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Adenylate Kinase
Adenylate Kinase H-Y Chang and C-Y Fu, National Tsing Hua University, Hsin Chu, Taiwan Ó 2014 Elsevier Ltd. All rights reserved. This article is a revision of the previous edition article by M.J. Murphy, D.J. Squirrell, volume 1, pp. 16–24, Ó 1999, Elsevier Ltd.
General Introduction of Adenylate Kinase Adenylate kinase (AK, adenosine triphosphate (ATP): adenosine monophosphate (AMP) phosphotransferase, EC 2.7.4.3) is a ubiquitous and abundant enzyme found in virtually all eukaryotic and prokaryotic cells. It catalyzes the reversible reaction: Mg2þ $ATP þ AMP4Mg2þ $ADP þ ADP where ATP, adenosine diphosphate (ADP), and AMP are the adenosine tri-, di-, and monophosphates, respectively. In vivo the reaction maintains the balance of adenylates in the cell, usually proceeding to the right to rephosphorylate the AMP into ADP. The ADP generated in the reaction can then be further phosphorylated to form ATP in major metabolic pathways, such as glycolysis. In eukaryotes, AK is found predominantly in the space between the inner and outer mitochondrial membranes. In Gram-negative bacteria, the enzyme is present primarily in the cytoplasm and the periplasmic space. Nevertheless, some extracellular AK can be found and has been implicated as a bacterial virulence factor causing macrophage death. It is the only enzyme produced by the cells for the purpose of phosphorylating AMP to ADP and, as such, is essential for life. It is a stable protein with a relatively long intracellular lifetime. The molecular mass of AK is typically 20–25 kDa. The bacterial AK usually is longer than its eukaryotic counterpart and is made up of approximately w210 or 220 amino acids. The Michaelis constant (KM) of the Escherichia coli AK for ADP is approximately 100 mM. Most AKs share similar tertiary features and are grouped into three functional subdomains. The core of the protein is composed of a central five-stranded parallel b-sheet surrounded by a number of ahelices. On the periphery of the core subdomain are the AMPbinding and the ATP-binding subdomains. Because of its essentiality and association with virulence, there is a profound interest in AK as a target for developing new drugs against infectious bacterial agents. The enzyme is also a good model of structural dynamics and catalysis research.
AK as the Detection Target in ATP Bioluminescence Assay Firefly luciferase catalyzes the following light-emitting reaction: ATP þ Luciferin þ O2 4AMP þ Oxyluciferin þ PPi þ Light Because of the high specificity of the firefly luciferase to ATP and the relative ease in light detection, the reaction is therefore convenient for quantifying ATP. The main reagents required in the assay, luciferase and luciferin, are commercially available and can be obtained in good purity. The reaction is simple to carry out and the light emitted can be measured easily by a luminometer that is generally inexpensive. Since ATP is present in all living organisms and is rapidly degraded
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following cell death, it can be used as a marker to monitor biomass, such as microorganisms. A recent study evaluated rapid microbiological monitoring methods based on detection of growth and found that ATP bioluminescence assay detected common microorganisms significantly faster than CO2 monitoring and turbidity assays. The ATP bioluminescence assay has gained acceptance by major regulatory authorities. For example, one of the commercial ATP bioluminescence assays, the Pallcheck Rapid Microbiology System (Pall Life Sciences, Hants, UK), has been granted approval by the Center for Drug Evaluation and Research at the US Food and Drug Administration for the release of certain nonsterile pharmaceuticals. Nevertheless, the sensitivity of ATP bioluminescence assay still has plenty of room to be improved. In typical assays, the limit of detection is about 103 bacteria or 1000 yeast cells, primarily due to background noise. The luminescence sensitivity can be improved partly by using a more sensitive optical sensor in the luminometer, although this is accompanied by adding up the instrument cost, and raising the background noises. An alternative way to improve the assay sensitivity is to change the detection target from ATP to ATP-producing enzyme such as AK. A medium-size bacterium normally contains about 1021 mol of AK in comparison with about 1018 mol of ATP. Escherichia coli AK has a kcat to ADP of around 300, which means that with just 1-min incubation the enzyme can generate 18 times more ATP for bioluminescent signal production than would be possible from the ATP naturally present on its own. The amplification reaction requires only a single substrate (i.e., ADP) and provides a linear increase in the amount of ATP over time. In theory, an AK assay should allow single bacterial cells to be detected in 10 min. Raised backgrounds from contaminating ATP and AK prevent this from being easily achieved, but it has been demonstrated. Kinases other than AK, such as pyruvate kinase, potentially could be used as ATP-generating cell markers. Unlike AK, which uses two ADP molecules to generate ATP, all the other ATP-generating kinases require two substrates: a phosphoryl donor and ADP. Obtaining a high degree of purity in the reagents is consequently made more difficult than when only one reagent is used in the assay. The approach based on AK detection thus has a unique advantage in terms of simplicity. Other reasons for the greater usefulness of AK over other kinases include its high catalytic activity, high robustness, and its ubiquitous nature due to its essential metabolic function.
General Considerations in AK-Based Bioluminescence Assay The procedure for AK detection is similar to the conventional ATP bioluminescence assay, except that an extended incubation time (of about 5 min) is needed for the AK assay. Certain requirements must be addressed in conducting and developing
Encyclopedia of Food Microbiology, Volume 1
http://dx.doi.org/10.1016/B978-0-12-384730-0.00003-3
Adenylate Kinase
the reagents for AK assays. First, different luminometers have different upper and lower detection limits and linear dynamic range in light detection. Appropriate tuning of the instrument into the right range is important. Second, there are both upper and lower limits to the concentrations of ADP substrate that can be used. A very high ADP concentration is inhibitory to the firefly luciferase reaction, reducing the light output for a given amount of AK, while a very low concentration of ADP provides too little substrate for conversion to ATP. Concentrations between 10 mM and 1 mM work out to be appropriate for most purposes. Third, the quality of the reagent must be in the highest obtainable purity to minimize the background signals that any extraneous ATP will cause. Certain batches of commercial ADP may require an additional purification step, frequently an anion exchange chromatography in which the ADP elutes before the contaminating ATP. Other components in the assay, in particular the luciferase, should be tested before the AK detection assay to ensure that the enzyme preparation contains minimal levels of contaminating AK to reduce background signals.
