Frc Gene Research Articles

Probiotic Oxalate-Degrading Bacteria: New Insight of Environmental Variables and Expression of the oxc and frc Genes on Oxalate Degradation Activity

Oxalate, a compound produced by many edible plants and as a terminal metabolite in the liver of mammals, is a toxin that has a detrimental role to human health. Humans and other mammals do possess enzymatic systems to degrade oxalate. Moreover, numerous oxalate-degrading bacteria reside in the mammalian gut and, thus, provide an important function for hosts. The current review focuses on the environmental factors that influence the efficacy of probiotic oxalate-degrading bacteria, relative to oxalate metabolism. We describe the mechanism of oxalate catabolism and its consumption by obligate and facultative anaerobic oxalate-degrading bacteria, in both in vitro and in vivo environments. We also explore the environmental variables that impact oxalate degradation. Studies on single species degrade oxalate have not shown a strong impact on oxalate metabolism, especially in high oxalate conditions such as consumption of foods high in oxalate (such as coffee and chocolate for humans or halogeton in animal feed). Considering effective variables which enhance oxalate degradation could be used in application of effective probiotic as a therapeutic tool in individuals with hyperoxaluria. This study indicates probiotics can be considered a good source of naturally occurring oxalate degrading agent in human colon.

The Effect of Oxalobacter formigenes Colonization in Patients with Calcium Oxalate Renal Stones in Comparison with Healthy People in Qom: A Case-Control Study

Background: Urinary stones are a major problem world, and their incidence has increased significantly in recent years. Objectives: This study aimed to develop a simple and rapid molecular method based on PCR and qPCR assays to detect Oxalobacter formigenes (which causes oxalate degradation in intestines) in fecal samples of healthy volunteers and patients with calcium oxalate nephrolithiasis, and determine the amount of urinary oxalate in the two groups. Methods: This study was performed on urine and fecal samples of 73 patients with kidney stones and 52 healthy individuals. After DNA extraction, PCR and qPCR assays were performed on two gene regions of O. formigenes, OXC, and FRC. Also, urine oxalate was measured in the study population using biochemical methods. Results: We found that the presence of O. formigenes could reduce the risk of kidney stones and calcium oxalate stones. In fact, both FRC and OXC genes were involved in the diagnosis of O. formigenes; however, the results based on the FRC gene showed higher efficiency. In addition, the presence or absence of stones did not affect the amount of urinary excretion of oxalate, rather it is affected by diet. Conclusions: Molecular identification of O. formigenes by PCR and qPCR assays allows rapid, specific, and reproducible detection in fecal samples, which also allows immediate processing of these samples in clinical conditions.

Distinct response patterns of soil bacteria to oxalate imply their role in buffering soil acidification: Evidence from red soils with long‐term fertilisation regimes

AbstractLong‐term chemical‐only fertilisation (NPK) decreases soil pH, but this soil acidification is rarely reported with manure fertilisation (M). Soil acidification always leads to a negative effect on sustainable development. As oxalate oxidation is accompanied by soil alkalisation, it is still unknown how and to what extent bacterial oxalate oxidation in soils with different fertilisation regimes contributes to buffering of soil acidification. To assess the potential role of oxalotrophy in buffering soil acidification, two soils that underwent contrasted fertilisation regimes for over 30 years were used. The oxalotrophic communities were characterised based on a biomarker of oxalotrophic taxa (the frc gene), and their responsiveness to calcium oxalate (CaOx) addition was assessed. In addition, stable isotope probing was employed to explore the active bacteria responding to CaOx addition. Despite similar abundances of oxalotrophic bacteria between NPK and M soils before microcosm incubation, the M soil harboured more oxalotrophic bacteria belonging to Deltaproteobacteria, and the NPK soil was colonised by more oxalotrophic bacteria affiliated with Actinobacteria, Alphaproteobacteria, and Betaproteobacteria. In the incubation experiments, the M soil took half of the time that the NPK soil needed to deplete the added CaOx. The faster CaOx consumption rate could be explained by the higher functional redundancy and higher proportion of fast‐response oxalotrophic taxa in M soil. The most dominant active oxalotrophic taxa were Oxalicibacterium and Burkholderia in the M and NPK soils, respectively. Furthermore, the better performance of oxalate oxidation was conjectured to contribute to a higher soil acidification buffering ability. This study obtained an important cue to the feasibility of active oxalotrophic taxa in buffering acidification in soils with different fertilisation regimes, which provides valuable evidence for exploring alternative ways for alleviating soil acidification.

