroflexia by 27.1%, Halobacterota by 22.9%, and Firmicutes by 6.2%, alongside increases in Actinobacteria by 46.6% and Proteobacteria by 23.8%. Combined application of TSP, NPK, and phosphate rock led to increases in Proteobacteria (24-40%) and Actinobacteria (13-39%), and decreases in Chloroflexia (5.2-22%) and Firmicutes (6-12.3%) compared to the control (T0). While the different phosphorus sources did not alter the composition of phosphorus cycle genes, they did modulate their abundance. NPK fertilizer did not significantly affect ppK genes (57-59%) but reduced gcd (100 to 69%), 3-phytase (74 to 34%), appA (91 to 63%), and phoD (83 to 67%). Phosphate rock reduced appA and gcd by 27 and 15%, respectively, while increasing 3-phytase by 19%. TSP decreased ppK and phoD by 42 and 40%, respectively, and gcd and appA by 34 and 56%, respectively. Combined fertilizers reduced appA (49 to 34%), 3-phytase (10 to 0%), and gcd (27 to 6%), while increasing ppK (72 to 100%). Among tested phosphorus sources, natural phosphate rock was best, causing moderate changes in bacterial composition and phosphorus genes, supporting balanced soil microbial activity. These findings highlight the complex interactions between fertilizers and soil microbial communities, underscoring the need for tailored fertilization strategies to maintain soil health and optimize agricultural productivity.

Different survival strategies of the phosphate-mineralizing bacterium Enterobacter sp. PMB-5 in response to cadmium stress: Biomineralization, biosorption, and bioaccumulation

The phosphate-mineralizing bacteria (PMBs) has shown great potential as a sustainable solution to support pollution remediation through its induced mineralization capacity. However, few studies have been conducted on the mechanism of cadmium (Cd) tolerance in PMBs. In this study, a PMB strain, Enterobacter sp. PMB-5, screened from Cd-contaminated rhizosphere soil, has high resistance to Cd (540 - 1220 mg/L) and solubilized phosphate (232.08 mg/L). The removal experiments showed that the strain PMB-5 removed 71.69–98.24% and 34.83–76.36% of Cd with and without biomineralization, respectively. The characterization result of SEM, EDS, TEM, XPS and XRD revealed that PMB-5 induced Cd to form amorphous phosphate precipitation through biomineralization and adopted different survival strategies, including biomineralization, bioaccumulation, and biosorption to resistance Cd in the microbial induced phosphate precipitation (MIPP) system and the non-MIPP system, respectively. Moreover, the results of whole genome sequencing and qRT-PCR indicated that phosphorus metabolism genes such as pst, pit, phn, ugp, ppk, etc. and heavy metal tolerance genes (including ion transport, ion efflux, redox, antioxidant stress), such as czcD, zntA, mgtA, mgtC, katE, SOD2, dsbA, cysM, etc. were molecular for the PMB-5 mineralization and Cd tolerance of PMB-5. Together, our findings suggested Enterobacter sp. PMB-5 is a potential target for developing more effective bioinoculants for Cd contamination remediation.

Combining protein and metabolic engineering strategies for high-level production of L-theanine in Corynebacterium glutamicum

L-theanine is a natural non-protein amino acid with wide applications. Thus, a high yield of L-theanine production is required on an industrial scale. Herein, an efficient L-theanine-producing strain of Corynebacterium glutamicum was constructed by combining protein and metabolic engineering. Firstly, a γ-glutamylmethylamide synthetase from Paracoccus aminovorans (PaGMAS) was isolated and engineered by computer-aided design, the resulting mutant E179K/N105R improved L-theanine yield by 36.61 %. Subsequently, to increase carbon flux towards L-theanine production, the gene ggt which degrades L-theanine, the gene alaT which participated in L-alanine synthesis, and the gene NCgl1221 which encodes glutamate-exporting protein were deleted. Finally, ppk gene was overexpressed to enhance intracellular ATP production. The reprogramed strain produced 44.12 g/L L-theanine with a yield of 57.11 % and productivity of 1.16 g/L/h, which is the highest L-theanine titer reported by Corynebacterium glutamicum. This study provides an efficient and economical biosynthetic pathway for the industrial production of L-theanine.