Release of ATP and AK from Cell by Detergents As in ATP bioluminescence, an extraction step is needed to release the intracellular AK. Extraction usually is carried out using a detergent. All of the manufacturers of ATP bioluminescence kits have proprietary formulations of extractants, and the suitability of the reagents for AK detection needs to be tested empirically to avoid inactivating either the AK or the luciferase. The concentration chosen for the detergent has to be a balance between maximizing disruption of cell membranes, and thus extraction of the AK, and minimizing inactivation of its enzymatic activity. Detergents, such as Triton X-100 and N, N0 ,N0 -polyoxyethylene (10)-N0 -tallow-1,3-diaminopropane at a concentration of 0.05%, have been found to produce reasonably good cell disruption effects without compromising the AK activity. Certain protocols (such as Promega’s ENLITENÒ ATP Assay System) employ trichloroacetic acid to extract ATP from bacterial and fungal cells. This approach is too harsh for AK and therefore is not suitable for AK assay even if a neutralization step is followed.
Effects of Reaction Time on AK Assay As explained, AK offers at least 10 times, and usually 100 times, greater sensitivity than the conventional ATP assays. Because the amplification kinetics is linear, it is therefore possible to increase the sensitivity of AK assays by extending the incubation time. Increasing the incubation time in the conventional ATP bioluminescence assays has no effect, because the majority of the ATP is extracted and consumed usually within the first 1-min incubation. The most convenient combination of speed and sensitivity for AK is achieved with a 5-min incubation. Using this, fewer than 100 cells of bacteria (such as E. coli or Pseudomonas aeruginosa) can be readily detected. The assay is reproducible and is relatively unaffected by changes in growth medium or conditions. By using small reaction volumes in combination with incubation times of up to 1 h, limits of detection approaching the single-cell level become possible. At this point, sampling statistics rather than assay sensitivity
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become limiting, and the technique compares favorably with polymerase chain reactions and growth assays. Such smallvolume assays, in effect, can be carried out on filter membranes, and the full capability of the AK approach may be realized in this format.
Effects of Exogenous AK on the Assay In industrial applications, the source of the sample on which the test is being carried out could have a large effect on the end result. AK is present in virtually all living matter, not just bacteria. This means that the samples to be tested could contribute toward the signal. This applies to plant sources (e.g., fruit or vegetable juices) as well as meat and dairy produce. The levels can be seen to vary greatly, with those in meat being especially high. When total microbial loading is to be determined, the nonmicrobial AK may either be removed by suitable sample pretreatment methods or accounted for by subtracting the results from control assays carried out on uncontaminated samples. In a practical sense, both of the approaches are difficult to carry out without compromising the microbial AK detection. As might be expected, physical inactivation techniques such as high temperature and ultraviolet treatment cause a reduction in extraneous AK levels, although the microbial AK levels also will be reduced. Subtracting the results from that of uncontaminated control assays is also difficult, particularly when the extraneous AK level is much higher than that of the microbial AK. Thus, the AK luminescence assay is better to be performed in samples naturally low of AK.
Correlation of AK Assay Results with Viable Cell Count The concentration of ATP typically fluctuates widely within bacteria according to their metabolic status, size, and stress. This leads to inaccuracies when attempting to quantify the bacterial loading of a sample. So caution must be exercised in setting pass and fail levels when using ATP bioluminescence assay in standard hygiene monitoring. In contrast, AK is present at relatively constant levels regardless of the energy status of the cell. It therefore provides a much more reliable measure of the number of bacteria present than ATP. Nevertheless, several factors can affect the correlation between cell numbers, determined as colony-forming units, and the net bioluminescent signal from an AK assay. First, the viable cell counts obtained will tend to be lower than the number of cells present in the sample. This may be due to the fact that the culture condition is not suitable for the growth of the target bacteria, the bacteria are in a viable but nonculturable state, and the bacterial cells have not been fully divided or are clumped together. Second, the AK assay may not always be in linear kinetics. This happens frequently when compounds inhibiting either AK or luciferase are present in the testing samples. The accumulation of the AK and luciferase reaction products can feedback inhibit these enzymes. Thus, whether AK assay readings or the number of live cells provide the better measure of contamination will depend on the reason for carrying out the assay in the first place. In general, AK may be particularly useful where safety critical monitoring is required.
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Adenylate Kinase
Potential Applications of AK Detection Assay The main application areas for AK-based microbiology assays in the food industry are rapid contamination and sterility testing, for example, hygiene monitoring for low-level contamination by microorganisms and other organic matter, such as air quality and food preparation surfaces. The materials to be tested should contain little endogenous AK or ATP (e.g., processing water) or samples in which endogenous free AK and ATP can be removed (e.g., beverages such as beer, cider, and soft drinks). With minor modification, the AK detection method may be used to monitor the presence of specific organisms such as foodborne bacterial pathogens. Such applications exploit the advantages that the high sensitivity of the AK approach can provide and will expand the areas of microbiological testing where rapid testing is feasible. Assays with AK as a marker should come to supplement rather than replace direct ATP measurements, which will remain appropriate in cases in which background contamination is inherently low, extreme sensitivity is not needed, or falsepositive results are more of a problem than false-negative results. Some application examples in using AK as a detection marker follow.
Hygiene Monitoring Using AK Bioluminescence Assay The procedure for hygiene monitoring using AK should be essentially the same as the widely used ATP bioluminescence method, lending itself equally well to the use of single-shot disposables. The only differences between the two assays are the addition of exogenous ADP and the need for an additional 5-min incubation time for AK detection. A typical AKbased luminescence method would include the following steps: 1. Swab area of interest with swab moistened with magnesium acetate buffer. 2. Place swab in a cuvette with ADP and extractant. 3. Incubate at room temperature for 5 min. 4. Add bioluminescence reagent. 5. Measure light output using a portable luminometer.
Specific Assays In cases in which identification of particular organisms is required rather than measurement of the total microbial loading, the AK assay may be used as the end point in immunoassays. One approach is to capture the target microorganism with magnetic beads coated with a specific antibody such as those against Listeria monocytogenes, E. coli O157, and Salmonella spp. The immunomagnetic separation procedures have been used to isolate bacterial cells from complex suspensions, such as food, before culture on agar plates or biochemical identification procedures to determine the level of contamination in the original sample. Nevertheless, the culture procedures are time consuming, taking 24–72 h to complete. The problem can be overcome by directly detecting the AK from the organisms captured on magnetic beads. Contamination from other organisms or the food materials is eliminated during the separation. Once immobilized, the bacteria can be lysed in the same way as a nonspecific assay. As illustrated in Figure 1, a typical AK-based pathogen-specific assay procedure is as follows: 1. Add antibody-conjugated beads to sample, resuspend, and incubate at room temperature. 2. Immobilize beads with a permanent magnet and remove supernatant. 3. Wash beads to remove any unbound material. 4. Resuspend beads in magnesium acetate buffer. 5. Add ADP plus extractant and incubate for 5 min. 6. Add bioluminescence reagent to sample extract. 7. Measure light output using a portable luminometer. The rate at which the bacteria will bind to the beads depends on the complexity of the testing sample. For typical liquid samples, binding of bacteria to the antibody-coated magnetic beads is rapid, taking a few minutes. In a more complex and viscous medium, the incubation time will need to be increased to allow the antibodies time to find the organisms. Nonetheless results will be obtained in approximately 1–2 h – a considerable time saving on conventional
Figure 1 Magnetic bead immunoassay for selective immobilization of microorganisms from food and beverage samples, prior to generic lysis and detection with AK bioluminescence. (a) Target cells are removed from suspension by mixing with magnetic beads pre-coated with an antibody specific to bacteria B. (b) Beads are immobilized by applying a magnetic field. Unwanted material is removed by washing. (c) The captured cells are lysed and ADP is added. (d) After incubation, bioluminescence reagent is added and the light output is measured.
