Leveraging the histidine kinase-phosphatase duality to sculpt two-component signaling.

Bacteria must constantly probe their environment for rapid adaptation, a crucial need most frequently served by two-component systems (TCS). As one component, sensor histidine kinases (SHK) control the phosphorylation of the second component, the response regulator (RR). Downstream responses hinge on RR phosphorylation and can be highly stringent, acute, and sensitive because SHKs commonly exert both kinase and phosphatase activity. With a bacteriophytochrome TCS as a paradigm, we here interrogate how this catalytic duality underlies signal responses. Derivative systems exhibit tenfold higher red-light sensitivity, owing to an altered kinase-phosphatase balance. Modifications of the linker intervening the SHK sensor and catalytic entities likewise tilt this balance and provide TCSs with inverted output that increases under red light. These TCSs expand synthetic biology and showcase how deliberate perturbations of the kinase-phosphatase duality unlock altered signal-response regimes. Arguably, these aspects equally pertain to the engineering and the natural evolution of TCSs.

Determination of Phosphoglycolate Phosphatase Activity via a Coupled Reaction Using Recombinant Glycolate Oxidase.

Phosphoglycolate phosphatase (PGLP) dephosphorylates 2-phosphoglycolate to glycolate that can be further metabolized to glyoxylate by glycolate oxidase (GOX) via an oxidative reaction that uses O2 and releases H2O2. The oxidation of o-dianisidine by H2O2 catalyzed by a peroxidase can be followed in real time by an absorbance change at 440 nm. Based on these reactions, a spectrophotometric method for measuring PGLP activity using a coupled reaction with recombinant Arabidopsis thaliana GOX is described. This protocol has been used successfully with either purified PGLP or total soluble proteins extracted from Arabidopsis rosette leaves.

Purification of Recombinant N-terminal Histidine-Tagged Arabidopsis thaliana Phosphoglycolate Phosphatase 1, Glycolate Oxidase 1 and 2, and Hydroxypyruvate Reductase 1.

To measure the kinetic properties of photorespiratory enzymes, it is necessary to work with purified proteins. Protocols to purify photorespiratory enzymes from leaves of various plant species require several time-consuming steps. It is now possible to produce large quantities of recombinant proteins in bacterial cells. They can be rapidly purified as histidine-tagged recombinant proteins by immobilized metal affinity chromatography using Ni2+-NTA-agarose. This chapter describes protocols to purify several Arabidopsis thaliana His-tagged recombinant photorespiratory enzymes (phosphoglycolate phosphatase, glycolate oxidase, and hydroxypyruvate reductase) from Escherichia coli cell cultures using two bacterial strain-plasmid systems: BL21(DE3)-pET and LMG194-pBAD.

An exchange of single amino acid between the phosphohydrolase modules of Escherichia coli MutT and Mycobacterium smegmatis MutT1 switches their cleavage specificities.

MutT proteins belong to the Nudix hydrolase superfamily that includes a diverse group of Mg2+ requiring enzymes. These proteins use a generalized substrate, nucleoside diphosphate linked to a chemical group X (NDP-X), to produce nucleoside monophosphate (NMP) and the moiety X linked with phosphate (XP). E. coli MutT (EcoMutT) and mycobacterial MutT1 (MsmMutT1) belong to the Nudix hydrolase superfamily that utilize 8-oxo-(d)GTP (referring to both 8-oxo-GTP or 8-oxo-dGTP). However, predominant products of their activities are different. While EcoMutT produces 8-oxo-(d)GMP, MsmMutT1 gives rise to 8-oxo-(d)GDP. Here, we show that the altered cleavage specificities of the two proteins are largely a consequence of the variation at the equivalent of Gly37 (G37) in EcoMutT to Lys (K65) in the MsmMutT1. Remarkably, mutations of G37K (EcoMutT) and K65G (MsmMutT1) switch their cleavage specificities to produce 8-oxo-(d)GDP, and 8-oxo-(d)GMP, respectively. Further, a time course analysis using 8-oxo-GTP suggests that MsmMutT1(K65G) hydrolyses 8-oxo-(d)GTP to 8-oxo-(d)GMP in a two-step reaction via 8-oxo-(d)GDP intermediate. Expectedly, unlike EcoMutT (G37K) and MsmMutT1, EcoMutT and MsmMutT1 (K65G) rescue an E. coli ΔmutT strain, better by decreasing A to C mutations.

