Specific Urease Activity Research Articles

Improving non-uniform gravelly sand using microbially induced carbonate precipitation: An outdoor cubic-meter scale trial by engineering contractors

Soil improvement using microbially induced carbonate precipitation (MICP) remains largely confined to the laboratory, with only a very small number of large-scale experiments having been completed under field conditions and none by engineering contractors. This study presents a cubic-meter scale improvement of heterogeneous natural sand collected from a local quarry, with a wide variation in grain size, via MICP. The MICP trial was conducted by engineering contractors in a cubic test cell under variable temperatures ranging from 5 °C to 19 °C. The upscaling of cultivation of Sporosarcina pasteurii (600 L for each treatment cycle) under non-sterile conditions, as performed by engineering contractors, achieved an optical density (OD600) of 0.89 and a specific urease activity of 2.5 mM urea/min/OD600. Post-MICP-treated sands were subjected to a series of coring, block sampling and laboratory tests. The block sampling process indicated that the majority of sand was effectively cemented, with a small region near a side wall forming a less well-cemented zone, likely induced by less effective fluid delivery in this region. The unconfined compressive strengths of three cores (diameter: 10 cm, length: 22 cm) were 3.6, 4.4, and 7.6 MPa. Consolidated-drained triaxial tests on sub-sampled cores also demonstrated rock-like material behaviour, with a peak friction angle of 43.6° and peak cohesion of 0.64 MPa, and ultimate state frictional angles of 42.0° and ultimate cohesion of 0.12 MPa. The increased shear parameters of the bio-cemented samples (relative to the untreated samples) have many implications for mitigation of geotechnical hazards such as soil liquefaction. The study marks a step forward industrial implementation of MICP for soil improvement by engineering contractors without prior knowledge of MICP.

Solidification of sodium sulfate saline loess by biomineralization of reactive magnesium oxide binder

This study aims to solidify sodium sulfate saline loess by biomineralization of reactive magnesium oxide (MgO) binder. The impact of Na2SO4 concentration on the viability and urease activity of S. pasteurii, the mechanism and products of biomineralization of MgO, and the effectiveness of the biomineralization-MgO binder in solidifying saline loess with varying salt content (1%, 3%, and 5%) were investigated. Results showed that low and moderate concentrations of Na2SO4 favored bacterial proliferation. However, the presence of Na2SO4 inhibited bacterial urease activity, and the inhibition was more significant at higher Na2SO4 concentrations. In addition, low and moderate concentrations of Na2SO4 decreased the specific urease activity, whereas high concentrations of Na2SO4 significantly increased specific urease activity. S. pasteurii was able to use carbonate ions formed by urea hydrolysis for the mineralization of MgO and to form magnesium carbonate minerals dominated by rosette-like dypingite and hydromagnesite crystals. The primary mechanism involves microbial cells and extracellular polymeric substances leading to partial dehydration of Mg2+ ions from the Mg2+-H2O complex and allowing for further association with carbonate anions to from Mg-bearing carbonates. Unconfined compressive strength tests conducted on the saline loess samples after 7 days of curing revealed a significant influence of urea concentration on the strength of the solidified soil. The optimal urea concentration to obtain a better 7-day UCS ranged from 4 mol/L to 5 mol/L. Furthermore, solidified soil with 5% salinity yielded the highest 7-day UCS and soil with 3% salinity exhibited the lowest 7-day UCS at the same urea concentration. XRD and SEM analysis of the solidified soil samples indicated that the formation of magnesium carbonate minerals in the soil matrix by the biomineralization-MgO binder was responsible for the UCS enhancement. The remarkable 7-day UCS of saline loess solidified with biomineralization-MgO binder demonstrates the effectiveness of this material in curing saline loess.

Feeding strategies for Sporosarcina pasteurii cultivation unlock more efficient production of ureolytic biomass for MICP.

The bacterium Sporosarcina pasteurii is the most commonly used microorganism for Microbial Induced Calcite Precipitation (MICP) due to its high urease activity. To date, no proper fed-batch cultivation protocol for S. pasteurii has been published, even though this cultivation method has a high potential for reducing costs of producing microbial ureolytic biomass. This study focusses on fed-batch cultivation of S. pasteurii DSM33. The study distinguishes between limited fed-batch cultivation and extended batch cultivation. Simply feeding glucose to a S. pasteurii culture does not seem beneficial. However, it was exploited that S. pasteurii is auxotrophic for two vitamins and amino acids. Limited fed-batch cultivation was accomplished by feeding the necessary vitamins or amino acids to a culture lacking them. Feeding nicotinic acid to a nicotinic acid deprived culture resulted in a 24% increase of the specific urease activity compared to a fed culture without nicotinic acid limitation. Also, extended batch cultivation was explored. Feeding a mixture of glucose and yeast extract results in OD600 of ≈70 at the end of cultivation, which is the highest value published in literature so far. These results have the potential to make MICP applications economically viable.