Adenylate Kinase
methods and without the need to start with a pure culture. A major drawback of this approach is that it is difficult to obtain an antibody with good specificity and sensitivity for many bacterial species. In addition to using regular extractant to disrupt the captured cells, a promising strategy is the use of test methods that have double specificity. This specificity is achieved by combining a specific capture step with a specific lysis step, which gives extra confidence in assays for a particular organism. This may be achieved using lytic bacteriophages or commercially available lysins, such as colicin for E. coli and lysostaphin for Staphylococcus aureus. These should allow significant opportunities for replacing traditional microbiological testing with rapid methods, because it is more sensitive and easier to carry out in comparison with techniques such as enzyme-linked immunosorbent assay. Bacteriophage-mediated lysis of target cells may be achieved by adding to a culture a phage specific for the organism of interest. If the target organisms are present, the phage will infect them and cause the cells to be lysed. This releases all the intracellular components, including AK, into suspension. By adding ADP (with no extractant), the activity of this released AK can be measured. The assay principle is illustrated in Figure 2 and a typical procedure is as follows: 1. Mix the sample with culture medium and incubate at 37 C for 1–2 h to activate bacteria growth and enhance their susceptibility to bacteriophage. 2. Split sample into two: add bacteriophage into one and leave the other as an uninfected control. 3. Incubate at 37 C for 30–90 min 4. At timed intervals, remove a sample from each culture into a cuvette containing magnesium acetate buffer. 5. Add ADP and incubate for 5 min. 6. Add bioluminescence reagent and measure light output. Using bacteriophages, fewer than 103 log phase, E. coli cells can be detected in around 2 h under laboratory conditions. The bacteria must be in log phase to be receptive to phage infection, so a short culture step would be required to activate stationary phase or otherwise stressed cells. The time course of
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the phage lysis is specific to the phagedhost combination used. Certain coliphage starts to induce bacteria lysis after about 20 min and may need 1 h to complete the lytic process. This means that the assay time will vary depending on the target organisms and bacteriophage chosen. Bacteriophages vary in their specificity. Some are capable of infecting bacteria from the same genus, whereas others are strain specific within a particular species. Because their bacterial host keep evolving in the natural environment, it is unlikely to identify a bacteriophage that can recognize all strains in a given species. Furthermore, the testing sample may not provide a suitable environment for the bacteriophage to complete their life cycle and hence release of AK. Together, these limitations prohibit wide application of bacteriophage in specific pathogen detection.
Application of AK in ATP Regeneration for Ultrasensitive Bioluminescence Assay Although AK can be a useful alternative cell marker to ATP in hygiene monitoring, ATP measurement remains a common and important test in biochemical research and medical diagnosis. The detection limit of ATP by conventional bioluminescence assay is approximately 1014–1012 M, which is suitable for most assays. Nevertheless, as highthroughput screening technologies become widely adopted and the testing sample volume is getting smaller, the increase of ATP bioluminescence assay sensitivity also has become important. In many testing samples, ATP is present in relatively low quantities and is depleted rapidly in the conventional luciferase assay. If the ATP consumed in the luciferase assay can be replenished, the assay sensitivity can be significantly enhanced. The replenishment of the ATP pool must be in a constant rate so the original ATP concentration can be determined reliably. A straightforward approach of ATP regeneration in the luciferase assay is to recycle the reaction products, either AMP or inorganic pyrophosphate (PPi), back to ATP. Because AK is the main kinase in the cells to
Figure 2 Specific bacteria detection assay based on selective lysis of target bacteria with bacteriophage. (a) Bacteriophage specific to bacteria B is added to the sample. (b) The phage causes bacteria B lysis and AK release. (c) ADP and bioluminescence reagents are then added to the sample and the light output is measured.
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Adenylate Kinase
Luciferase ATP + Luciferin + O2 Acetate Acetate kinase Acetyl-P
Figure 3 assay.
AMP + Oxyluciferin + PPi + Light UTP
Adenylate kinase
ADP
UDP
Schematic presentation of AMP-regeneration bioluminescence
phosphorylate AMP into ADP, the enzyme was tested for its potential to replenish the ATP pool in the luciferase assay. It is obvious that exogenous ATP and ADP cannot be used as the phosphoryl donor in the recycling reaction because they will change the original ATP concentration and compromise the quantitative assay. Luckily, the substrate specificity of AK is remarkably high for AMP as a phosphoryl acceptor and relatively low for ATP as a phosphoryl donor. Thus, uridine triphosphate (UTP), which can be used as the phosphoryl donor in AK reaction but not in the luciferase reaction, is used in the AMP-recycling reaction. The reaction is as follows: Mg2þ $UTP þ AMP4Mg2þ $UDP þ ADP In theory, the ADP generated in this reaction can be further converted into ATP by AK. An additional kinase, such as acetate kinase, pyruvate kinase, and polyphosphate kinase, and their respective phosphoryl donor (acetyl phosphate, phosphoenolpyruvate, and polyphosphate) often are added to improve the efficiency of ADP to ATP conversion. In the presence of excess UTP, AK together with the luciferase reaction forms a cyclic loop of ATP regeneration. Ideally, this ATP regeneration approach enables the light signal output in a constant rate when other substrates are at excess. Thus, the assay sensitivity can be enhanced simply by increasing the measurement time. This approach is much more sensitive than the conventional bioluminescence assay, allowing for the detection of as low as one colony-forming unit of bacterium per assay. Further improvement of the ATP bioluminescence assay by recycling both PPi and AMP into ATP can result in exponential amplification of ATP. The exponential ATP amplification assay provides an extremely sensitive mean for ATP and bacteria detection. It therefore will be useful in situations in which the highest standard of hygiene is required, such as pharmaceutical
Table 1
Conclusion Firefly luciferase-based ATP bioluminescence assay is a fastdeveloping technique in rapid microbiological testing. Recently, several attempts have been made to apply AK in the bioluminescence assay to improve the assay sensitivity and to better correlate the results with the bacterial cell counts in the sample. AK can be used as the cell marker that generates ATP for detection or a tool to form a cyclic loop for ATP amplification. Certain types of samples inevitably may contain their own AK, which will tend to swamp that from contaminating microorganisms. The applicability of AK detection in rapid testing is thus restricted to cleanliness monitoring (i.e., testing for the absence of AK) and testing samples that have an inherently low level of AK or that can be treated easily to remove endogenous AK. On the other hand, the AK-mediated ATP regeneration approach offers high sensitivity in ATP detection and gives reliable results in ATP quantification. A comparison among the bioluminescent assays based on direct ATP detection, AK detection, and ATP-regeneration for microorganism monitoring is shown in Table 1. The primary advantage of using AK-based luminescence assays in hygiene monitoring over the conventional viable cell count method is their short assay time. This makes on-site monitoring possible and allows for decisions, such as whether to sterilize the processing facility or whether a food product can be shipped out, to be made without significant delay. In the future, the AK-based luminescence assays likely will become
Comparison of direct ATP detection, AK detection, and ATP-regeneration bioluminescent assays for microorganism detection
Nature of cell marker Ubiquitous in living cells Amount/average bacterial cell Intracellular levels Approximate assay time Incubation time dependent Correlation with cell number Assay detection limit (colony-forming unit/0.1 ml sample) a
manufacturing. On the other hand, the AMP recycling assay is preferred in situations in which accurate ATP quantification is more desirable, such as in enzyme kinetic studies and metabolic pathway analysis (Figure 3). Although the modification can significantly improve the sensitivity of an ATP bioluminescence assay, such an approach is more difficult to perform because several additional reagents need to be included in the assay. To reduce the assay background, the highest purity of the reagents must be used in the reaction. The concentration of the reagents also needs to be adjusted carefully to optimize the assay performance without adding too much reagent cost. Apparently, identifying how to simplify the assay components while maintaining the high sensitivity of the modified ATP bioluminescence reaction is the direction in which to go.