Sur7 mediates a novel pathway for PI4,5P2 regulation in C. albicans that promotes stress resistance and cell wall morphogenesis.

The human fungal pathogen Candida albicans can cause lethal systemic infections due to its ability to resist stress from the host and to undergo invasive hyphal growth. Previous studies showed that plasma membrane MCC/eisosome domains were important for virulence by promoting the ability of Sur7 to mediate normal cell wall morphogenesis and stress resistance. The sur7Δ mutant displayed abnormal clusters of PI4,5P2, suggesting that misregulation of this lipid underlies the sur7Δ phenotype. To test this, we increased PI4,5P2 levels by deleting combinations of the three PI4,5P2 5' phosphatase genes (INP51, INP52, and INP54) and found that some combinations, such as inp51Δ inp52Δ, gave phenotypes similar the sur7Δ mutant. In contrast, deleting one copy of MSS4, the gene that encodes the 5' kinase needed to create PI4,5P2, reduced the abnormal PI4,5P2 clusters and also decreased the abnormal cell wall and stress sensitive phenotypes of the sur7Δ mutant. Additional studies support a model that the abnormal PI4,5P2 patches recruit septin proteins, which in turn promote aberrant cell wall growth. These results identify Sur7 as a novel regulator of PI4,5P2 and highlight the critical role of PI4,5P2 in the regulation of C. albicans virulence properties.

Enhanced Iturin A Production of Engineered Bacillus amyloliquefaciens by Knockout of Endogenous Plasmid and Rap Phosphatase Genes.

Iturin A biosynthesis has garnered considerable interest, yet bottlenecks persist in its low productivity in wild strains and the ability to engineer Bacillus amyloliquefaciens producers. This study reveals that deleting the endogenous plasmid, plas1, from the wild-type B. amyloliquefaciens HM618 notably enhances iturin A synthesis, likely related to the effect of the Rap phosphatase gene within plas1. Furthermore, inactivating Rap phosphatase-related genes (rapC, rapF, and rapH) in the genome of the strain also improved the iturin A level and specific productivity while reducing cell growth. Strategic rap genes and plasmid elimination achieved a synergistic balance between cell growth and iturin A production. Engineered strain HM-DR13 exhibited an increase in iturin A level to 849.9 mg/L within 48 h, significantly shortening the production period. These insights underscore the critical roles of endogenous plasmids and Rap phosphatases in iturin A biosynthesis, presenting a novel engineering strategy to optimize iturin A production in B. amyloliquefaciens.

Plausible role of INPP4A dysregulation in idiopathic pulmonary fibrosis.

INPP4A has been shown to be involved in the regulation of cell proliferation and apoptosis of multiple cell types including fibroblasts. Previous reports from our group have demonstrated the role of inositol polyphosphate 4-phosphatase Type I A (INPP4A) in these functions. Though existing evidences suggest a critical role for INPP4A in the maintenance of lung homeostasis, its role in chronic lung diseases is relatively under explored. In the current study, we made an attempt to understand the regulation of INPP4A in idiopathic pulmonary fibrosis (IPF). Through integration of relevant INPP4A gene expression data from public repositories with our results from in vitro experiments and mouse models, we show that INPP4A is altered in IPF. Interestingly, the direction of the change is dependent both on the disease stage and the region of the lung used. INPP4A was found to be upregulated when analyzed in lung sample representative of the whole lung, but was downregulated in the fibrotic regions of the lung. Similarly, INPP4A was found to be high, compared to controls, only in the early stage of the disease. Though the observed increase in INPP4A was found to be negatively correlated to physiological indices, FVC, and DLCO, of lung function, treatment with anti-INPP4A antibody worsened the condition in bleomycin treated mice. These contrasting results taken together are suggestive of a nuanced regulation of INPP4A in IPF which is dependent on the disease stage, cellular state and extent of fibrosis in the lung region being analyzed.

Nontraditional Roles of Magnesium Ions in Modulating Sav2152: Insight from a Haloacid Dehalogenase-like Superfamily Phosphatase from Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg2+ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg2+ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg2+ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg2+ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity.

Clinical features and genetic analysis of 15 Chinese children with dent disease.