Revisiting the urease production of MICP-relevant bacterium Sporosarcina pasteurii during cultivation

The ureolytic bacterium Sporosarcina pasteurii is commonly used for Microbial Induced Calcium Carbonate Precipitation (MICP). This process has a variety of applications in the fields of construction, geotechnical engineering, and environmental remediation. However, the factors influencing urease activity of S. pasteurii during cultivation are not yet fully understood. Even though there is debate over whether higher urease activity is in fact beneficial for MICP applications, knowledge about urease production can help provide ureolytic biomass more cost-effectively, reducing overall production costs. This study revisits the effect of various cultivation conditions on growth and urease activity by using an automated high-throughput microbioreactor system combined with a novel system for high-throughput urease activity quantification. This experimental set-up allows for unprecedented data density and enables new insights into urease production of S. pasteurii.Several factors were tested, including oxygen limitation, the feeding of urea, choice of ammonium salt, the proportion of ammonium salt in the medium, and lowering the pH during cultivation; none of these had a notable impact on urease production. Only the addition of a broad spectrum of nutrients through regular feeding with highly concentrated yeast extract and glucose led to a 70 % increased specific urease activity compared to a batch culture.These results can improve the understanding of the regulatory mechanisms governing urease expression in S. pasteurii during cultivation, and allow to adapt the cultivation process accordingly. Additionally, the new system for automated high-throughput enzyme activity determination presented in this study could be applied to optimize bioprocesses involving other ion producing enzymes.

Experimental Study on the Effective Production of Biocement for Soil Solidification and Wind Erosion Control

Biocement can be achieved through the microbially induced carbonate precipitation (MICP) process. Such a method can potentially be utilized as an eco-friendly method for civil and environmental engineering applications such as soil ground improvement and wind erosion control of surface soil. In this method, one key step is the effective production of ureolytic bacteria. In previous laboratory and field studies, the cultivation and production of the bacteria used for the MICP were usually expensive and time-consuming. The purpose of this study was to optimize the cultivation method of the ureolytic bacteria (Sporosarcina pasteurii), and soil stabilization tests were conducted to verify the effectiveness of the cultured bacteria used to strengthen soil against the wind-induced erosion. Bacterial cultivation methods were studied by investigating the effects of different cultivation media and conditions. Testing variables included the types and concentrations of nitrogen sources (urea or NH4Cl), pH values (7.5–9.5), cultivation conditions (batch or chemostat condition), and different carbon sources. It was found that, with the same amount of nitrogen source, the test with pure urea had the highest biomass yield, urease activity, and specific urease activity than the other tests with pure NH4Cl or both NH4Cl and urea. The use of urea as the nitrogen source in the media also led to an increase in pH, which was not found in the test with pure NH4Cl. As for the factor of urea concentration, the tests with a higher urea concentration had a higher biomass yield, urease activity, and pH. The factor of pH values also played an important role. The test with an 8.5 initial pH value had a higher biomass yield, urease activity, and specific urease activity than the tests with 7.5 and 9.5 initial pH values. In the chemostat condition, the ureolytic bacteria could be effectively produced with urease activity up to 7 mmol/L/min, as compared with around 12 mmol/L/min activity in the batch condition. Thus, the optimum nitrogen source, pH value, and cultivation condition for the cultivation of Sporosarcina pasteurii was urea, 8.5, and batch condition, respectively. In addition, when soybean milk powder or milk powder was used as the carbon source, the urease activity was around 2.5 mmol/L/min, which is also high enough to be used for biocement.

Optimization of biocementation responses by artificial neural network and random forest in comparison to response surface methodology.