Dependent on the size and energy status of the bacterial cell.
Direct ATP
AK
ATP regeneration
Metabolite Yes 1018 mol Variable 1 min No Approximate 1000–10 000a
Enzyme Yes 1021 mol Constant 5 min Yes Good w100
Metabolite Yes 1018 mol Variable 5 min Yes Approximate <10
Adenylate Kinase
Table 2 Comparison of AK-based luminescence assay and conventional viable cell count assay in hygiene monitoring
Time required On-site monitoring Major equipment required Interference by food Microbial species identification
AK luminescence assay
Viable cell count assay
w5 min Yes Luminometer High Difficult
Overnight No Incubator Low Possible
a common supplement to the current ATP bioluminescent assay in situations in which high sensitivity is needed (Table 2).
See also: Application in Meat Industry; Bacteriophage-Based Techniques for Detection of Foodborne Pathogens; Biophysical Techniques for Enhancing Microbiological Analysis; Rapid Methods for Food Hygiene Inspection; Total Viable Counts: Metabolic Activity Tests; Water Quality Assessment: Routine Techniques for Monitoring Bacterial and Viral Contaminants.
Further Reading Blasco, B.R., Murphy, M.J., Sanders, M.F., Squirrell, D.J., 1998. Specific assays for bacteria using bacteriophage mediated release of adenylate kinase. Journal of Applied Microbiology 84, 661–666. Brokaw, J.B., Chu, J.W., 2010. On the roles of substrate binding and hinge unfolding in conformational changes of adenylate kinase. Biophysical Journal 99, 3420–3429. Brolin, S.E., Borglund, E., Agren, M.J., 1979. Photokinetic microassay of adenylate kinase using the firefly luciferase reaction. Journal of Biochemical and Biophysical Methods 1, 163–169. Buchko, G.W., Robinson, H., Abendroth, J., Staker, B.L., Myler, P.J., 2010. Structural characterization of Burkholderia pseudomallei adenylate kinase (Adk): profound asymmetry in the crystal structure of the ‘open’ state. Biochemical and Biophysical Research Communication 394, 1012–1017. Ceresa, L., Ball, P., 2006. Using ATP bioluminescence for microbiological measurement in pharmaceutical facturing. In: Miller, M.J. (Ed.), Encyclopedia of Rapid Microbiological Methods, vol. 2. Paranteral Drug Association/Davis Healthcare International Publishing LLC, pp. 233–249.
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Gilles, A.M., Saint-Girons, I., Monnot, M., Fermandjian, S., Michelson, S., Bârzu, O., 1986. Substitution of a serine residue for proline-87 reduces catalytic activity and increases susceptibility to proteolysis of Escherichia coli adenylate kinase. Proceedings of the National Academy of Sciences of the United States of America 83, 5798–5802. Jacobs, A.C., Didone, L., Jobson, J., Sofia, M.K., Krysan, D., Dunman, P.M., 2012. Adenylate kinase release as a high throughput screening compatible reporter of bacterial lysis for the identification of antibacterial agents. Antimicrobial Agents and Chemotherapy [Epub ahead of print]. Lee, H.J., Ho, M.R., Tseng, C.S., Hsu, C.Y., Huang, M.S., Peng, H.L., Chang, H.Y., 2011. Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria. Analytical Biochemistry 418, 19–23. Markaryan, A., Zaborina, O., Punj, V., Chakrabarty, A.M., 2001. Adenylate kinase as a virulence factor of Pseudomonas aeruginosa. Journal of Bacteriology 183, 3345–3352. Murphy, M.J., Squirrell, D.J., 1999. Adenylate kinase. In: Robinson, R.K. (Ed.), Encyclopedia of Food Microbiology. Elsevier Ltd, pp. 16–24. Murphy, M.J., Squirrell, D.J., Sanders, M.F., Blasco, R., 1995. The use of adenylate kinase for the detection and identification of low numbers of microorganisms. In: Hastings, J.W., Kricka, L.J., Stanley, P.E. (Eds.), Bioluminescence and Chemiluminescence: Molecular Reporting with Photons. John Wiley, Chichester, pp. 319–322. Parveen, S., Kaur, S., David, S.A., Kenney, J.L., McCormick, W.M., Gupta, R.K., 2011. Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products. Vaccine 29, 8012–8023. Satoh, T., Kato, J., Takiguchi, N., Ohtake, H., Kuroda, A., 2004. ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell. Bioscience Biotechnology and Biochemistry 68, 1216–1220. Schrank, T.P., Bolen, D.W., Hilser, V.J., 2009. Rational modulation of conformational fluctuations in adenylate kinase reveals a local unfolding mechanism for allostery and functional adaptation in proteins. Proceedings of the National Academy of Sciences of the United States of America 106, 16984–16989. Schulz, G., Muller, C.W., Diederichs, K., 1990. Induced-fit movements in adenylate kinases. Journal of Molecular Biology 213, 627–630. Squirrell, D.J., Murphy, M.J., 1994. Adenylate kinase as a cell marker in bioluminescent assays. In: Campbell, A.K., Kricka, L.J., Stanley, P.E. (Eds.), Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects. John Wiley, Chichester, pp. 486–489. Squirrell, D.J., Murphy, M.J., 1997. Rapid detection of very low numbers of micro-organisms using adenylate kinase as a cell marker. In: Stanley, P.E., Simpson, W.J., Smither, R. (Eds.), A Practical Guide to Industrial Uses of ATPluminescence in Rapid Microbiology. Cara Technology, Lingfield. Wills, K., 2003. ATP bioluminescence and its use in pharmaceutical microbiology. In: Easter, M.C., Raton, B. (Eds.), Rapid Microbiological Methods in the Pharmaceutical Industry. Interpharm/CRC Press, Florida.