A functional analysis of the formyl-coenzyme A (frc) gene fromLactobacillus reuteri100-23C

To examine the role of the Lactobacillus reuteri 100-23C frc gene product in oxalate metabolism, host colonization and the acid stress response. Genes encoding putative formyl-CoA transferase (frc) and oxalyl-CoA decarboxylase (oxc) enzymes are present in the genome sequences of Lact.reuteri strains. Two strains isolated from humans harboured an IS200 insertion sequence in the frc ORF and a group 2 intron-associated transposase downstream of the frc gene, both of which were lacking in two strains of animal origin, which contained intact frc and oxc genes. An frc(-) insertional mutant of Lact.reuteri 100-23C was compared with the parent strain with respect to oxalate degradation, colonization of an RLF-mouse host model and growth in the presence of acids. Neither parent nor mutant degraded oxalate in vitro or in vivo. However, the parent outcompeted the frc(-) mutant in the mouse intestine during co-colonization and the frc(-) mutant showed a reduced growth rate in the presence of hydrochloric acid. Intact oxc and frc genes do not ensure oxalate degradation under the conditions tested. The frc gene product is important during host colonization and survival of acid stress by Lact.reuteri 100-23C. Oxalate metabolism by oxalate-degrading intestinal bacterial strains may be important in preventing urolithiasis and might lead to the derivation of probiotic products. To produce safe and efficacious probiotics, however, an understanding of the genetic characteristics of potential oxalate degraders must be obtained, together with knowledge of their functional ramifications.

Identification of active oxalotrophic bacteria by Bromodeoxyuridine DNA labeling in a microcosm soil experiments

The oxalate-carbonate pathway (OCP) leads to a potential carbon sink in terrestrial environments. This process is linked to the activity of oxalotrophic bacteria. Although isolation and molecular characterizations are used to study oxalotrophic bacteria, these approaches do not give information on the active oxalotrophs present in soil undergoing the OCP. The aim of this study was to assess the diversity of active oxalotrophic bacteria in soil microcosms using the Bromodeoxyuridine (BrdU) DNA labeling technique. Soil was collected near an oxalogenic tree (Milicia excelsa). Different concentrations of calcium oxalate (0.5%, 1%, and 4% w/w) were added to the soil microcosms and compared with an untreated control. After 12 days of incubation, a maximal pH of 7.7 was measured for microcosms with oxalate (initial pH 6.4). At this time point, a DGGE profile of the frc gene was performed from BrdU-labeled soil DNA and unlabeled soil DNA. Actinobacteria (Streptomyces- and Kribbella-like sequences), Gammaproteobacteria and Betaproteobacteria were found as the main active oxalotrophic bacterial groups. This study highlights the relevance of Actinobacteria as members of the active bacterial community and the identification of novel uncultured oxalotrophic groups (i.e. Kribbella) active in soils.

