Urease Gene Research Articles

Polymerase chain reaction–hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation

In this article, we discuss the polymerase chain reaction (PCR)–hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA–BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase–streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR–hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR–hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications.

Structural and transcriptional characterization of a novel member of the soybean urease gene family

In plants, ureases have been related to urea degradation, to defense against pathogenic fungi and phytophagous insects, and to the soybean−Bradyrhizobium japonicum symbiosis. Two urease isoforms have been described for soybean: the embryo-specific, encoded by Eu1 gene, and the ubiquitous urease, encoded by Eu4. A third urease-encoding locus exists in the completed soybean genome. The gene was designated Eu5 and the putative product of its ORF as SBU-III. Phylogenetic analysis shows that 41 plant, moss and algal ureases have diverged from a common ancestor protein, but ureases from monocots, eudicots and ancient species have evolved independently. Genomes of ancient organisms present a single urease-encoding gene and urease-encoding gene duplication has occurred independently along the evolution of some eudicot species. SBU-III has a shorter amino acid sequence, since many gaps are found when compared to other sequences. A mutation in a highly conserved amino acid residue suggests absence of ureolytic activity, but the overall protein architecture remains very similar to the other ureases. The expression profile of urease-encoding genes in different organs and developmental stages was determined by RT-qPCR. Eu5 transcripts were detected in seeds one day after dormancy break, roots of young plants and embryos of developing seeds. Eu1 and Eu4 transcripts were found in all analyzed organs, but Eu4 expression was more prominent in seeds one day after dormancy break whereas Eu1 predominated in developing seeds. The evidence suggests that SBU-III may not be involved in nitrogen availability to plants, but it could be involved in other biological role(s).

Prevalence of Ureaplasma urealyticum and Mycoplasma hominis in High Vaginal Swab Samples of Infertile Females.

Background:Mycoplasma hominis and Ureaplasma urealyticum are important causative agents of vaginitis, cervicitis, postpartum sepsis, reproductive infections and infertility in both males and females.Objectives:According to the uncertain prevalence of U. urealyticum and M. hominis in Iranian infertile females, the present study was carried out to determine the prevalence of U. urealyticum and M. hominis in high vaginal swab samples of fertile and infertile females.Patients and Methods:A total of 350 high vaginal swab specimens were taken from fertile and infertile females. Samples were cultured and those that were positive for bacteria were subjected to the polymerase chain reaction (PCR) for further confirmation.Results:Of the 350 collected samples, eleven were positive for M. hominis (3.14%), fifteen were positive for U. urealyticum (4.28%) and five were positive for both of them (1.42%). Prevalence of U. urealyticum and M. hominis in the high vaginal parts of infertile females was higher than fertile females (P < 0.05). The results of traditional method were also confirmed, using the PCR amplification of urease gene of U. urealyticum and 16SrRNA gene of the M. hominis. Ureaplasma urealyticum and M. hominis had a higher prevalence in the high vaginal samples collected during the summer season.Conclusions:Considerable prevalence of M. hominis and U. urealyticum in the high vaginal swab samples of infertile females compared to the low prevalence in fertile females may suggest that these two pathogens can be cause infertility. Application of the PCR method is recommended for rapid and sensitive detection of M. hominis and U. urealyticum in high vaginal swab samples.

Factors influencing nitrification rates and the abundance and transcriptional activity of ammonia‐oxidizing microorganisms in the dark northeast P acific O cean