General Introduction of Adenylate Kinase Adenylate kinase (AK, adenosine triphosphate (ATP): adenosine monophosphate (AMP) phosphotransferase, EC 2.7.4.3) is a ubiquitous and abundant enzyme found in virtually all eukaryotic and prokaryotic cells. It catalyzes the reversible reaction: Mg2þ $ATP þ AMP4Mg2þ $ADP þ ADP where ATP, adenosine diphosphate (ADP), and AMP are the adenosine tri-, di-, and monophosphates, respectively. In vivo the reaction maintains the balance of adenylates in the cell, usually proceeding to the right to rephosphorylate the AMP into ADP. The ADP generated in the reaction can then be further phosphorylated to form ATP in major metabolic pathways, such as glycolysis. In eukaryotes, AK is found predominantly in the space between the inner and outer mitochondrial membranes. In Gram-negative bacteria, the enzyme is present primarily in the cytoplasm and the periplasmic space. Nevertheless, some extracellular AK can be found and has been implicated as a bacterial virulence factor causing macrophage death. It is the only enzyme produced by the cells for the purpose of phosphorylating AMP to ADP and, as such, is essential for life. It is a stable protein with a relatively long intracellular lifetime. The molecular mass of AK is typically 20–25 kDa. The bacterial AK usually is longer than its eukaryotic counterpart and is made up of approximately w210 or 220 amino acids. The Michaelis constant (KM) of the Escherichia coli AK for ADP is approximately 100 mM. Most AKs share similar tertiary features and are grouped into three functional subdomains. The core of the protein is composed of a central five-stranded parallel b-sheet surrounded by a number of ahelices. On the periphery of the core subdomain are the AMPbinding and the ATP-binding subdomains. Because of its essentiality and association with virulence, there is a profound interest in AK as a target for developing new drugs against infectious bacterial agents. The enzyme is also a good model of structural dynamics and catalysis research.
AK as the Detection Target in ATP Bioluminescence Assay Firefly luciferase catalyzes the following light-emitting reaction: ATP þ Luciferin þ O2 4AMP þ Oxyluciferin þ PPi þ Light Because of the high specificity of the firefly luciferase to ATP and the relative ease in light detection, the reaction is therefore convenient for quantifying ATP. The main reagents required in the assay, luciferase and luciferin, are commercially available and can be obtained in good purity. The reaction is simple to carry out and the light emitted can be measured easily by a luminometer that is generally inexpensive. Since ATP is present in all living organisms and is rapidly degraded
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following cell death, it can be used as a marker to monitor biomass, such as microorganisms. A recent study evaluated rapid microbiological monitoring methods based on detection of growth and found that ATP bioluminescence assay detected common microorganisms significantly faster than CO2 monitoring and turbidity assays. The ATP bioluminescence assay has gained acceptance by major regulatory authorities. For example, one of the commercial ATP bioluminescence assays, the Pallcheck Rapid Microbiology System (Pall Life Sciences, Hants, UK), has been granted approval by the Center for Drug Evaluation and Research at the US Food and Drug Administration for the release of certain nonsterile pharmaceuticals. Nevertheless, the sensitivity of ATP bioluminescence assay still has plenty of room to be improved. In typical assays, the limit of detection is about 103 bacteria or 1000 yeast cells, primarily due to background noise. The luminescence sensitivity can be improved partly by using a more sensitive optical sensor in the luminometer, although this is accompanied by adding up the instrument cost, and raising the background noises. An alternative way to improve the assay sensitivity is to change the detection target from ATP to ATP-producing enzyme such as AK. A medium-size bacterium normally contains about 1021 mol of AK in comparison with about 1018 mol of ATP. Escherichia coli AK has a kcat to ADP of around 300, which means that with just 1-min incubation the enzyme can generate 18 times more ATP for bioluminescent signal production than would be possible from the ATP naturally present on its own. The amplification reaction requires only a single substrate (i.e., ADP) and provides a linear increase in the amount of ATP over time. In theory, an AK assay should allow single bacterial cells to be detected in 10 min. Raised backgrounds from contaminating ATP and AK prevent this from being easily achieved, but it has been demonstrated. Kinases other than AK, such as pyruvate kinase, potentially could be used as ATP-generating cell markers. Unlike AK, which uses two ADP molecules to generate ATP, all the other ATP-generating kinases require two substrates: a phosphoryl donor and ADP. Obtaining a high degree of purity in the reagents is consequently made more difficult than when only one reagent is used in the assay. The approach based on AK detection thus has a unique advantage in terms of simplicity. Other reasons for the greater usefulness of AK over other kinases include its high catalytic activity, high robustness, and its ubiquitous nature due to its essential metabolic function.
General Considerations in AK-Based Bioluminescence Assay The procedure for AK detection is similar to the conventional ATP bioluminescence assay, except that an extended incubation time (of about 5 min) is needed for the AK assay. Certain requirements must be addressed in conducting and developing
Encyclopedia of Food Microbiology, Volume 1
http://dx.doi.org/10.1016/B978-0-12-384730-0.00003-3
Adenylate Kinase
the reagents for AK assays. First, different luminometers have different upper and lower detection limits and linear dynamic range in light detection. Appropriate tuning of the instrument into the right range is important. Second, there are both upper and lower limits to the concentrations of ADP substrate that can be used. A very high ADP concentration is inhibitory to the firefly luciferase reaction, reducing the light output for a given amount of AK, while a very low concentration of ADP provides too little substrate for conversion to ATP. Concentrations between 10 mM and 1 mM work out to be appropriate for most purposes. Third, the quality of the reagent must be in the highest obtainable purity to minimize the background signals that any extraneous ATP will cause. Certain batches of commercial ADP may require an additional purification step, frequently an anion exchange chromatography in which the ADP elutes before the contaminating ATP. Other components in the assay, in particular the luciferase, should be tested before the AK detection assay to ensure that the enzyme preparation contains minimal levels of contaminating AK to reduce background signals.