In vitro hepatic differentiation of oxalate-degrading human bone mesenchymal stem cells

Objective To induce the differentiation of human bone mesenchmal stem cells with oxalate-degrading genes (hMSCs-frc-oxc) into hepatocyte-like cells in vitro.Methods The hMSCs-frc-oxc in passage 4 were cultured and induced to differentiate into hepatocyte-like cells in differentiation medium supplemented with 40 μg/L hepatocyte growth factor (HGF) and 10 μg/L fibroblast growth factors-4 (FGF-4).The morphological changes of cells were observed microscopically.The RNA and protein levels of hepatic markers [alpha fetal protein (AFP),cytokeratin-18 (CK-18) and albumin (ALB)] and oxalate-degrading genes (frc and oxc) were detected by using immunocytochemistry,Western blotting and real-time quantitative polymerase chain reaction (Real-time PCR).L-02 cells and transfected mock-vehicle hMSCs (hMSCsvector) were used as positive and negative controls,respectively.Results The hMSCs-frc-oxc were cultured successfully in vitro.After induction,hMSCs-frc-oxc gradually transformed into polygonal cells.Real-time PCR revealed the expression of hepatic markers (AFP,ALB and CK-18) and oxalate-degrading genes (frc and oxc) in hMSCs-frc-oxc after inducting for 14 days and 28 days,whereas hMSCs cultured alone did not show liver specific gene expression.Immunocytochemistry revealed positive ALB expression in cells differentiating for 28 days.Western blotting revealed that the expression levels of AFP and CK-18 in differentiated cells were higher than in undifferentiated cells (P ＜ 0.05).The differentiated hMSCs-frc-oxc also expressed the oxalate-degrading genes products (myc-FCOAT and flag-OCOAD).Conclusion The hMSCs with oxalate-degrading genes could be induced to differentiate into hepatocyte-like cell by HGF and FGF-4 at certain concentrations and the hepatic differentiated hMSCs-frc-oxc could express the oxalate-degrading genes and their products. Key words: Bone mesenchymal stem cells ; Cell differentiation ; Hepatocyte ; Frc gene ; Oxc gene

The construction of an oxalate-degrading intestinal stem cell population in mice: a potential new treatment option for patients with calcium oxalate calculus

About 80% of all urological stones are calcium oxalate, mainly caused by idiopathic hyperoxaluria (IH). The increased absorption of oxalate from the intestine is the major factor underlying IH. The continuous self-renewal of the intestinal epithelium is due to the vigorous proliferation and differentiation of intestinal stem cells. If the intestinal stem cell population can acquire the ability to metabolize calcium oxalate by means of oxc and frc transgenes, this will prove a promising new therapy option for IH. In our research, the oxalate-degrading genes of Oxalobacter formigenes (Oxf)-the frc gene and oxc gene-were cloned and transfected into a cultured mouse-derived intestinal SC population to give the latter an oxalate-degrading function. Oxf was isolated and cultivated and the oxalate-degrading genes-frc and oxc-were cloned. The dicistronic eukaryotic expression vector pIRES-oxc-frc was constructed and transferred into the mouse stem cell population. After selection with G418, the expression of the genes was identified. The oxalate-degrading function of transfected cells was determined by transfection into the intestinal stem cell population of the mouse. The change in oxalate concentration was determined with an ion chromatograph. The recombinant plasmid containing oxc and frc genes was transfected into the stem cell population of the mouse and the expression of the genes found normal. The cell population had acquired an oxalate-degrading function. The oxc and frc genes could be transfected into the intestinal stem cell population of the mouse and the cells acquired an oxalate-degrading function.

Use of the frc gene as a molecular marker to characterize oxalate-oxidizing bacterial abundance and diversity structure in soil

