Development of a M9-based urea medium (M9U) for sensitive and real-time monitoring of ureolytic activity of bacteria and cell-free urease.

Jens Jakob Sigurdarson,Simon Svane,Henrik Karring

doi:10.1002/mbo3.976

Abstract

The enzyme urease is widespread in nature and catalyzes the hydrolysis of urea to form ammonia and carbonic acid. The high proficiency of the enzyme is associated with a wide range of societal challenges. In agriculture, bacterial urease activity leads to loss of fertilizer through NH3 emission, which has a negative impact on the environment and human health. Urease is also an essential virulence factor for several pathogenic bacteria. To screen for potential urease inhibitors, efficient, sensitive, and accurate urease activity assays are needed. However, most urease activity assays are labor‐intensive and become time‐consuming when used to screen multiple samples. Based on systematic optimization, we have developed a urea‐containing growth medium and method for continuous real‐time monitoring and screening of urease activity from both bacterial cells and pure urease in a plate reader setup. The defined M9‐based urea (M9U) medium was found to be more sensitive and suitable for a plate reader setup than both Christensen's urea broth (CUB) and Stuart's urea broth (SUB), which are established and well‐known complex urea media that formed the principle foundation of M9U. Furthermore, we show that urease activity measurements using the M9U medium in our plate reader‐based method allow reliable high‐throughput screening of urease inhibitors.

Highlights

The potential of using the M9-based urea (M9U) and urease activity assay for screening compounds for antiureolytic properties were investigated with the urease inhibitors hydroxyurea, NBPT, and PPDA (Figure 3 and Appendix 4; Figures A4–A6)
Since urease activity depends on nickel atoms in the active site of the enzyme, nickel was added to the M9U medium to ensure that ureolytic bacteria in the assay are not limited in their synthesis of active urease
It was found that manure slurry from pigs contains 11.54 μM Ni2+, which is more than enough to maintain ureolysis.(Svane & Karring, 2019) to avoid toxicity/inhibition of urease, only 0.34 μM NiCl2 was included in the M9U medium

Summary

Introduction

The dinickel enzyme urease catalyzes the hydrolysis of urea, resulting in the overall formation of two ammonia molecules (NH3) and one carbonic acid molecule (H2CO3) per urea.(Mobley, Island, & Hausinger, 1995; Sigurdarson, Svane, & Karring, 2018) It is a common enzyme in nature and is found among plants, bacteria, algae, fungi, and invertebrates.(Bekheet & Syrett, 1977; Booth & Vishniac, 1987; Cook, 1976; Frankenberger & Tabatabai, 1982; Hanlon, 1975; Sumner, 1926) Urease in livestock animal feces is a concern in agriculture because it hydrolyzes the urea found in livestock animal urine, causing emissions of NH3 that can damage the environment, reduce air quality, and represent a loss of fertilizer nitrogen. (Sigurdarson et al, 2018) Urease is an important virulence factor of several pathogens. A. Stuart developed urea broth, or Stuart's urea broth (SUB), (Rustigian & Stuart, 1941) which is a complex growth medium designed to recognize ureolytic bacteria by incorporating both urea and the pH indicator phenol red. Ureolytic bacteria growing in SUB at pH 6.8 will convert the urea into NH3, which will cause the pH to increase and the media to change color from yellow/orange to red/ pink. B. Christensen developed a different complex growth medium for identifying ureolytic bacteria based on the same principles.(Christensen, 1946) One of the changes that W. By reducing the buffer capacity, less NH3 is required to overcome the buffer capacity of the medium and increase pH, thereby making the assay sensitive to lower levels of urease activity

Methods

Results

Conclusion