In this article, the optimization of the specific urease activity (SUA) and the calcium carbonate (CaCO3) using microbially induced calcite precipitation (MICP) was compared to optimization using three algorithms based on machine learning: random forest regressor, artificial neural networks (ANNs), and multivariate linear regression. This study applied the techniques in two existing response surface method (RSM) experiments involving MICP technique. Random forest-based models and artificial neural network-based models were submitted through the optimization of hyperparameters via cross-validation technique and grid search, to select the best-optimized model. For this study, the random forest-based algorithm is aimed at having the best performance of 0.9381 and 0.9463 in comparison to the original r2 of 0.9021 and 0.8530, respectively. This study is aimed at exploring the capability of using machine learning-based models in small datasets for the purpose of optimization of experimental variables in MICP technique and the meaningfulness of the models by their specificities in the small experimental datasets applied to experimental designs. This study is aimed at exploring the capability of using machine learning-based models in small datasets for experimental variable optimization in MICP technique. The use of these techniques can create prerogatives to scale and mitigate costs in future experiments associated to the field.

Effects of resveratrol on the urease-positive urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis

Resveratrol (3, 5, 4’-trihydroxy-Trans-stilbene) is a polyphenolic plant metabolite with antibacterial, antioxidant, and anti-inflammatory activities. Staphylococcus saprophyticus and Proteus mirabilis were inhibited by pure resveratrol with minimum inhibitory concentrations of 250 to 500 µg ml-1. Urease activity in cell-free extracts and whole cells was partially inhibited by resveratrol but a concentration of 5 mmol l-1 was required to reduce it by more than 60%. Inhibition was competitive for S. saprophyticus but noncompetitive for P. mirabilis. Addition of resveratrol to the growth medium did not affect the specific activity of urease or prevent the increase in pH that occurs as a result of urease activity. Resveratrol reduced biofilm formation by S. saprophyticus at concentrations >125 µg ml-1 and partially inhibited it by P. mirabilis in rich media containing urea. While resveratrol has some potential for the treatment of urinary tract infections, it may need to be modified or combined with other urease inhibitors.

Use of recycled concrete aggregates as carriers for self-healing of concrete cracks by bacteria with high urease activity

Microbial induced carbonate precipitation (MICP) has become one of the most attractive strategies to achieve self-healing of concrete cracks in recent decades. In order to provide a moderate space for bacteria in the harsh environment of concrete, the incorporation of protective carriers is a necessity. In this study, the microbial urease activity at different generation of propagation in the selected culture medium was first investigated, followed by exploring the potential of using recycled concrete aggregates (RCAs) as microbial carriers, considering the porous nature of RCAs. The specific urease activity of bacteria at 48 h kept as high as 2.4 × 10−8 mM·min−1·cells−1 even after 5 generations of propagation in the selected culturing medium. RCAs would not result in a loss of ureolytic activity after immobilization and were capable of retaining bacterial viability during concrete mixing. Compared with concrete without any healing agents or with only substrates and calcium source, the one with bio-agents loaded in RCAs completed most of the crack healing in the first week. When the initial crack widths were less than 0.6 mm, the average crack healing ratio and the crack area healing ratio were 71% and 84%, respectively, for microbial self-healing. The depth of crack healing was 17.8 mm as indicated by the microbial precipitates on the crack wall. After microbial self-healing, the compressive strength regain ratio achieved 99.7% while the water tightness regain ratio attained 99.2%, and the cracks were filled with densely packed irregular rhombohedral-shaped crystals consisting of calcite and a small amount of vaterite.

Kinetic modeling and quasi-economic analysis of fermentative glycolipopeptide biosurfactant production in a medium co-optimized by statistical and neural network approaches

This study presents the kinetics of production of a glycolipopeptide biosurfactant in a medium previously co-optimized by response surface and neural network methods to gain some insight into its volumetric and specific productivities for possible scale-up towards industrial production. Significant kinetic parameters including maximum specific growth rate, µmax, specific substrate consumption rate, qs and specific biosurfactant yield, Yp/x were determined from logistic model parameters after comparison with other kinetic models. Results showed that bio-catalytic rates of lipase and urease reached exponential values within the first 12 h of fermentation leading to high specific rates of substrate consumption and bacterial growth. Volumetric biosurfactant production reached significantly high levels during prolonged stationary growth and specific urease activity. This suggests that glycolipopeptide biosynthesis may proceed through stationary phase transpeptidation of the glycolipid base. A high cross-correlation coefficient of 0.950 confirmed that substrate consumption and glycolipopeptide production occurred contemporaneously during the 66-h fermentation. The maximum biosurfactant concentration of 132.52 g/L, µmax of 0.292 h-1, qp of 1.674 g/gDCW/h, rp of 2.008 g/(Lh) and Yp/x of 4.413 g/g predicted by the selected logistic model and a unit cost of €0.57/g glycolipopeptide in the optimized medium may lead to technical and economic benefits.

