Search Results (10)

Phosphorus is essential for crop growth, but excessive use of chemical fertilizers can lead to environmental issues. The incorporation of organic materials has the potential to enhance phosphorus availability and promote soil phosphorus cycling. This study investigated the effects of chemical fertilizer co-application with two organic materials on soil properties and functions. Four treatments were established: (1) chemical fertilizer alone (SC, consisting of urea, ammonium phosphate, and potassium sulfate), (2) chemical fertilizer with corn-straw-derived biochar (SCB), (3) chemical fertilizer with composted manure-based organic fertilizer (SCF), and (4) chemical fertilizer with both biochar and organic fertilizer (SCBF). This study focused on changes in soil properties, bioavailable phosphorus, phosphorus cycling functional genes, and related microbial communities. Compared to SC, the combined application of organic materials significantly increased available phosphorus (AP), alkaline hydrolysis nitrogen (AN), and available potassium (AK), with the SCBF exhibiting the highest increases of 78.76%, 47.47%, and 336.61%, respectively. However, applying organic materials reduced alkaline phosphatase (ALP) and acid phosphatase (ACP) activities, except for the increase in ACP in SCBF. Additionally, bioavailable phosphorus increased by up to 157.00% in SCBF. Adding organic materials significantly decreased organic phosphorus mineralization genes (phoA, phoD, phnP) and phosphate degradation genes (ppk2), while increasing inorganic phosphorus solubilization genes (pqqC, gcd), which subsequently increased CaCl₂-P and Citrate-P contents in SCB and in SCBF. In summary, organic material application significantly enhances phosphorus bioavailability by improving soil physicochemical properties and phosphorus-related gene abundance. These findings provide new insights into sustainable soil fertility management and highlight the potential of integrating organic materials with chemical fertilizers to improve soil nutrient availability, thereby contributing to increased soybean yield. Moreover, this study advances our understanding of the underlying mechanisms driving phosphorus cycling under combined fertilization strategies, offering a scientific basis for optimizing fertilization practices in agroecosystems. Full article

Crop rotation and microbial driving force significantly influence soil phosphorus (P) bioavailability and crop yield. However, differences in underlying microbial mechanisms in rotations remain unclear. We examined rice yield, P uptake, soil and microbial P contents, enzyme activity, and P functional genes over six years (2016–2022) to elucidate microbial mechanisms driving rice yield in rice–wheat (RW) and rice–oilseed rape (RO) rotations. RO significantly increased rice yield and plant P uptake by 9.17% and 20.70%, respectively, compared to RW. Soil total (TP) and available (AP) P contents were significantly lower (4.83% and 18.31%, respectively) under RO than RW, whereas microbial biomass phosphorus (MBP) and acid phosphatase activity (EP) were greater (39.40% and 128.45%, respectively). PICRUSt2 results revealed that RO increased phoA phoB (alkaline phosphatase), phnX (phosphonoacetaldehyde hydrolase [EC:3.11.1.1]), gcd (Quinoprotein glucose dehydrogenase [EC:1.1.5.2]), and ppaC (manganese-dependent inorganic pyrophosphatase) and decreased phnD (phosphonate transport system substrate-binding protein), ugpE (sn-glycerol 3-phosphate transport system permease protein), ugpA (sn-glycerol 3-phosphate transport system permease protein), and phnO ((aminoalkyl)phosphonate N-acetyltransferase [EC:2.3.1.280]) abundance. Random forest analysis showed that ppaC, phnD, gcd, and phnX were important for rice yield and plant P uptake. Partial least squares analysis revealed that RO indirectly increased rice yield by influencing MBP and affecting plant P uptake through P functional genes. Overall, RO improves rice yield and P bioavailability by altering P functional genes (ppaC, phnD, gcd, and phnX), providing new perspectives on crop–microorganism interactions and resource use efficiency. Full article