Pelagic marine Thaumarchaea play a primary role in ammonia oxidation, an integral part of nitrification and the nitrogen cycle. This study examines how physicochemical and biological variables influence rates of nitrification and the distribution, abundance and activity of ammonia oxidizers throughout the dark northeast Pacific Ocean. Nitrification rates are highest near the epipelagic-upper mesopelagic transition and decrease with depth according to a Martin-like power function, suggestive of a coupling to the organic matter flux. In contrast, archaeal and bacterial ammonia monooxygenase (amoA) gene abundance remains fairly constant throughout the upper mesopelagic. Density-based composites reveal nitrification to be highest in the upper pycnocline, within the nitrate:silica and ammonium maxima, while ammonia-oxidizing archaea (AOA) abundances are highest in the lower pycnocline. Water column group A (WCA) and B (WCB) AOA amoA genes are present throughout the dark ocean but have no relationship to nitrification rates. WCA comprise the majority of the AOA community above 200 m and WCB comprise the majority of it below 500 m, largely because WCA abundances decrease precipitously from 200 m to 500 m. WCA and WCB amoA genes are actively transcribed throughout the dark ocean, irrespective of conditions. Thaumarchaeal urease (ureC) genes are also present throughout, implying a widespread capacity for mixotrophy; however, unlike amoA, their expression is not detectable. Together, the results support a strong linkage between organic matter flux and nitrification rates, identify density as an important control over AOA distributions, and suggest that WCA and WCB distributions are influenced by the availability of their preferred substrates in the dark ocean.

해수에서 분리한 Photobacterium sp. Strain HA-2가 보유하는 요소분해효소 유전자의 유전적 특징

In this study, we cloned and sequenced the 15,204-bp DNA region containing the gene cluster for urease production from the chromosome of the environmental Photobacterium sp. strain HA-2. We identified 15 open reading frames (ORFs) and the G+C content was 40.3%. The urease gene cluster of Photobacterium sp. strain HA-2 consisted of seven genes, namely, ureDABCEF and ureG. There were five ORFs of urease genes in the opposite direction, which were homologous to the nickel transport operons (nik) of Vibrio parahaemolyticus and Escherichia coli. The genetic organization and sequences of the urease genes of Photobacterium sp. strain HA-2 resembled those found in Vibrio fischeri and V. parahaemolyticus.

Expanded metabolic versatility of ubiquitous nitrite-oxidizing bacteria from the genus Nitrospira

Nitrospira are a diverse group of nitrite-oxidizing bacteria and among the environmentally most widespread nitrifiers. However, they remain scarcely studied and mostly uncultured. Based on genomic and experimental data from Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II, we identified ecophysiological traits that contribute to the ecological success of Nitrospira. Unexpectedly, N. moscoviensis possesses genes coding for a urease and cleaves urea to ammonia and CO2. Ureolysis was not observed yet in nitrite oxidizers and enables N. moscoviensis to supply ammonia oxidizers lacking urease with ammonia from urea, which is fully nitrified by this consortium through reciprocal feeding. The presence of highly similar urease genes in Nitrospira lenta from activated sludge, in metagenomes from soils and freshwater habitats, and of other ureases in marine nitrite oxidizers, suggests a wide distribution of this extended interaction between ammonia and nitrite oxidizers, which enables nitrite-oxidizing bacteria to indirectly use urea as a source of energy. A soluble formate dehydrogenase lends additional ecophysiological flexibility and allows N. moscoviensis to use formate, with or without concomitant nitrite oxidation, using oxygen, nitrate, or both compounds as terminal electron acceptors. Compared with Nitrospira defluvii from lineage I, N. moscoviensis shares the Nitrospira core metabolism but shows substantial genomic dissimilarity including genes for adaptations to elevated oxygen concentrations. Reciprocal feeding and metabolic versatility, including the participation in different nitrogen cycling processes, likely are key factors for the niche partitioning, the ubiquity, and the high diversity of Nitrospira in natural and engineered ecosystems.

Elongated Helicobacter pylori in gastric mucosa of children associated with gastric disease

Two human gastric pathogens, Helicobacter pylori and Helicobacter heilmannii are apparently different genetically as well as morphologically. H. pylori have short spiral morphology while H. heilmannii is a long spiral bacterium with four or more turns. In this study, we observed elongated, spiral, Gram-negative organisms in crush smears of antral biopsies from two children with upper abdominal pain and one with celiac disease; all these biopsies were positive for rapid urease test. Histologically the children had mild to moderate gastritis. Ultra-structurally, the organisms were more elongated than H. pylori and spiral shaped with periplasmic space. They were grown on chocolate Brucella agar and colonies were identified as H. pylori biochemically and by urease gene based polymerase chain reaction. Nuclear magnetic resonance study of sonicated extracts of these organisms showed similar metabolic peaks as H. pylori. 16S-rDNA sequencing results confirmed that the isolated organisms were H. pylori. The study suggests that a morphologically different (elongated) form of H. pylori strain exists in the stomach of children and is associated with gastric pathology.