Co-inoculation of phosphate-solubilizing bacteria and phosphate accumulating bacteria in phosphorus-enriched composting regulates phosphorus transformation by facilitating polyphosphate formation

This study aimed to explore the impact of co-inoculating phosphate-solubilizing bacteria (PSB) and phosphate accumulating bacteria (PAB) on phosphorus forms transformation, microbial biomass phosphorus (MBP) and polyphosphate (Poly-P) accumulation, bacterial community composition in composting, using high throughput sequencing, PICRUSt 2, network analysis, structural equation model (SEM) and random forest (RF) analysis. The results demonstrated PSB-PAB co-inoculation (T1) reduced Olsen-P content (1.4 g) but had higher levels of MBP (74.2 mg/kg) and Poly-P (419 A.U.) compared to PSB-only (T0). The mantel test revealed a significantly positive correlation between bacterial diversity and both bioavailable P and MBP. Halocella was identified as a key genus related to Poly-P synthesis by network analysis. SEM and RF analysis showed that pH and bacterial community had the most influence on Poly-P synthesis, and PICRUSt 2 analysis revealed inoculation of PAB increased ppk gene abundance in T1. Thus, PSB-PAB co-inoculation provides a new idea for phosphorus management.

Effects of ppk1 deletion on the drug susceptibility of uropathogenic Escherichia coli producing ESBLs

To investigate the effect and the mechanism of ppk1 gene deletion on the drug susceptibility of uropathogenic Escherichia coli producing extended-spectrum beta-lactamases (ESBLs-UPEC). The study was an experimental study. From March to April 2021, a strain of ESBLs-UPEC (genotype was TEM combined with CTX-M-14) named as UE210113, was isolated from urine sample of the patient with urinary tract infection in the Laboratory Department of Guangzhou Eighth People's Hospital, meanwhile its ppk1 gene knock-out strain Δpk1 and complemented strain Δpk1-C were constructed by suicide plasmid homologous recombination technique, which was used to study the effect of ppk1 gene on ESBLs-UPEC drug sensitivity and its mechanism. The drug susceptibility of UE210113, Δpk1, and Δpk1-C were measured by Vitek2 Compact System and broth microdilution method. The quantitative expression of ESBLs, outer membrane protein and multidrug efflux systems encoding genes of UE210113, Δpk1 and Δpk1-C were performed by using qRT-PCR analysis. By using two independent sample Mann-Whitney U test, the drug susceptibility results showed that, compared with UE210113 strain, the sensitivities of Δpk1 to ceftazidime, cefepime, tobramycin, minocycline and cotrimoxazole were enhanced (Z=-2.121,P<0.05;Z=-2.236,P<0.05;Z=-2.236,P<0.05;Z=-2.121,P<0.05), and the drug susceptibility of Δpk1-C restored to the same as which of UE210113 (Z=0,P>0.05). The expression levels of ESBLs-enconding genes blaTEM and blaCTX-M-14 in Δpk1 were significantly down-regulated compared with UE210113, but the expression was not restored in Δpk1-C. The expression of outer membrane protein gene omp F in Δpk1 was significantly up-regulated, while the expression of omp A and omp C were down-regulated. The results showed that the expression of multidrug efflux systems encoding genes tol C, mdt A and mdtG were down-regulated in Δpk1 compared with UE210113. The expression of all of the outer membrane protein genes and the multidrug efflux systems genes were restored in Δpk1-C. In conclusion,the lost of ppk1 gene can affect the expression of the outer membrane protein and multidrug efflux systems encoding genes of ESBLs-UPEC, which increase the sensitivity of ESBLs-UPEC to various drugs.

Study of the biosynthesis and functionality of polyphosphate in Bifidobacterium longum KABP042

Polyphosphate (poly-P) biosynthesis in bacteria has been linked to many physiological processes and has been characterized as an interesting functional molecule involved in intestinal homeostasis. We determined the capacity for poly-P production of 18 probiotic strains mainly belonging to Bifidobacterium and former Lactobacillus genera, showing that poly-P synthesis varied widely between strains and is dependent on the availability of phosphate and the growth phase. Bifidobacteria were especially capable of poly-P synthesis and poly-P kinase (ppk) genes were identified in their genomes together with a repertoire of genes involved in phosphate transport and metabolism. In Bifidobacterium longum KABP042, the strain we found with highest poly-P production, variations in ppk expression were linked to growth conditions and presence of phosphate in the medium. Moreover, the strain produced poly-P in presence of breast milk and lacto-N-tetraose increased the amount of poly-P synthesized. Compared to KABP042 supernatants low in poly-P, exposure of Caco-2 cells to KABP042 supernatants rich in poly-P resulted in decreased epithelial permeability and increased barrier resistance, induction of epithelial protecting factors such as HSP27 and enhanced expression of tight junction protein genes. These results highlight the role of bifidobacteria-derived poly-P as a strain-dependent functional factor acting on epithelial integrity.