Alkaline phosphatase (ALP) of the PhoA family is an important enzyme in mammals, microalgae, and certain marine bacteria. It plays a crucial role in the dephosphorylation of lipopolysaccharides (LPS) and nucleotides, which overstimulate cell signaling pathways and cause tissue inflammation in animals and humans. Insufficient ALP activity and expression levels have been linked to various disorders. This study aims to produce recombinant ALP from the marine bacterium Cobetia amphilecti KMM 296 (CmAP) in transformed leaves and calli of Nicotiana tabacum and to elucidate the influence of the plant host on its physical and chemical properties. N. tabacum has proven to be versatile and is extensively used as a heterologous host in molecular farming. The alp gene encoding for CmAP was cloned into the binary vectors pEff and pHREAC and transformed into N. tabacum leaves through agroinfiltration and the leaf disc method for callus induction using Agrobacterium tumefaciens strain EHA105. Transformed plants were screened for recombinant CmAP (rCmAP) production by its enzymatic activity and protein electrophoresis, corresponding to 55 kDa of mature CmAP. A higher rCmAP activity (14.6 U/mg) was detected in a homogenate of leaves bearing the pEFF-CmAP construct, which was further purified 150-fold using metal affinity, followed by anion exchange chromatography. Enzymatic activity and stability were assessed at different temperatures (15–75 °C) and exposure times (≤1 h), with different buffers, pHs, divalent metal ions, and salt concentrations. The results show that rCmAP is relatively thermostable, retaining its activity at 15–45 °C for up to 1 h. Its activity is highest in Tris HCl (pH 9.0–11.0) at 35 °C for 40 min. rCmAP shows higher salt-tolerance and divalent metal-dependence than obtained in Escherichia coli. This can be further explored for cost-effective and massively scalable production of LPS-free CmAP for possible biomedical and agricultural applications. Full article

The natural 5-azaindoles, marine sponge guitarrin C and D, were observed to exert inhibitory activity against a highly active alkaline phosphatase (ALP) CmAP of the PhoA family from the marine bacterium Cobetia amphilecti, with IC₅₀ values of 8.5 and 110 µM, respectively. The superimposition of CmAP complexes with p-nitrophenyl phosphate (pNPP), a commonly used chromogenic aryl substrate for ALP, and the inhibitory guitarrins C, D, and the non-inhibitory guitarrins A, B, and E revealed that the presence of a carboxyl group at C6 together with a hydroxyl group at C8 is a prerequisite for the inhibitory effect of 5-azaindoles on ALP activity. The 10-fold more active guitarrin C could compete with pNPP for binding sites in the ALP active site due to similarities in size, three-dimensional structure, and the orientation of the COOH group along the phosphate group. However, the inhibition of CmAP and calf intestinal ALP (CIAP) by guitarrin C was observed to occur via a non-competitive mode of action, as evidenced by a twofold decrease in V_max and an unchanged K_m. In contrast, the kinetic model with guitarrin D, with an additional OH group at C7, reflected a mixed type of inhibition, with a decrease in both values. The sensitivity of CIAP to guitarrins C and D was shown to be slightly lower than that of CmAP, with IC₅₀ values of 195 and 230 µM, respectively. Nevertheless, these findings prompted the prediction of complexes of human ALP isoenzymes with guitarrins C and D. Full article

A highly active alkaline phosphatase (ALP) of the protein structural family PhoA, from a mussel gut-associated strain of the marine bacterium Cobetia amphilecti KMM 296 (CmAP), was found to effectively dephosphorylate lipopolysaccharides (LPS). Therefore, the aim of this work was to perform a comprehensive bioinformatics analysis of the structure, and to suggest the physiological role of this enzyme in marine bacteria of the genus Cobetia. A scrutiny of the CmAP-like sequences in 36 available Cobetia genomes revealed nine homologues intrinsic to the subspecies C. amphilecti, whereas PhoA of a distant relative Cobetia crustatorum JO1^T carried an inactive mutation. However, phylogenetic analysis of all available Cobetia ALP sequences showed that each strain of the genus Cobetia possesses several ALP variants, mostly the genes encoding for PhoD and PhoX families. The C. amphilecti strains have a complete set of four ALP families’ genes, namely: PhoA, PafA, PhoX, and two PhoD structures. The Cobetia marina species is distinguished by the presence of only three PhoX and PhoD genes. The Cobetia PhoA proteins are clustered together with the human and squid LPS-detoxifying enzymes. In addition, the predicted PhoA biosynthesis gene cluster suggests its involvement in the control of cellular redox balance, homeostasis, and cell cycle. Apparently, the variety of ALPs in Cobetia spp. indicates significant adaptability to phosphorus-replete and depleted environments and a notable organophosphate destructor in eco-niches from which they once emerged, including Zostera spp. The ALP clusterization and degree of similarity of the genus-specific biosynthetic genes encoding for ectoine and polyketide cluster T1PKS, responsible for sulfated extracellular polysaccharide synthesis, coincide with a new whole genome-based taxonomic classification of the genus Cobetia. The Cobetia strains and their ALPs are suggested to be adaptable for use in agriculture, biotechnology and biomedicine. Full article