OBJECTIVE:  The clinical characteristics, genetic mutation spectrum, treatment strategies and prognoses of 15 children with Dent disease were retrospectively analyzed to improve pediatricians' awareness of and attention to this disease. METHODS:  We analyzed the clinical and laboratory data of 15 Chinese children with Dent disease who were diagnosed and treated at our hospital between January 2017 and May 2023 and evaluated the expression of the CLCN5 and OCRL1 genes. RESULTS:  All 15 patients were male and complained of proteinuria, and the incidence of low-molecular-weight proteinuria (LMWP) was 100.0% in both Dent disease 1 (DD1) and Dent disease 2 (DD2) patients. The incidence of hypercalciuria was 58.3% (7/12) and 66.7% (2/3) in DD1 and DD2 patients, respectively. Nephrocalcinosis and nephrolithiasis were found in 16.7% (2/12) and 8.3% (1/12) of DD1 patients, respectively. Renal biopsy revealed focal segmental glomerulosclerosis (FSGS) in 1 patient, minimal change lesion in 5 patients, and small focal acute tubular injury in 1 patient. A total of 11 mutations in the CLCN5 gene were detected, including 3 missense mutations (25.0%, c.1756C > T, c.1166T > G, and c.1618G > A), 5 frameshift mutations (41.7%, c.407delT, c.1702_c.1703insC, c.137delC, c.665_666delGGinsC, and c.2200delG), and 3 nonsense mutations (25.0%, c.776G > A, c.1609C > T, and c.1152G > A). There was no significant difference in age or clinical phenotype among patients with different mutation types (p > 0.05). All three mutations in the OCRL1 gene were missense mutations (c.1477C > T, c.952C > T, and c.198A > G). CONCLUSION:  Pediatric Dent disease is often misdiagnosed. Protein electrophoresis and genetic testing can help to provide an early and correct diagnosis.

L-serine deficiency: on the properties of the Asn133Ser variant of human phosphoserine phosphatase.

The non-essential amino acid L-serine is involved in a number of metabolic pathways and in the brain its level is largely due to the biosynthesis from the glycolytic intermediate D-3-phosphoglycerate by the phosphorylated pathway (PP). This cytosolic pathway is made by three enzymes proposed to generate a reversible metabolon named the "serinosome". Phosphoserine phosphatase (PSP) catalyses the last and irreversible step, representing the driving force pushing L-serine synthesis. Genetic defects of the PP enzymes result in strong neurological phenotypes. Recently, we identified the homozygous missense variant [NM_004577.4: c.398A > G p.(Asn133Ser)] in the PSPH, the PSP encoding gene, in two siblings with a neurodevelopmental syndrome and a myelopathy. The recombinant Asn133Ser enzyme does not show significant alterations in protein conformation and dimeric oligomerization state, as well as in enzymatic activity and functionality of the reconstructed PP. However, the Asn133Ser variant is less stable than wild-type PSP, a feature also apparent at cellular level. Studies on patients' fibroblasts also highlight a strong decrease in the level of the enzymes of the PP, a partial nuclear and perinuclear localization of variant PSP and a stronger perinuclear aggregates formation. We propose that these alterations contribute to the formation of a dysfunctional serinosome and thus to the observed reduction of L-serine, glycine and D-serine levels (the latter playing a crucial role in modulating NMDA receptors). The characterization of patients harbouring the Asn133Ser PSP substitution allows to go deep into the molecular mechanisms related to L-serine deficit and to suggest treatments to cope with the observed amino acids alterations.

Hydrophobic residues in S1 modulate enzymatic function and voltage sensing in voltage-sensing phosphatase.

The voltage-sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage-sensing proteins, the VSDs do not interact with one another, and the S1-S3 helices are considered mainly scaffolding, except in the voltage-sensing phosphatase (VSP) and the proton channel (Hv). To investigate its contribution to VSP function, we mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134, and L137), individually or in combination. Most of these mutations shifted the voltage dependence of activity to higher voltages; however, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered, with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions was consistently shifted to lower voltages and indicated a second voltage-dependent motion. Additionally, none of the mutations broke the VSP dimer, indicating that the S1 impact could stem from intra- and/or intersubunit interactions. Lastly, when the same mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzyme's conformational response to membrane potential transients and influencing the function of the VSD.

Visualization of oxidized guanine nucleotides accumulation in living cells with split MutT.