Integrative transcriptome and proteome revealed high-yielding mechanisms of epsilon-poly-L-lysine by Streptomyces albulus.

ε-poly-L-lysine (ε-PL) is a high value, widely used natural antimicrobial peptide additive for foods and cosmetic products that is mainly produced by Streptomyces albulus. In previous work, we developed the high-yield industrial strain S. albulus WG-608 through successive rounds of engineering. Here, we use integrated physiological, transcriptomic, and proteomics association analysis to resolve the complex mechanisms underlying high ε-PL production by comparing WG-608 with the progenitor strain M-Z18. Our results show that key genes in the glycolysis, pentose phosphate pathway, glyoxylate pathway, oxidative phosphorylation, and L-lysine biosynthesis pathways are differentially upregulated in WG-608, while genes in the biosynthetic pathways for fatty acids, various branched amino acids, and secondary metabolite by-products are downregulated. This regulatory pattern results in the introduction of more carbon atoms into L-lysine biosynthesis and ε-PL production. In addition, significant changes in the regulation of DNA replication, transcription, and translation, two component systems, and quorum sensing may facilitate the adaptability to environmental pressure and the biosynthesis of ε-PL. Overexpression of ppk gene and addition of polyP6 further enhanced the ε-PL production. This study enables comprehensive understanding of the biosynthetic mechanisms of ε-PL in S. albulus WG-608, while providing some genetic modification and fermentation strategies to further improve the ε-PL production.

Polyphosphate Accelerates Transformation of Nonstructural Carbohydrates to Improve Growth of ppk-Expressing Transgenic Rice in Phosphorus Deficiency Culture

Crop yield and quality are often limited by the amount of phosphate fertilizer added to infertile soils, a key limiting factor for sustainable development in modern agriculture. The polyphosphate kinase (ppk) gene -expressing transgenic rice with a single-copy line (ETRS) is constructed to improve phosphate fertilizer utilization efficiency for phosphorus resource conservation. To investigate the potential mechanisms of the increased biomass in ETRS in low phosphate culture, ETRS was cultivated in a low inorganic phosphate (Pi) culture medium (15 μmol/L Pi, LP) and a normal Pi culture medium (300 μmol/L Pi, CP), respectively. After 89 d of cultivation in different concentrations of phosphate culture media, the total phosphorus, polyphosphate (polyP), biomass, photosynthetic rate, nonstructural carbohydrate (NSC) contents, related enzyme activities, and related gene expression levels were analyzed. The results showed that ETRS had a high polyP amount to promote the photosynthetic rate in LP, and its biomass was almost the same as the wild type (WT) in CP. The NSC content of ETRS in LP was higher than that of WT in LP, but slightly lower than that of WT in CP. PolyP notably promoted the sucrose phosphate synthase activities of ETRS and significantly down-regulated the expression levels of sucrose transporter genes (OsSUT3 and OsSUT4), resulting in inhibiting the transport of sucrose from shoot to root in ETRS. It was concluded that polyP can stimulate the synthesis of NSCs in LP, which improved the growth of ETRS and triggered the biological activities of ETRS to save phosphate fertilizer. Our study provides a new way to improve the utilization rate of phosphate fertilizer in rice production.

Genome-wide screen in human plasma identifies multifaceted complement evasion of Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is a leading cause of bacteremia with a high mortality rate. We recently reported that P. aeruginosa forms a persister-like sub-population of evaders in human plasma. Here, using a gain-of-function transposon sequencing (Tn-seq) screen in plasma, we identified and validated previously unknown factors affecting bacterial persistence in plasma. Among them, we identified a small periplasmic protein, named SrgA, whose expression leads to up to a 100-fold increase in resistance to killing. Additionally, mutants in pur and bio genes displayed higher tolerance and persistence, respectively. Analysis of several steps of the complement cascade and exposure to an outer-membrane-impermeable drug, nisin, suggested that the mutants impede membrane attack complex (MAC) activity per se. Electron microscopy combined with energy-dispersive X-ray spectroscopy (EDX) revealed the formation of polyphosphate (polyP) granules upon incubation in plasma of different size in purD and wild-type strains, implying the bacterial response to a stress signal. Indeed, inactivation of ppk genes encoding polyP-generating enzymes lead to significant elimination of persisting bacteria from plasma. Through this study, we shed light on a complex P. aeruginosa response to the plasma conditions and discovered the multifactorial origin of bacterial resilience to MAC-induced killing.