Draft Genome Sequence of Strain ATCC 33958, Reported To Be Elizabethkingia miricola.

We report the draft genome of Elizabethkingia strain ATCC 33958, which has been classified as Elizabethkingia miricola. Similar to other Elizabethkingia species, the ATCC 33958 draft genome contains numerous β-lactamase genes. ATCC 33958 also harbors a urease gene cluster which supports classification as E. miricola.

Engineering the gut microbiota to treat hyperammonemia.

Increasing evidence indicates that the gut microbiota can be altered to ameliorate or prevent disease states, and engineering the gut microbiota to therapeutically modulate host metabolism is an emerging goal of microbiome research. In the intestine, bacterial urease converts host-derived urea to ammonia and carbon dioxide, contributing to hyperammonemia-associated neurotoxicity and encephalopathy in patients with liver disease. Here, we engineered murine gut microbiota to reduce urease activity. Animals were depleted of their preexisting gut microbiota and then inoculated with altered Schaedler flora (ASF), a defined consortium of 8 bacteria with minimal urease gene content. This protocol resulted in establishment of a persistent new community that promoted a long-term reduction in fecal urease activity and ammonia production. Moreover, in a murine model of hepatic injury, ASF transplantation was associated with decreased morbidity and mortality. These results provide proof of concept that inoculation of a prepared host with a defined gut microbiota can lead to durable metabolic changes with therapeutic utility.

Phosphorylation-dependent and Phosphorylation-independent Regulation of Helicobacter pylori Acid Acclimation by the ArsRS Two-component System.

The pH-sensitive Helicobacter pylori ArsRS two-component system (TCS) aids survival of this neutralophile in the gastric environment by directly sensing and responding to environmental acidity. ArsS is required for acid-induced trafficking of urease and its accessory proteins to the inner membrane, allowing rapid, urea-dependent cytoplasmic and periplasmic buffering. Expression of ArsR, but not its phosphorylation, is essential for bacterial viability. The aim of this study was to characterize the roles of ArsS and ArsR in the response of H. pylori to acid. Wild-type H. pylori and an arsR(D52N) phosphorylation-deficient strain were incubated at acidic or neutral pH. Gene and protein expression, survival, membrane trafficking of urease proteins, urease activity, and internal pH were studied. Phosphorylation of ArsR is not required for acid survival. ArsS-driven trafficking of urease proteins to the membrane in acid, required for recovery of internal pH, is independent of ArsR phosphorylation. ArsR phosphorylation increases expression of the urease gene cluster, and the loss of negative feedback in a phosphorylation-deficient mutant leads to an increase in total urease activity. ArsRS has a dual function in acid acclimation: regulation of urease trafficking to UreI at the cytoplasmic membrane, driven by ArsS, and regulation of urease gene cluster expression, driven by phosphorylation of ArsR. ArsS and ArsR work through phosphorylation-dependent and phosphorylation-independent regulatory mechanisms to impact acid acclimation and allow gastric colonization. Furthering understanding of the intricacies of acid acclimation will impact the future development of targeted, nonantibiotic treatment regimens.

Subcutaneous immunization with a novel immunogenic candidate (urease) confers protection against Brucella abortus and Brucella melitensis infections.