Cancer cells produce vast quantities of reactive oxygen species, leading to the accumulation of toxic nucleotides as 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP). The human MTH1 protein catalyzes the hydrolysis of 8-oxo-dGTP, and cancer cells are dependent on MTH1 for their survival. MTH1 inhibitors are possible candidates for a class of anticancer drugs; however, a reliable screening system using live cells has not been developed. Here we report a visualization method for 8-oxo-dGTP and its related nucleotides in living cells. Escherichia coli MutT, a functional homologue of MTH1, is divided into the N-terminal (1-95) and C-terminal (96-129) parts (Mu95 and 96tT, respectively). Mu95 and 96tT were fused to Ash (assembly helper tag) and hAG (Azami Green), respectively, to visualize the nucleotides as fluorescent foci formed upon the Ash-hAG association. The foci were highly increased when human cells expressing Ash-Mu95 and hAG-96tT were treated with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-dGTP. The foci formation by 8-oxo-dG(TP) was strikingly enhanced by the MTH1 knockdown. Moreover, known MTH1 inhibitors and oxidizing reagents also increased foci. This is the first system that visualizes damaged nucleotides in living cells, provides an excellent detection method for the oxidized nucleotides and oxidative stress, and enables high throughput screening for MTH1 inhibitors.

Rhizobial-induced phosphatase GmPP2C61A positively regulates soybean nodulation.

Symbiotic nitrogen fixation (SNF) is crucial for legumes, providing them with the nitrogen necessary for plant growth and development. Nodulation is the first step in the establishment of SNF. However, the determinant genes in soybean nodulation and the understanding of the underlying molecular mechanisms governing nodulation are still limited. Herein, we identified a phosphatase, GmPP2C61A, which was specifically induced by rhizobia inoculation. Using transgenic hairy roots harboring GmPP2C61A::GUS, we showed that GmPP2C61A was mainly induced in epidermal cells following rhizobia inoculation. Functional analysis revealed that knockdown or knock-out of GmPP2C61A significantly reduced the number of nodules, while overexpression of GmPP2C61A promoted nodule formation. Additionally, GmPP2C61A protein was mainly localized in the cytoplasm and exhibited conserved phosphatase activity in vitro. Our findings suggest that phosphatase GmPP2C61A serves as a critical regulator in soybean nodulation, highlighting its potential significance in enhancing symbiotic nitrogen fixation.

CgSHIP2 negatively regulates the mRNA expressions of CgIL-17s in response to Vibrio splendidus stimulation in Crassostrea gigas.

SH2 domain containing inositol polyphosphate5-phosphatase-2 (SHIP2) is a member of the 5-phosphatase family, acting as a vital negative regulator of immune response in vertebrates. In the present study, a SHIP2 homologue (designed as CgSHIP2) was identified from Pacific oyster, Crassostrea gigas. There was a SH2 domain, an IPPc domain and a SAM domain in CgSHIP2. The mRNA transcripts of CgSHIP2 were widely expressed in all the tested tissues with the highest expression in haemolymph. The mRNA expressions of CgSHIP2 in haemocytes increased significantly at 6, 12, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgSHIP2 protein were mainly located in cytoplasm of haemocytes. After the expression of CgSHIP2 was inhibited by RNA interference, the mRNA transcripts of interleukin 17s (CgIL-17-1, CgIL-17-2, CgIL-17-3 and CgIL-17-6) in the haemocytes increased significantly at 24 h after V. splendidus stimulation, which were 8.15-fold (p < 0.001), 3.44-fold (p < 0.05), 2.15-fold (p < 0.01) and 4.63-fold (p < 0.05) compared with that in NC-RNAi group, respectively. Obvious branchial swelling and cilium shedding in gills were observed in CgSHIP2-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgSHIP2 played an important role in controlling inflammatory response induced by bacteria in oysters.

Base editing correction of OCRL in Lowe syndrome: ABE-mediated functional rescue in patient-derived fibroblasts.

Lowe syndrome, a rare X-linked multisystem disorder presenting with major abnormalities in the eyes, kidneys, and central nervous system, is caused by mutations in OCRL gene (NG_008638.1). Encoding an inositol polyphosphate 5-phosphatase, OCRL catalyzes the hydrolysis of PI(4,5)P2 into PI4P. There are no effective targeted treatments for Lowe syndrome. Here, we demonstrate a novel gene therapy for Lowe syndrome in patient fibroblasts using an adenine base editor (ABE) that can efficiently correct pathogenic point mutations. We show that ABE8e-NG-based correction of a disease-causing mutation in a Lowe patient-derived fibroblast line containing R844X mutation in OCRL gene, restores OCRL expression at mRNA and protein levels. It also restores cellular abnormalities that are hallmarks of OCRL dysfunction, including defects in ciliogenesis, microtubule anchoring, α-actinin distribution, and F-actin network. The study indicates that ABE-mediated gene therapy is a feasible treatment for Lowe syndrome, laying the foundation for therapeutic application of ABE in the currently incurable disease.