Oxalate catabolism, which can have both medical and environmental implications, is performed by phylogenetically diverse bacteria. The formyl-CoA-transferase gene was chosen as a molecular marker of the oxalotrophic function. Degenerated primers were deduced from an alignment of frc gene sequences available in databases. The specificity of primers was tested on a variety of frc-containing and frc-lacking bacteria. The frc-primers were then used to develop PCR-DGGE and real-time SybrGreen PCR assays in soils containing various amounts of oxalate. Some PCR products from pure cultures and from soil samples were cloned and sequenced. Data were used to generate a phylogenetic tree showing that environmental PCR products belonged to the target physiological group. The extent of diversity visualised on DGGE pattern was higher for soil samples containing carbonate resulting from oxalate catabolism. Moreover, the amount of frc gene copies in the investigated soils was detected in the range of 1.64 × 10 7 to 1.75 × 10 8/g of dry soil under oxalogenic tree (representing 0.5 to 1.2% of total 16S rRNA gene copies), whereas the number of frc gene copies in the reference soil was 6.4 × 10 6 (or 0.2% of 16S rRNA gene copies). This indicates that oxalotrophic bacteria are numerous and widespread in soils and that a relationship exists between the presence of the oxalogenic trees Milicia excelsa and Afzelia africana and the relative abundance of oxalotrophic guilds in the total bacterial communities. This is obviously related to the accomplishment of the oxalate–carbonate pathway, which explains the alkalinization and calcium carbonate accumulation occurring below these trees in an otherwise acidic soil. The molecular tools developed in this study will allow in-depth understanding of the functional implication of these bacteria on carbonate accumulation as a way of atmospheric CO 2 sequestration.

Stable expression of the oxc and frc genes from Oxalobacter formigenes in human embryo kidney 293 cells: Implications for gene therapy of hyperoxaluria

Hyperoxaluria can lead to multiple pathologic conditions such as recurrent urolithiasis, oxalosis, nephrocalcinosis and even renal failure, but there is no known oxalate-degrading pathway in the human body, and current therapeutic options for patients with hyperoxaluria are limited. Oxalyl-CoA decarboxylase and formyl-CoA transferase are the key enzymes in the oxalate catabolism of Oxalobacter formigenes which dwell in the intestine of vertebrates and have an important symbiotic relationship with their hosts. The aim of this study was to insert the oxalate-degrading enzyme genes into human embryo kidney (HEK) 293 cells and to evaluate if the oxalate-degrading enzymes could be expressed in these cells and keep their enzyme activity. We present here the cloning of the two genes from O. formigenes and their subsequent expression in HEK293 cells. The results showed that the expression of oxalyl-CoA decarboxylase and formyl-CoA transferase was confirmed by RT-PCR and Western blotting, and the proteins were located in the cytoplasm of transfected cells. Furthermore, the transfected cells were capable to a certain degree of degrading oxalate. These findings suggest that the transfer of oxalate-degrading enzyme genes into human cells is possibly a potential candidate for the gene therapy of hyperoxaluria.

Cloning and identification of frc gene from Oxalobacter frmigenes

The cloning and identification of frc gene from Oxalobacter formigenes in the intestines of Chinese people were conducted. The genomic DNA of Oxalobacter formigenes was extracted. frc gene fragment was amplified by polymerase chain reaction (PCR) and linked with pEGFP-C1. The recombinant plasmid was designated pEGFP-frc and was identified by restriction-enzyme digestion and sequencing. Human embryo kidney 293 cells were transfected with pEGFP-frc, then RT-PCR and Western blotting were performed to detect the expression of frc gene. The length of frc gene was found to be 1287 bp, and the homology of nucleotides and amino-acid residue with the sequence in GenBank was 95.88% and 99.07%. Bright green fluorescent light could be observed in 293 cells transfected with the pEGFP-frc. frc mRNA and fusion protein FCoAT-EGFP were detected in the cells. It is concluded that frc gene cloned from the Oxalobacter formigenes in the intestines of Chinese people can be expressed in eucaryotic 293 cells and keep its enzyme activity.

Detection and identification of oxalate-degrading bacteria in human feces.