Release of ATP and AK from Cell by Detergents As in ATP bioluminescence, an extraction step is needed to release the intracellular AK. Extraction usually is carried out using a detergent. All of the manufacturers of ATP bioluminescence kits have proprietary formulations of extractants, and the suitability of the reagents for AK detection needs to be tested empirically to avoid inactivating either the AK or the luciferase. The concentration chosen for the detergent has to be a balance between maximizing disruption of cell membranes, and thus extraction of the AK, and minimizing inactivation of its enzymatic activity. Detergents, such as Triton X-100 and N, N0 ,N0 -polyoxyethylene (10)-N0 -tallow-1,3-diaminopropane at a concentration of 0.05%, have been found to produce reasonably good cell disruption effects without compromising the AK activity. Certain protocols (such as Promega’s ENLITENÒ ATP Assay System) employ trichloroacetic acid to extract ATP from bacterial and fungal cells. This approach is too harsh for AK and therefore is not suitable for AK assay even if a neutralization step is followed.
Effects of Reaction Time on AK Assay As explained, AK offers at least 10 times, and usually 100 times, greater sensitivity than the conventional ATP assays. Because the amplification kinetics is linear, it is therefore possible to increase the sensitivity of AK assays by extending the incubation time. Increasing the incubation time in the conventional ATP bioluminescence assays has no effect, because the majority of the ATP is extracted and consumed usually within the first 1-min incubation. The most convenient combination of speed and sensitivity for AK is achieved with a 5-min incubation. Using this, fewer than 100 cells of bacteria (such as E. coli or Pseudomonas aeruginosa) can be readily detected. The assay is reproducible and is relatively unaffected by changes in growth medium or conditions. By using small reaction volumes in combination with incubation times of up to 1 h, limits of detection approaching the single-cell level become possible. At this point, sampling statistics rather than assay sensitivity
19
become limiting, and the technique compares favorably with polymerase chain reactions and growth assays. Such smallvolume assays, in effect, can be carried out on filter membranes, and the full capability of the AK approach may be realized in this format.
Effects of Exogenous AK on the Assay In industrial applications, the source of the sample on which the test is being carried out could have a large effect on the end result. AK is present in virtually all living matter, not just bacteria. This means that the samples to be tested could contribute toward the signal. This applies to plant sources (e.g., fruit or vegetable juices) as well as meat and dairy produce. The levels can be seen to vary greatly, with those in meat being especially high. When total microbial loading is to be determined, the nonmicrobial AK may either be removed by suitable sample pretreatment methods or accounted for by subtracting the results from control assays carried out on uncontaminated samples. In a practical sense, both of the approaches are difficult to carry out without compromising the microbial AK detection. As might be expected, physical inactivation techniques such as high temperature and ultraviolet treatment cause a reduction in extraneous AK levels, although the microbial AK levels also will be reduced. Subtracting the results from that of uncontaminated control assays is also difficult, particularly when the extraneous AK level is much higher than that of the microbial AK. Thus, the AK luminescence assay is better to be performed in samples naturally low of AK.
Correlation of AK Assay Results with Viable Cell Count The concentration of ATP typically fluctuates widely within bacteria according to their metabolic status, size, and stress. This leads to inaccuracies when attempting to quantify the bacterial loading of a sample. So caution must be exercised in setting pass and fail levels when using ATP bioluminescence assay in standard hygiene monitoring. In contrast, AK is present at relatively constant levels regardless of the energy status of the cell. It therefore provides a much more reliable measure of the number of bacteria present than ATP. Nevertheless, several factors can affect the correlation between cell numbers, determined as colony-forming units, and the net bioluminescent signal from an AK assay. First, the viable cell counts obtained will tend to be lower than the number of cells present in the sample. This may be due to the fact that the culture condition is not suitable for the growth of the target bacteria, the bacteria are in a viable but nonculturable state, and the bacterial cells have not been fully divided or are clumped together. Second, the AK assay may not always be in linear kinetics. This happens frequently when compounds inhibiting either AK or luciferase are present in the testing samples. The accumulation of the AK and luciferase reaction products can feedback inhibit these enzymes. Thus, whether AK assay readings or the number of live cells provide the better measure of contamination will depend on the reason for carrying out the assay in the first place. In general, AK may be particularly useful where safety critical monitoring is required.
20
Adenylate Kinase
Potential Applications of AK Detection Assay The main application areas for AK-based microbiology assays in the food industry are rapid contamination and sterility testing, for example, hygiene monitoring for low-level contamination by microorganisms and other organic matter, such as air quality and food preparation surfaces. The materials to be tested should contain little endogenous AK or ATP (e.g., processing water) or samples in which endogenous free AK and ATP can be removed (e.g., beverages such as beer, cider, and soft drinks). With minor modification, the AK detection method may be used to monitor the presence of specific organisms such as foodborne bacterial pathogens. Such applications exploit the advantages that the high sensitivity of the AK approach can provide and will expand the areas of microbiological testing where rapid testing is feasible. Assays with AK as a marker should come to supplement rather than replace direct ATP measurements, which will remain appropriate in cases in which background contamination is inherently low, extreme sensitivity is not needed, or falsepositive results are more of a problem than false-negative results. Some application examples in using AK as a detection marker follow.
Hygiene Monitoring Using AK Bioluminescence Assay The procedure for hygiene monitoring using AK should be essentially the same as the widely used ATP bioluminescence method, lending itself equally well to the use of single-shot disposables. The only differences between the two assays are the addition of exogenous ADP and the need for an additional 5-min incubation time for AK detection. A typical AKbased luminescence method would include the following steps: 1. Swab area of interest with swab moistened with magnesium acetate buffer. 2. Place swab in a cuvette with ADP and extractant. 3. Incubate at room temperature for 5 min. 4. Add bioluminescence reagent. 5. Measure light output using a portable luminometer.