Calcifying Bacteria Flexibility in Induction of CaCO3 Mineralization.

Microbially induced CaCO3 precipitation (MICP) is considered as an alternative green technology for cement self-healing and a basis for the development of new biomaterials. However, some issues about the role of bacteria in the induction of biogenic CaCO3 crystal nucleation, growth and aggregation are still debatable. Our aims were to screen for ureolytic calcifying microorganisms and analyze their MICP abilities during their growth in urea-supplemented and urea-deficient media. Nine candidates showed a high level of urease specific activity, and a sharp increase in the urea-containing medium pH resulted in efficient CaCO3 biomineralization. In the urea-deficient medium, all ureolytic bacteria also induced CaCO3 precipitation although at lower pH values. Five strains (B. licheniformis DSMZ 8782, B. cereus 4b, S. epidermidis 4a, M. luteus BS52, M. luteus 6) were found to completely repair micro-cracks in the cement samples. Detailed studies of the most promising strain B. licheniformis DSMZ 8782 revealed a slower rate of the polymorph transformation in the urea-deficient medium than in urea-containing one. We suppose that a ureolytic microorganism retains its ability to induce CaCO3 biomineralization regardless the origin of carbonate ions in a cell environment by switching between mechanisms of urea-degradation and metabolism of calcium organic salts.

Non-sterile corn steep liquor a novel, cost effective and powerful culture media for Sporosarcina pasteurii cultivation for sand improvement.

Microbial induced calcium carbonate precipitation (MICP) is one of the bio-cementation methods for improving granular soils. This study evaluate the feasibility of obtaining a bacterial solution with high optical density and urease activity by an inexpensive corn steep liquor (CSL) medium in non-sterile conditions in order to achieve sand improvement. Corn steep liquor media with different concentrations (different dilution rates) were prepared and, without any autoclaving (non-sterile conditions), different percentage of the inoculum solutions were added to them and incubated. Effect of inoculum solution percentage and CSL dilution rates on specifications of bacterial solution was evaluated. Urease activity and scanning electron microscope (SEM) and X-Ray Diffraction (XRD) were used to efficiency of CLS media in sand improvement. The considerable urease activity was measured as 5·7mScm-1 min-1 using nonsterile CLS. By using CYNU (CSL-Yeast extract-NH4Cl-Urea) bacterial solution, the urease activity of 5·5mScm-1 min-1 for the OD600 (optical density at 600nm) of 1·88 and, consequently, specific urease activity of 2·93 mScm-1 min-1 OD600 -1 was obtained. The highest unconfined compressive strength (811kPa) was obtained for the CYNU. XRD revealed new calcite peaks next to the quartz peaks. Production of inexpensive bacterial solution using diluted CSL as the inexpensive, effective and powerful culture media for Sporosarcina pasteurii cultivation in nonsterile conditions, allows geotechnical and biotechnological engineers to use MICP technology more widely in land improvement and field-scale bio-cementation and bioremediation projects. Obtaining high urease activity of inexpensive microbial solution using diluted CSL as the culture medium in nonsterile conditions, as the unique results of this study, can be significant in the field of bioremediation studies using MICP.

Different amounts of sugarcane trash left on the soil: Effects on microbial and enzymatic indicators in a short‐term experiment

AbstractWhen using mechanical harvesting, the sugarcane trash can be partially recovered for use as feedstock for bioenergy. However, the amount of trash that can be removed without prejudicing the sustainability of the sugarcane production system has still been little studied. The aim of this study was to evaluate the short‐term impact of removing part of the sugarcane trash using enzymatic and microbiological soil quality indicators. We tested three rates of trash removal: 0% (T0), 50% (T50) and 100% (T100). The microbial biomass C (Cmic) and N (Nmic), β‐glucosidase activity (GA) and the ratios Cmic as a per cent of Ctot and Nmic as a per cent of Ntot were highest for T100. The urease activity decreased with an increase in trash removal. The specific activity of β‐glucosidase did not differ between the treatments, while the specific activity of urease showed the same pattern as the absolute activity. The metabolic quotients (qCO2) were higher for T50 and T100 with no significant difference between them. The principal component analysis showed that the first component, which explained 77.66% of the total variance, was significantly correlated with Nmic, basal respiration and GA and discriminated the three treatments. The results showed that the removal of part of the trash adversely affected some of the soil quality attributes. This suggests the need for monitoring over a period of years in sugarcane areas where part of the trash is removed for industrial purposes, in order to better understand the cumulative impact on soil quality and to ensure the sustainability of bioenergy production.