Oxalate is detoxified (catabolized) via the action of two enzymatic proteins, formyl coenzyme A transferase (encoded by the frc gene) and oxalyl coenzyme A decarboxylase (encoded by the oxc gene), contained in the cytosol of Oxalobacter formigenes that colonizes the human intestinal tract. It is speculated that oxalate-degrading bacteria decrease oxalate absorption from the intestines and their absence in the gastrointestinal tract correlates with the formation of calcium-oxalate urolithiasis. Two methods of detection and identification of this bacterial strain were studied in human fecal samples collected from Japanese subjects. Genomic DNA was isolated from bacterial culture, and specific 16S rDNA was amplified by polymerase chain reaction (PCR) followed by sequencing. The oxc gene was amplified directly from human feces by PCR using the specific primers. Oxalate-degrading bacteria were identified by comparing the sequences of 16S rDNA. The oxc gene was directly detected from human feces by PCR. It was ascertained that a combined PCR detection method using both 16S rDNA and the oxc gene allows for identification of O. formigenes in human fecal samples. This detection and identification method of oxalate-degrading bacteria using 16S rDNA and oxc gene should be applied in examination of clinical samples.

UDP-sugar transporter implicated in glycosylation and processing of Notch.

Glycosylation modifies protein activities in various biological processes. Here, we report the functions of a novel UDP-sugar transporter (UST74C, an alternative name for Fringe connection (Frc)) localized to the Golgi apparatus in cellular signalling of Drosophila. Mutants in the frc gene exhibit phenotypes resembling wingless and Notch mutants. Both Fringe-dependent and Fringe-independent Notch pathways are affected, and both glycosylation and proteolytic maturation of Notch are defective in mutant larvae. The results suggest that changes in nucleotide-sugar levels can differently affect Wingless and two distinct aspects of Notch signalling.

Fusion of two subunits does not impair the function of a [NiFeSe]-hydrogenase in the archaeon Methanococcus voltae.

[NiFe]-hydrogenases generally carry the bimetallic Ni-Fe reaction center on their largest subunit. The [NiFeSe]-hydrogenase Vhu from Methanococcus voltae has an unusual subunit composition. Some of the amino acids participating in the formation of the reaction center are within a separate, very small subunit, called VhuU. It consists of only 25 amino acids and contains the selenocysteinyl residue, a ligand to the Ni atom. We have tested whether the special configuration of the Vhu-hydrogenase is of particular biochemical relevance. We have constructed a fusion subunit derived from the VhuA and VhuU subunits by generating a gene fusion which was inserted into the chromosome of M. voltae by gene replacement. The enzyme was purified and shown to be as active as the wild-type enzyme. M. voltae carries the genetic information for four different [NiFe]-hydrogenases. In addition to the Vhu-hydrogenase, a second selenium-containing enzyme, Fru, has been purified. Two selenium-free enzymes, Vhc and Frc, are homologues of Vhu and Fru, respectively. Their gene groups, vhc and frc are transcribed only upon selenium depletion. The selenium-containing subunit VhuU has been implicated in their negative regulation. However, cells containing the fusion hydrogenase still exhibited normal regulation of the vhc andfrc promoter activities as tested in reporter gene constructs. This indicates that the free VhuU polypeptide is not required for the negative regulation of the vhc or frc genes.

Identification and classification of Oxalobacter formigenes strains by using oligonucleotide probes and primers.

Genomic DNAs of various strains of Oxalobacter formigenes were subjected to restriction endonuclease fragment length polymorphism- and PCR-based amplification analyses with DNA probes and primers complementary to sequences within either the oxc gene, encoding oxalyl coenzyme A (oxalyl-CoA) decarboxylase, or the frc gene, encoding formyl-CoA transferase. Oligonucleotide probes based on nonconserved sequences of oxc or frc were able to divide O. formigenes strains into at least two groups, consistent with the current separation of O. formigenes strains into groups I and II on the basis of 16S rRNA sequence similarities and lipid content. In contrast, an oligonucleotide probe based on the conserved 5' end of oxc appeared to bind all group I and the majority of group II strains. PCR amplification of the oxc gene showed even greater sensitivity in detecting O. formigenes and provided support for further division of the strains into subgroups. In addition, these oligonucleotides failed to hybridize to or amplify PCR products from whole fecal DNA isolated from fresh stool samples from an individual not colonized with O. formigenes, indicating unique specificity. Thus, these DNA analyses permit both detection as well as classification of O. formigenes strains.