Specific Assays In cases in which identification of particular organisms is required rather than measurement of the total microbial loading, the AK assay may be used as the end point in immunoassays. One approach is to capture the target microorganism with magnetic beads coated with a specific antibody such as those against Listeria monocytogenes, E. coli O157, and Salmonella spp. The immunomagnetic separation procedures have been used to isolate bacterial cells from complex suspensions, such as food, before culture on agar plates or biochemical identification procedures to determine the level of contamination in the original sample. Nevertheless, the culture procedures are time consuming, taking 24–72 h to complete. The problem can be overcome by directly detecting the AK from the organisms captured on magnetic beads. Contamination from other organisms or the food materials is eliminated during the separation. Once immobilized, the bacteria can be lysed in the same way as a nonspecific assay. As illustrated in Figure 1, a typical AK-based pathogen-specific assay procedure is as follows: 1. Add antibody-conjugated beads to sample, resuspend, and incubate at room temperature. 2. Immobilize beads with a permanent magnet and remove supernatant. 3. Wash beads to remove any unbound material. 4. Resuspend beads in magnesium acetate buffer. 5. Add ADP plus extractant and incubate for 5 min. 6. Add bioluminescence reagent to sample extract. 7. Measure light output using a portable luminometer. The rate at which the bacteria will bind to the beads depends on the complexity of the testing sample. For typical liquid samples, binding of bacteria to the antibody-coated magnetic beads is rapid, taking a few minutes. In a more complex and viscous medium, the incubation time will need to be increased to allow the antibodies time to find the organisms. Nonetheless results will be obtained in approximately 1–2 h – a considerable time saving on conventional
Figure 1 Magnetic bead immunoassay for selective immobilization of microorganisms from food and beverage samples, prior to generic lysis and detection with AK bioluminescence. (a) Target cells are removed from suspension by mixing with magnetic beads pre-coated with an antibody specific to bacteria B. (b) Beads are immobilized by applying a magnetic field. Unwanted material is removed by washing. (c) The captured cells are lysed and ADP is added. (d) After incubation, bioluminescence reagent is added and the light output is measured.
Adenylate Kinase
methods and without the need to start with a pure culture. A major drawback of this approach is that it is difficult to obtain an antibody with good specificity and sensitivity for many bacterial species. In addition to using regular extractant to disrupt the captured cells, a promising strategy is the use of test methods that have double specificity. This specificity is achieved by combining a specific capture step with a specific lysis step, which gives extra confidence in assays for a particular organism. This may be achieved using lytic bacteriophages or commercially available lysins, such as colicin for E. coli and lysostaphin for Staphylococcus aureus. These should allow significant opportunities for replacing traditional microbiological testing with rapid methods, because it is more sensitive and easier to carry out in comparison with techniques such as enzyme-linked immunosorbent assay. Bacteriophage-mediated lysis of target cells may be achieved by adding to a culture a phage specific for the organism of interest. If the target organisms are present, the phage will infect them and cause the cells to be lysed. This releases all the intracellular components, including AK, into suspension. By adding ADP (with no extractant), the activity of this released AK can be measured. The assay principle is illustrated in Figure 2 and a typical procedure is as follows: 1. Mix the sample with culture medium and incubate at 37 C for 1–2 h to activate bacteria growth and enhance their susceptibility to bacteriophage. 2. Split sample into two: add bacteriophage into one and leave the other as an uninfected control. 3. Incubate at 37 C for 30–90 min 4. At timed intervals, remove a sample from each culture into a cuvette containing magnesium acetate buffer. 5. Add ADP and incubate for 5 min. 6. Add bioluminescence reagent and measure light output. Using bacteriophages, fewer than 103 log phase, E. coli cells can be detected in around 2 h under laboratory conditions. The bacteria must be in log phase to be receptive to phage infection, so a short culture step would be required to activate stationary phase or otherwise stressed cells. The time course of
21
the phage lysis is specific to the phagedhost combination used. Certain coliphage starts to induce bacteria lysis after about 20 min and may need 1 h to complete the lytic process. This means that the assay time will vary depending on the target organisms and bacteriophage chosen. Bacteriophages vary in their specificity. Some are capable of infecting bacteria from the same genus, whereas others are strain specific within a particular species. Because their bacterial host keep evolving in the natural environment, it is unlikely to identify a bacteriophage that can recognize all strains in a given species. Furthermore, the testing sample may not provide a suitable environment for the bacteriophage to complete their life cycle and hence release of AK. Together, these limitations prohibit wide application of bacteriophage in specific pathogen detection.
Application of AK in ATP Regeneration for Ultrasensitive Bioluminescence Assay Although AK can be a useful alternative cell marker to ATP in hygiene monitoring, ATP measurement remains a common and important test in biochemical research and medical diagnosis. The detection limit of ATP by conventional bioluminescence assay is approximately 1014–1012 M, which is suitable for most assays. Nevertheless, as highthroughput screening technologies become widely adopted and the testing sample volume is getting smaller, the increase of ATP bioluminescence assay sensitivity also has become important. In many testing samples, ATP is present in relatively low quantities and is depleted rapidly in the conventional luciferase assay. If the ATP consumed in the luciferase assay can be replenished, the assay sensitivity can be significantly enhanced. The replenishment of the ATP pool must be in a constant rate so the original ATP concentration can be determined reliably. A straightforward approach of ATP regeneration in the luciferase assay is to recycle the reaction products, either AMP or inorganic pyrophosphate (PPi), back to ATP. Because AK is the main kinase in the cells to
Figure 2 Specific bacteria detection assay based on selective lysis of target bacteria with bacteriophage. (a) Bacteriophage specific to bacteria B is added to the sample. (b) The phage causes bacteria B lysis and AK release. (c) ADP and bioluminescence reagents are then added to the sample and the light output is measured.
22
Adenylate Kinase
Luciferase ATP + Luciferin + O2 Acetate Acetate kinase Acetyl-P
Figure 3 assay.