Optimization of cost effective culture medium for Sporosarcina pasteurii as biocementing agent using response surface methodology: Up cycling dairy waste and seawater

Biological precipitation of calcium carbonate using Sporosarcina pasteurii, has been developed in the recent decades for the civil applications such as improvement of soil engineering parameters, crack repair in concrete and remediation of contaminated soil. In order to facilitate the application of this method, cost optimization and production simplification are the main challenges. In this study, optimization of culture media in order to reducing the cost of production of urease bacteria and achieving an ecofriendly process were investigated. Therefore, inexpensive nutrients and water resources were used in composition of the culture media and sanitized media were examined instead of sterilized ones. A central composite design was used to design required tests to investigate yeast extract, whey, heating temperature and the Caspian Sea water effects. The response surface method predicated that the maximum specific urease activity (16.50 mm urea.min−1.OD−1) could be achieved at 9.94 g l−1 yeast extract, 23.43 g l−1 whey, heating temperature of 128.6 °C and 0% seawater. When the medium contained 12.31 g l−1 yeast extract, 23.43 g l−1 whey and 0% seawater was sanitized by heating at 100.0 °C, 64% of optimum specific urease activity was reserved; and it could improve the unconfined compressive strength of a poorly graded sand up to 520 kPa. In case of using the Caspian Sea water as the solvent of culture media, the specific urease activity changes could be ignored. Application of whey as a nutrient source has shown promising results that could relieve some environmental and economic concerns. This approach is promising to achieve an ecofriendly biocementation technology for large scale applications.

A Novel Approach to Enhance the Urease Activity of Sporosarcina pasteurii and its Application on Microbial-Induced Calcium Carbonate Precipitation for Sand

Urease is involved in the formation of carbonate sediments by microbial-induced calcium carbonate precipitation (MICP), and Sporosarcina pasteurii used extensively in this technique owing to its high urease production. In this study, a simple two-step culture method with the appropriate medium was developed to enhance the urease activity of S. pasteurii. Urea played an important role in the culture process, particularly during the pre-cultivation step and the newly developed method improved both urease activity and specific urease activity. Furthermore, the increase in urease activity by MICP resulted in increased production of calcium carbonate and better strength of bio-cemented sand.

Experimental optimisation of various cultural conditions on urease activity for isolated Sporosarcina pasteurii strains and evaluation of their biocement potentials

Microbially induced carbonate precipitation (MICP) is a process that has emerged as an attractive alternative ground improvement technique in Geotechnical and Civil Engineering using ureolytic bacteria for soil strengthening and stabilisation. Locally isolated Sporosarcina pasteurii from limestone cave samples of Sarawak were found to have high urease-producing abilities. Optimisation of various cultural conditions (incubation temperature, initial pH medium, incubation period and urea concentration) were performed using conductivity and optical density measurements to determine the maximum specific urease activity. In addition, an in vitro biocement test was done to define the prospect of using these bacterial isolates in civil engineering work for the improvement of soils with inferior properties. The experimental results showed that urease activities were optimum at 25–30°C, pH 6.5–8.0, 24h incubation and 6–8% (w/v) urea concentration. It was also demonstrated that biocementation using the local ureolytic bacteria can improve the strength of poorly graded soils. However, the efficiency of the MICP process in improving the soil’s strength varied among samples treated with different bacterial cultures.

Caries-free subjects have high levels of urease and arginine deiminase activity.

ObjectivesThis study investigated the relationship between urease and arginine deiminase system (ADS) activities and dental caries through a cross-sectional study.Material and MethodsUrease and ADS activities were measured in saliva and plaque samples from 10 caries-free subjects and 13 caries-active. Urease activity was obtained from the ammonia produced by incubation of plaque and saliva samples in urea. ADS activity was obtained from the ammonia generated by the arginine-HCl and Tris-maleate buffer. Specific activity was defined as micromoles of ammonia per minute per milligram of protein. Shapiro-Wilk statistical test was used to analyze the distribution of the data, and Mann-Whitney test was used to determine the significance of the data.ResultsThe specific urease activity in saliva and plaque was significantly higher in individuals with low DMFT scores. ADS activity in saliva (6.050 vs 1.350, p=0.0154) and plaque (8.830 vs 1.210, p=0.025) was also higher in individuals with low DMFT scores.ConclusionsCaries-free subjects had a higher ammonia generation activity by urease and arginine deiminase system for both saliva and plaque samples than low caries-active subjects. High levels of alkali production in oral environment were related to caries-free subjects.