Cancer-associated point mutations within the extracellular domain of PTPRD affect protein stability and HSPG interaction.

PTPRD, a well-established tumor suppressor gene, encodes the protein tyrosine phosphatase-type D. This protein consists of three immunoglobulin-like (Ig) domains, four to eight fibronectin type 3 (FN) domains, a single transmembrane segment, and two cytoplasmic tandem tyrosine phosphatase domains. PTPRD is known to harbor various cancer-associated point mutations. While it is assumed that PTPRD regulates cellular functions as a tumor suppressor through the tyrosine phosphatase activity in the intracellular region, the function of its extracellular domain (ECD) in cancer is not well understood. In this study, we systematically examined the impact of 92 cancer-associated point mutations within the ECD. We found that 69.6% (64 out of 92) of these mutations suppressed total protein expression and/or plasma membrane localization. Notably, almost all mutations (20 out of 21) within the region between the last FN domain and transmembrane segment affected protein expression and/or localization, highlighting the importance of this region for protein stability. We further found that some mutations within the Ig domains adjacent to the glycosaminoglycan-binding pocket enhanced PTPRD's binding ability to heparan sulfate proteoglycans (HSPGs). This interaction is proposed to suppress phosphatase activity. Our findings therefore suggest that HSPG-mediated attenuation of phosphatase activity may be involved in tumorigenic processes through PTPRD dysregulation.

Inhibition of signaling protein ERN1 increases the sensitivity of serine synthesis gene expressions to glucose and glutamine deprivations in U87MG glioblastoma cells.

Objective. Glucose and glutamine supply as well as serine synthesis and endoplasmic reticulum (ER) stress are important factors of glioblastoma growth. Previous studies showed that the knockdown of ERN1 (ER to nucleus signaling 1) suppressed glioblastoma cell proliferation and modified the sensitivity of numerous gene expressions to nutrient deprivations. The present study is aimed to investigate the impact of glucose and glutamine deprivations on the expression of serine synthesis genes in U87MG glioblastoma cells in relation to ERN1 knockdown with the intent to reveal the role of ERN1 signaling pathway on the ER stress-dependent regulation of these gene expressions. Clarification of the regulatory mechanisms of serine synthesis is a great significance for glioblastoma therapy. Methods. The control U87MG glioblastoma cells (transfected by empty vector) and ERN1 knockdown cells (transfected by dominant-negative ERN1) were exposed under glucose and glutamine deprivation conditions for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of PHGDH (phosphoglycerate dehydrogenase), PSAT1 (phosphoserine amino-transferase 1), PSPH (phosphoserine phosphatase), ATF4 (activating transcription factor 4), and SHMT1 (serine hydroxymethyltransferase 1) genes was studied by real-time qPCR and normalized to ACTB. Results. It was found that the expression level of genes responsible for serine synthesis such as PHGDH, PSAT1, PSPH, and transcription factor ATF4 was up-regulated in U87MG glioblastoma cells under glucose and glutamine deprivations. Furthermore, inhibition of ERN1 significantly enhances the impact of glucose and especially glutamine deprivations on these gene expressions. At the same time, the expression of the SHMT1 gene, which is responsible for serine conversion to glycine, was down-regulated in both nutrient deprivation conditions with more significant changes in ERN1 knockdown glioblastoma cells. Conclusion. Taken together, the results of present study indicate that the expression of genes responsible for serine synthesis is sensitive to glucose and glutamine deprivations in gene-specific manner and that suppression of ERN1 signaling significantly modifies the impact of both glucose and glutamine deprivations on PHGDH, PSAT1, PSPH, ATF4, and SHMT1 gene expressions and reflects the ERN1-mediated genome reprograming introduced by nutrient deprivation condition.

Atypical phenotypes and novel OCRL variations in southern Chinese patients with Lowe syndrome.