Nutrients removal by Olivibacter jilunii immobilized on activated carbon for aquaculture wastewater treatment: ppk1 gene and bacterial community structure

In this study, immobilized biological activated carbon (IBAC) mediated with Olivibacter jilunii (strain PAO-9) was utilized to treat aquaculture wastewater for nutrients removal. IBAC with strain PAO-9 could load the greatest ppk1 gene copy numbers (129524.6) per gram on activated carbon at 28 °C for 2 d in 120 rpm of stirring speed and 2 d in stationary condition. Moreover, the results about the nutrients removal and microbiology community structure showed that strain PAO-9 on IBAC could alter the structure and diversity of microbial communities and then promoted to remove the total phosphorus and total nitrogen of eel aquaculture wastewater. The highest total phosphorus, chemical oxygen demand, ammonia and total nitrogen of the wastewater treated by strain PAO-9 on IBAC were 96.1 %, 98.0 %, 100.0 % and 97.4 %, respectively. In all, O. jilunii PAO-9 immobilized activated carbon was a potential and effective approach to remove the nutrients of eel aquaculture wastewater.

Identification of genes associated with environmental persistence in Campylobacter jejuni and Campylobacter coli isolates from processing in a broiler abattoir

The aim of this study was to determine the prevalence of the htrA, htrB and ppk1 genes -all of which are related to environmental persistence- in C. jejuni and C. coli isolates obtained from abattoir samples at the arrival of broilers (initial stage) and in meat products after processing (final stage). A total of 119 DNA extracts (55 C. jejuni and 64 C. coli) were included in the study. Identification of genes was performed by conventional PCR (one for each gene). The overall prevalence was 40.3%, 93.3% and 68.9% for the htrA, htrB and ppk1 genes, respectively. Statistically significant differences were found (p < 0.05) between prevalence of C. jejuni and C. coli for all three genes. In C. coli the prevalence was significantly higher for the htrA (p = 0.007) and htrB (p = 0.015) genes, while ppk1 gene prevalence was significantly higher in C. jejuni (p < 0.001). In addition, statistically significant increase in the frequency of htrA (p = 0.007) and htrB (p = 0.013) genes in the final product compared to broilers on arrival at the abattoir was observed in C. jejuni, but not in C. coli. These results suggest that htrA and htrB genes are involved in environmental persistence of Campylobacter jejuni.

Microbial behaviours inside alternating anaerobic-anoxic environment of a sulfur cycle–driven EBPR system: A metagenomic investigation

Denitrifying sulfur conversion-assisted enhanced biological phosphorus removal (DS-EBPR) was recently developed for saline wastewater treatment. However, the main functional bacteria and the interrelationship of functional bacteria of the DS-EBPR have not been defined and identified so far. This study used metagenomics and multivariate statistics to deduce the functional microbial community and distribution of functional genes associated with the critical metabolic pathways of carbon (C), nitrogen (N), phosphorus (P) and sulfur (S), particularly regarding how they would behave under the alternating anaerobic-anoxic conditions inside a long-term DS-EBPR system. An analysis of the metagenomics and metabolic functions identified 11 major microbial species which were classifiable into four groups: sulfate reducing bacteria (SRB, 0.8–2.2%), sulfur oxidizing bacteria (SOB, 31.9–37.7%), denitrifying phosphate accumulating organisms (DPAOs, 10.0–15.8%) and glycogen accumulating organisms (GAOs, 3.7–7.7%). The four groups of microorganisms performed their respective metabolisms synergistically. In terms of distribution of functional genes, SRB (Desulfococcus and Desulfobacter) and SOB (Chromatiaceae and Thiobacillus) are not only encoded by the related sulfur conversion genes (sqr, dsrAB, aprAB and sat), but also encoded by the necessary ppx and ppk1 gene for P removal that they can be considered as the potential S-related PAOs. Between the anaerobic and anoxic conditions, the metagenome-based microbial community remained structurally similar, but the functional genes, which encode various key enzymes for the P, N, and S pathways, changed in abundance. This study contributes to our understanding on the interactions and competition between the SRB, SOB, DPAOs, and GAOs in a DS-EBPR system.