A strictly aerobic, Gram-stain-negative, rod-shaped, and motile bacterium, designated strain KMM 296, isolated from the coelomic fluid of the mussel Crenomytilus grayanus, was investigated in detail due to its ability to produce a highly active alkaline phosphatase CmAP of the structural family PhoA. A previous taxonomic study allocated the strain to the species Cobetia marina, a member of the family Halomonadaceae of the class Gammaproteobacteria. However, 16S rRNA gene sequencing showed KMM 296’s relatedness to Cobetia amphilecti NRIC 0815^T. The isolate grew with 0.5–19% NaCl at 4–42 °C and hydrolyzed Tweens 20 and 40 and L-tyrosine. The DNA G+C content was 62.5 mol%. The prevalent fatty acids were C_18:1 ω7c, C_12:0 3-OH, C_18:1 ω7c, C_12:0, and C_17:0 cyclo. The polar lipid profile was characterized by the presence of phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, and also an unidentified aminolipid, phospholipid, and a few unidentified lipids. The major respiratory quinone was Q-8. According to phylogenomic and chemotaxonomic evidence, and the nearest neighbors, the strain KMM 296 represents a member of the species C. amphilecti. The genome-based analysis of C. amphilecti NRIC 0815^T and C. litoralis NRIC 0814^T showed their belonging to a single species. In addition, the high similarity between the C. pacifica NRIC 0813^T and C. marina LMG 2217^T genomes suggests their affiliation to one species. Based on the rules of priority, C. litoralis should be reclassified as a later heterotypic synonym of C. amphilecti, and C. pacifica is a later heterotypic synonym of C. marina. The emended descriptions of the species C. amphilecti and C. marina are also proposed. Full article

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. Full article

The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. Protein export pathways are attractive potential targets. The Sec pathway is essential for bacterial viability and includes components that are absent from eukaryotes. Here, we used a new high-throughput in vivo screen based on the secretion and activity of alkaline phosphatase (PhoA), a Sec-dependent secreted enzyme that becomes active in the periplasm. The assay was optimized for a luminescence-based substrate and was used to screen a ~240K small molecule compound library. After hit confirmation and analoging, 14 HTS secretion inhibitors (HSI), belonging to eight structural classes, were identified with IC₅₀ < 60 µM. The inhibitors were evaluated as antibacterials against 19 Gram-negative and Gram-positive bacterial species (including those from the WHO’s top pathogens list). Seven of them—HSI#6, 9; HSI#1, 5, 10; and HSI#12, 14—representing three structural families, were bacteriocidal. HSI#6 was the most potent hit against 13 species of both Gram-negative and Gram-positive bacteria with IC₅₀ of 0.4 to 8.7 μM. HSI#1, 5, 9 and 10 inhibited the viability of Gram-positive bacteria with IC₅₀ ~6.9–77.8 μM. HSI#9, 12, and 14 inhibited the viability of E. coli strains with IC₅₀ < 65 μM. Moreover, HSI#1, 5 and 10 inhibited the viability of an E. coli strain missing TolC to improve permeability with IC₅₀ 4 to 14 μM, indicating their inability to penetrate the outer membrane. The antimicrobial activity was not related to the inhibition of the SecA component of the translocase in vitro, and hence, HSI molecules may target new unknown components that directly or indirectly affect protein secretion. The results provided proof of the principle that the new broad HTS approach can yield attractive nanomolar inhibitors that have potential as new starting compounds for optimization to derive potential antibiotics. Full article

Marine bacteria of the genus Cobetia, which are promising sources of unique enzymes and secondary metabolites, were found to be complicatedly identified both by phenotypic indicators due to their ecophysiology diversity and 16S rRNA sequences because of their high homology. Therefore, searching for the additional methods for the species identification of Cobetia isolates is significant. The species-specific coding sequences for the enzymes of each functional category and different structural families were applied as additional molecular markers. The 13 closely related Cobetia isolates, collected in the Pacific Ocean from various habitats, were differentiated by the species-specific PCR patterns. An alkaline phosphatase PhoA seems to be a highly specific marker for C. amphilecti. However, the issue of C. amphilecti and C. litoralis, as well as C. marina and C. pacifica, belonging to the same or different species remains open. Full article

Screening antibody libraries is an important step in establishing recombinant monoclonal antibodies. The colony assay can identify positive clones without almost any false-positives; however, its antibody library is smaller than those used in other recombinant screening methods such as phage display. Thus, to improve the efficiency of colony assays, it is necessary to increase library size per screening. Here, we report developing a colony assay with single-chain variable fragment (scFv) fused to the N-terminus of bacterial alkaline phosphatase (scFv-PhoA). The scFv-PhoA library was constructed in an expression vector specifically designed for this study. Use of this library allowed the successful and direct detection of positive clones exhibiting PhoA activity, without the need for a secondary antibody. Colony assay screening with scFv-PhoA is simple, rapid, offers a higher success rate than previous methods based on scFv libraries, and—most importantly—it enables high-throughput procedures. Full article