BACKGROUND: Lowe syndrome is characterized by the presence of congenital cataracts, psychomotor retardation, and dysfunctional proximal renal tubules. This study presents a case of an atypical phenotype, investigates the genetic characteristics of eight children diagnosed with Lowe syndrome in southern China, and performs functional analysis of the novel variants. METHODS: Whole-exome sequencing was conducted on eight individuals diagnosed with Lowe syndrome from three medical institutions in southern China. Retrospective collection and analysis of clinical and genetic data were performed, and functional analysis was conducted on the five novel variants. RESULTS: In our cohort, the clinical symptoms of the eight Lowe syndrome individuals varied. One patient was diagnosed with Lowe syndrome but did not present with congenital cataracts. Common features among all patients included cognitive impairment, short stature, and low molecular weight proteinuria. Eight variations in the OCRL gene were identified, encompassing three previously reported and five novel variations. Among the novel variations, three nonsense mutations were determined to be pathogenic, and two patients harboring novel missense variations of uncertain significance exhibited severe typical phenotypes. Furthermore, all novel variants were associated with altered protein expression levels and impacted primary cilia formation. CONCLUSION: This study describes the first case of an atypical Lowe syndrome patient without congenital cataracts in China and performs a functional analysis of novel variants in the OCRL gene, thereby expanding the understanding of the clinical manifestations and genetic diversity associated with Lowe syndrome.

Systematic Analysis of the Effect of Genomic Knock-Out of Non-Essential Promiscuous HAD-Like Phosphatases YcsE, YitU and YwtE on Flavin and Adenylate Content in Bacillus Subtilis.

Studying the metabolic role of non-essential promiscuous enzymes is a challenging task, as genetic manipulations usually do not reveal at which point(s) of the metabolic network the enzymatic activity of such protein is beneficial for the organism. Each of the HAD-like phosphatases YcsE, YitU and YwtE of Bacillus subtilis catalyzes the dephosphorylation of 5-amino-6-ribitylamino-uracil 5'-phosphate, which is essential in the biosynthesis of riboflavin. Using CRISPR technology, we have found that the deletion of these genes, individually or in all possible combinations failed to cause riboflavin auxotrophy and did not result in significant growth changes. Analysis of flavin and adenylate content in B. subtilis knockout mutants showed that (i) there must be one or several still unidentified phosphatases that can replace the deleted proteins; (ii) such replacements, however, cannot fully restore the intracellular content of any of three flavins studied (riboflavin, FMN, FAD); (iii) whereas bacterial fitness was not significantly compromised by mutations, the intracellular balance of flavins and adenylates did show some significant changes.

Insight into the function of voltage-sensing phosphatase in hindgut-derived pseudoplacenta of a viviparous teleost Xenotoca eiseni.

Nutrient absorption is essential for animal survival and development. Our previous study on zebrafish reported that nutrient absorption in lysosome-rich enterocytes (LREs) is promoted by the voltage-sensing phosphatase (VSP), which regulates phosphoinositide (PIP) homeostasis via electrical signaling in biological membranes. However, it remains unknown whether this VSP function is shared by different absorptive tissues in other species. Here, we focused on the function of VSP in a viviparous teleost Xenotoca eiseni, whose intraovarian embryos absorb nutrients from the maternal ovarian fluid through a specialized hindgut-derived pseudoplacental structure called trophotaenia. Xenotoca eiseni VSP (Xe-VSP) is expressed in trophotaenia epithelium, an absorptive tissue functionally similar to zebrafish LREs. Notably, the apical distribution of Xe-VSP in trophotaenia epithelial cells closely resembles zebrafish VSP (Dr-VSP) distribution in zebrafish LREs, suggesting a shared role for VSP in absorptive tissues between the two species. Electrophysiological analysis using a heterologous expression system revealed that Xe-VSP preserves functional voltage sensors and phosphatase activity with the leftward shifted voltage sensitivity compared with zebrafish VSP (Dr-VSP). We also identified a single amino acid variation in the S4 helix of Xe-VSP as one of the factors contributing to the leftward shifted voltage sensitivity. This study highlights the biological variation and significance of VSP in various animal species, as well as hinting at the potential role of VSP in nutrient absorption in X. eiseni trophotaenia.NEW & NOTEWORTHY We investigate the voltage-sensing phosphatase (VSP) in Xenotoca eiseni, a viviparous fish whose intraovarian embryos utilize trophotaenia for nutrient absorption. Although X. eiseni VSP (Xe-VSP) shares key features with known VSPs, its distinct voltage sensitivity arises from species-specific amino acid variation. Xe-VSP in trophotaenia epithelium suggests its involvement in nutrient absorption, similar to VSP in zebrafish enterocytes and potentially in species with similar absorptive cells. Our findings highlight the potential role of VSP across species.