Role of Helicobacter pylori methionine sulfoxide reductase in urease maturation

The persistence of the gastric pathogen Helicobacter pylori is due in part to urease and Msr (methionine sulfoxide reductase). Upon exposure to relatively mild (21% partial pressure of O2) oxidative stress, a Δmsr mutant showed both decreased urease specific activity in cell-free extracts and decreased nickel associated with the partially purified urease fraction as compared with the parent strain, yet urease apoprotein levels were the same for the Δmsr and wild-type extracts. Urease activity of the Δmsr mutant was not significantly different from the wild-type upon non-stress microaerobic incubation of strains. Urease maturation occurs through nickel mobilization via a suite of known accessory proteins, one being the GTPase UreG. Treatment of UreG with H2O2 resulted in oxidation of MS-identified methionine residues and loss of up to 70% of its GTPase activity. Incubation of pure H2O2-treated UreG with Msr led to reductive repair of nine methionine residues and recovery of up to full enzyme activity. Binding of Msr to both oxidized and non-oxidized UreG was observed by cross-linking. Therefore we conclude Msr aids the survival of H. pylori in part by ensuring continual UreG-mediated urease maturation under stress conditions.

콘크리트 포장도로에서 분리한 탄산칼슘형성미생물의 다양한 환경 스트레스반응

Microbially induced calcite precipitation (MICP) has been explored for protection and consolidation of construction materials such as concrete. In this study, we isolated 54 calcite forming bacteria from concrete pavement and selected 5 isolates which showed high specific urease activity. Also response of the 5 strains against various environmental stresses was examined. BC 4 and BC 5 showed 35% and 26% viability at heat stress (), respectively. BC 1 and BC 4 maintained 60.4% and 70.4% viability upon osmotic stress (1 M NaCl), respectively. Among the 5 isolates BC 4 had the highest viability upon alkaline stress (pH 10).

Urease Activity of Ureolytic Bacteria Isolated from Six Soils in which Calcite was Precipitated by Indigenous Bacteria

Ten distinct and phylogenetically diverse bacterial strains able to produce urease constitutively, even in the presence of high levels of ammonia, were isolated. Each isolate was characterized with respect to morphology, growth conditions, 16S rRNA sequence identity and the urease specific activity each isolate was measured. At least one bacteria that was able to hydrolyze urea in the presence of high concentration of ammonia was isolated from each soil tested. These results indicate that constitutive production of urease is a common trait in soil bacteria. This makes the general application of biomineralization process based on stimulation of urea hydrolysis possible and potentially useful for a variety of soil bioengineering and environmental bioremediation applications.

Complex Regulation of Urease Formation from the Two Promoters of the ure Operon of Klebsiella pneumoniae

Klebsiella pneumoniae can use urea as the sole source of nitrogen, thanks to a urease encoded by the ureDABCEFG operon. Expression of this operon is independent of urea and is regulated by the supply of nitrogen in the growth medium. When cells were growth rate limited for nitrogen, the specific activity of urease was about 70 times higher than that in cells grown under conditions of excess nitrogen. Much of this nitrogen regulation of urease formation depended on the nitrogen regulatory system acting through the nitrogen assimilation control protein, NAC. In a strain deleted for the nac gene, nitrogen limitation resulted in only a 7-fold increase in the specific activity of urease, in contrast to the 70-fold increase seen in that of the wild type. The ure operon was transcribed from two promoters. The proximal promoter (P1) had an absolute requirement for NAC; little or no transcription was seen in the absence of NAC. The distal promoter (P2) was independent of NAC, but its activity increased about threefold when the growth rate of the cells was limited by the nitrogen source. Transcriptional regulation of P1 and P2 accounted for most of the changes in urease activity seen under various nitrogen conditions. However, when transcription of ureDABCEFG was less than 20% of its maximum, the amount of active urease formed per transcript of ure decreased almost linearly with decreasing transcription. This may reflect a defect in the assembly of active urease and accounted for as much as a threefold activity difference under the conditions tested here. Thus, the ure operon was transcribed from a NAC-independent promoter (P2) and the most strongly NAC-dependent promoter known (P1). Most of the regulation of urease formation was transcriptional, but when ure transcription was low, assembly of active urease also was defective.