Brucellosis is a world prevalent endemic illness that is transmitted from domestic animals to humans. Brucella spp. exploits urease for survival in the harsh conditions of stomach during the gastrointestinal infection. In this study, we examined the immune response and the protection elicited by using recombinant Brucella urease (rUrease) vaccination in BALB/c mice. The urease gene was cloned in pET28a and the resulting recombinant protein was employed as subunit vaccine. Recombinant protein was administered subcutaneously and intraperitoneally. Dosage reduction was observed with subcutaneous (SC) vaccination when compared with intraperitoneal (IP) vaccination. rUrease induced mixed Th1-Th2 immune responses with high titers of specific IgG1 and IgG2a. In lymphocyte proliferation assay, splenocytes from IP and SC-vaccinated mice displayed a strong recall proliferative response with high amounts of IL-4, IL-12 and IFN-γ production. Vaccinated mice were challenged with virulent Brucella melitensis, B.abortus and B.suis. The SC vaccination route exhibited a higher degree of protection than IP vaccination (p value≤0.05). Altogether, our results indicated that rUrease could be a useful antigen candidate for the development of subunit vaccines against brucellosis.

Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor.

Azotobacter vinelandii is a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an obligate aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a free-living bacterium, A. vinelandii has evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies of A. vinelandii producing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability of A. vinelandii to recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter gene amtB that resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green alga Chlorella sorokiniana confirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of the A. vinelandii in coculture. The nature and quantities of nitrogen released by urease and amtB disruptions were further compared to strains reported in previous efforts that altered the nifLA regulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes.

Complete Genome Sequences of Two Helicobacter pylori Strains from a Canadian Arctic Aboriginal Community.

We report here the complete genome sequences of two Amerind Helicobacter pylori strains from Aklavik, Northwest Territories, Canada. One strain contains extra iron-cofactored urease genes and ~140 rearrangements in its chromosome relative to other described strains (typically differing from one another by <10 rearrangements), suggesting that it represents a novel lineage of H. pylori.

Coccidioides species determination: does sequence analysis agree with restriction fragment length polymorphism?

Fifteen Coccidioides isolates were previously examined for genetic diversity using restriction fragment length polymorphism (RFLP); two fragment patterns were observed. Two isolates demonstrated one banding pattern (designated RFLP group I), while the remaining 13 isolates demonstrated a second pattern (designated RFLP group II). Recently, molecular studies supported the division of the genera Coccidioides into two species: Coccidioides posadasii and Coccidioides immitis. It has been assumed that the species division corresponds to the RFLP grouping. We tested this hypothesis by amplifying the ribosomal DNA internal transcribed spacer region as well as the dioxygenase, serine proteinase, and urease genes from 13 isolates previously examined by RFLP and then sequencing the PCR products. The appropriate species for each isolate was assigned using phylogenetically informative sites. The RFLP grouping agreed with the Coccidioides species assignment for all but one isolate, which may represent a hybrid. In addition, polymorphic sites among the four genes examined were in agreement for species assignment such that analysis of a single gene may be sufficient for species assignment.

Complete Genome Sequence of Corynebacterium ureicelerivorans DSM 45051, a Lipophilic and Urea-Splitting Isolate from the Blood Culture of a Septicemia Patient.

Corynebacterium ureicelerivorans is an opportunistic pathogen with a lipophilic lifestyle and an exceptionally high urease activity. The genome sequence of the type strain revealed that lipophilism is caused by the lack of a fatty acid synthase gene. The ureABCEFGD genes are similar to the urease gene region of Corynebacterium glucuronolyticum.

Detection of the tetM resistance determinant among phenotypically sensitive Ureaplasma species by a novel real-time PCR method

The study aimed to identify the proportion of tetM-positive Ureaplasma spp. isolates phenotypically susceptible to tetracycline by real-time PCR. Ureaplasma spp. strains of urogenital origin were isolated from 100 female or male adults on A7 agar plates. The presence of Ureaplasma was confirmed by the presence of urease gene by a novel real-time PCR method. Genotyping and sensitivity to tetracyclines were examined using commercial methods. The tetM gene was detected by a novel real-time PCR method especially designed for this study. Ureaplasma parvum was isolated from 87 of the specimens; Ureaplasma urealyticum, from 12; and both species were isolated from a single specimen. All isolates were phenotypically susceptible to tetracyclines. Thirty-five strains were tetM carriers; 29 (82.9%), U. parvum; 5 (14.3%), U. urealyticum; and 1 (2.9%), U. parvum/U. urealyticum. No statistically significant difference was observed between the 3 groups. Four (40%) tetM carriers were isolated from 10 symptomatic men; 11 (32.4%), from 34 symptomatic women; and 20 (35.7%), from 56 asymptomatic women. No statistically significant difference was observed between the 3 groups. The tetM determinant is detected in 35% of phenotypically susceptible to tetracycline Ureaplasma spp. Greek isolates. The use of a real-time PCR technique is particularly helpful, as it makes its detection easy; cost-effective; rapid; and, therefore, more convenient for the surveillance of the dissemination of the tetM resistance gene.

Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis.

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.

Genome analysis of NDM-1 producing Morganella morganii clinical isolate

Objective: To analyze the resistome and virulence genes of Morganella morganii F675, a multidrug-resistant clinical isolate using whole genome sequencing (WGS). Methods: M. morganii F675 was isolated from a patient from Jerusalem, Israel. WGS was performed using both 454 and SOLiD sequencing technologies. Analyses of the bacterial resistome and other virulence genes were performed in addition to comparison with other available M. morganii genomes. Results: The assembled sequence had a genome size of 4,127,528 bp with G+C content of 51%. The resistome consisted of 13 antibiotic resistance genes including blaNDM-1 located in a plasmid likely acquired from Acinetobacter spp. Moreover, we characterized for the first time the whole lipid A biosynthesis pathway in this species along with the O-antigen gene cluster, the urease gene cluster and several other virulence genes. Conclusion: The WGS analysis of this pathogen further provides insight into its pathogenicity and resistance to antibiotics.

High-level expression and characterization of recombinant acid urease for enzymatic degradation of urea in rice wine.

Ethylcarbamate, a carcinogenic compound, is formed from urea and ethanol in rice wine, and enzymatic elimination of urea is always attractive. In the present work, we amplified the acid urease gene cluster ureABCEFGD from Lactobacillus reuteri CICC6124 and constructed robust Lactococcus lactis cell factories for the production of acid urease. The titer of the recombinant acid urease was increased from 1,550 to 11,560 U/L by optimization of the cultivation process. Meanwhile, the enzyme showed satisfied properties toward urea elimination in the rice wine model system. By incubating the enzyme (50 U/L) at 20 °C for 60 h, about 95.8% of urea in rice wine was removed. Interestingly, this acid urease also exhibited activity toward ethylcarbamate. The results demonstrated that this recombinant acid urease has great potential in the elimination of urea in rice wine.

Su1184 A Randomised Study Comparing 10 Days Concomitant and Sequential Treatments for the Eradication of Helicobacter pylori, in a High Clarithromycin Resistance Area

G A A b st ra ct s assay on research rhesus macaques. At necropsy, saline gastric wash and gastric tissues were collected from 15 NHPs from two colonies and were evaluated by 16S rRNA and urease genes PCR and sequence analysis for Helicobacter genus and species. Formalin-fixed paraffin embedded tissues were evaluated using H&E,Warthin-Starry, and fluorescent in situ hybridization (FISH). Histologically, all 15 NHPs had mild to moderate lymphoplasmacytic gastric inflammation. Fourteen of 15 NHPs were considered positive for H. spp. infection based on genus-level PCR products of gastric tissue. Of these, two NHPs were negative for H. spp. infection via PCR of gastric wash, demonstrating an assay sensitivity of 93%. Urease gene PCR and 16S rRNA sequence analysis of gastric wash and gastric tissues identified one NHP co-infected with both H. pylori and H. suis, and seven and five NHPs were mono-infected with H. pylori and H. suis, respectively. Gastric wash was 100% sensitive and specific for speciation of H. suis by species-specific PCR assays. Given that PCR based assays are reliable and gastric wash is a sensitive, easily performed, non-invasive procedure, this technique can be easily incorporated into routine exams of captive NHPs to evaluate gastric H. spp. infection. In addition, this technique may be useful in the human clinic as a cost-efficient and effective way to diagnosis and speciate gastric helicobacteriosis.