AMP + Oxyluciferin + PPi + Light UTP
Adenylate kinase
ADP
UDP
Schematic presentation of AMP-regeneration bioluminescence
phosphorylate AMP into ADP, the enzyme was tested for its potential to replenish the ATP pool in the luciferase assay. It is obvious that exogenous ATP and ADP cannot be used as the phosphoryl donor in the recycling reaction because they will change the original ATP concentration and compromise the quantitative assay. Luckily, the substrate specificity of AK is remarkably high for AMP as a phosphoryl acceptor and relatively low for ATP as a phosphoryl donor. Thus, uridine triphosphate (UTP), which can be used as the phosphoryl donor in AK reaction but not in the luciferase reaction, is used in the AMP-recycling reaction. The reaction is as follows: Mg2þ $UTP þ AMP4Mg2þ $UDP þ ADP In theory, the ADP generated in this reaction can be further converted into ATP by AK. An additional kinase, such as acetate kinase, pyruvate kinase, and polyphosphate kinase, and their respective phosphoryl donor (acetyl phosphate, phosphoenolpyruvate, and polyphosphate) often are added to improve the efficiency of ADP to ATP conversion. In the presence of excess UTP, AK together with the luciferase reaction forms a cyclic loop of ATP regeneration. Ideally, this ATP regeneration approach enables the light signal output in a constant rate when other substrates are at excess. Thus, the assay sensitivity can be enhanced simply by increasing the measurement time. This approach is much more sensitive than the conventional bioluminescence assay, allowing for the detection of as low as one colony-forming unit of bacterium per assay. Further improvement of the ATP bioluminescence assay by recycling both PPi and AMP into ATP can result in exponential amplification of ATP. The exponential ATP amplification assay provides an extremely sensitive mean for ATP and bacteria detection. It therefore will be useful in situations in which the highest standard of hygiene is required, such as pharmaceutical
Table 1
Conclusion Firefly luciferase-based ATP bioluminescence assay is a fastdeveloping technique in rapid microbiological testing. Recently, several attempts have been made to apply AK in the bioluminescence assay to improve the assay sensitivity and to better correlate the results with the bacterial cell counts in the sample. AK can be used as the cell marker that generates ATP for detection or a tool to form a cyclic loop for ATP amplification. Certain types of samples inevitably may contain their own AK, which will tend to swamp that from contaminating microorganisms. The applicability of AK detection in rapid testing is thus restricted to cleanliness monitoring (i.e., testing for the absence of AK) and testing samples that have an inherently low level of AK or that can be treated easily to remove endogenous AK. On the other hand, the AK-mediated ATP regeneration approach offers high sensitivity in ATP detection and gives reliable results in ATP quantification. A comparison among the bioluminescent assays based on direct ATP detection, AK detection, and ATP-regeneration for microorganism monitoring is shown in Table 1. The primary advantage of using AK-based luminescence assays in hygiene monitoring over the conventional viable cell count method is their short assay time. This makes on-site monitoring possible and allows for decisions, such as whether to sterilize the processing facility or whether a food product can be shipped out, to be made without significant delay. In the future, the AK-based luminescence assays likely will become
Comparison of direct ATP detection, AK detection, and ATP-regeneration bioluminescent assays for microorganism detection
Nature of cell marker Ubiquitous in living cells Amount/average bacterial cell Intracellular levels Approximate assay time Incubation time dependent Correlation with cell number Assay detection limit (colony-forming unit/0.1 ml sample) a
manufacturing. On the other hand, the AMP recycling assay is preferred in situations in which accurate ATP quantification is more desirable, such as in enzyme kinetic studies and metabolic pathway analysis (Figure 3). Although the modification can significantly improve the sensitivity of an ATP bioluminescence assay, such an approach is more difficult to perform because several additional reagents need to be included in the assay. To reduce the assay background, the highest purity of the reagents must be used in the reaction. The concentration of the reagents also needs to be adjusted carefully to optimize the assay performance without adding too much reagent cost. Apparently, identifying how to simplify the assay components while maintaining the high sensitivity of the modified ATP bioluminescence reaction is the direction in which to go.
Dependent on the size and energy status of the bacterial cell.
Direct ATP
AK
ATP regeneration
Metabolite Yes 1018 mol Variable 1 min No Approximate 1000–10 000a
Enzyme Yes 1021 mol Constant 5 min Yes Good w100
Metabolite Yes 1018 mol Variable 5 min Yes Approximate <10
Adenylate Kinase
Table 2 Comparison of AK-based luminescence assay and conventional viable cell count assay in hygiene monitoring
Time required On-site monitoring Major equipment required Interference by food Microbial species identification
AK luminescence assay
Viable cell count assay
w5 min Yes Luminometer High Difficult
Overnight No Incubator Low Possible
a common supplement to the current ATP bioluminescent assay in situations in which high sensitivity is needed (Table 2).
See also: Application in Meat Industry; Bacteriophage-Based Techniques for Detection of Foodborne Pathogens; Biophysical Techniques for Enhancing Microbiological Analysis; Rapid Methods for Food Hygiene Inspection; Total Viable Counts: Metabolic Activity Tests; Water Quality Assessment: Routine Techniques for Monitoring Bacterial and Viral Contaminants.
Further Reading Blasco, B.R., Murphy, M.J., Sanders, M.F., Squirrell, D.J., 1998. Specific assays for bacteria using bacteriophage mediated release of adenylate kinase. Journal of Applied Microbiology 84, 661–666. Brokaw, J.B., Chu, J.W., 2010. On the roles of substrate binding and hinge unfolding in conformational changes of adenylate kinase. Biophysical Journal 99, 3420–3429. Brolin, S.E., Borglund, E., Agren, M.J., 1979. Photokinetic microassay of adenylate kinase using the firefly luciferase reaction. Journal of Biochemical and Biophysical Methods 1, 163–169. Buchko, G.W., Robinson, H., Abendroth, J., Staker, B.L., Myler, P.J., 2010. Structural characterization of Burkholderia pseudomallei adenylate kinase (Adk): profound asymmetry in the crystal structure of the ‘open’ state. Biochemical and Biophysical Research Communication 394, 1012–1017. Ceresa, L., Ball, P., 2006. Using ATP bioluminescence for microbiological measurement in pharmaceutical facturing. In: Miller, M.J. (Ed.), Encyclopedia of Rapid Microbiological Methods, vol. 2. Paranteral Drug Association/Davis Healthcare International Publishing LLC, pp. 233–249.
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Gilles, A.M., Saint-Girons, I., Monnot, M., Fermandjian, S., Michelson, S., Bârzu, O., 1986. Substitution of a serine residue for proline-87 reduces catalytic activity and increases susceptibility to proteolysis of Escherichia coli adenylate kinase. Proceedings of the National Academy of Sciences of the United States of America 83, 5798–5802. Jacobs, A.C., Didone, L., Jobson, J., Sofia, M.K., Krysan, D., Dunman, P.M., 2012. Adenylate kinase release as a high throughput screening compatible reporter of bacterial lysis for the identification of antibacterial agents. Antimicrobial Agents and Chemotherapy [Epub ahead of print]. Lee, H.J., Ho, M.R., Tseng, C.S., Hsu, C.Y., Huang, M.S., Peng, H.L., Chang, H.Y., 2011. Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria. Analytical Biochemistry 418, 19–23. Markaryan, A., Zaborina, O., Punj, V., Chakrabarty, A.M., 2001. Adenylate kinase as a virulence factor of Pseudomonas aeruginosa. Journal of Bacteriology 183, 3345–3352. Murphy, M.J., Squirrell, D.J., 1999. Adenylate kinase. In: Robinson, R.K. (Ed.), Encyclopedia of Food Microbiology. Elsevier Ltd, pp. 16–24. Murphy, M.J., Squirrell, D.J., Sanders, M.F., Blasco, R., 1995. The use of adenylate kinase for the detection and identification of low numbers of microorganisms. In: Hastings, J.W., Kricka, L.J., Stanley, P.E. (Eds.), Bioluminescence and Chemiluminescence: Molecular Reporting with Photons. John Wiley, Chichester, pp. 319–322. Parveen, S., Kaur, S., David, S.A., Kenney, J.L., McCormick, W.M., Gupta, R.K., 2011. Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products. Vaccine 29, 8012–8023. Satoh, T., Kato, J., Takiguchi, N., Ohtake, H., Kuroda, A., 2004. ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell. Bioscience Biotechnology and Biochemistry 68, 1216–1220. Schrank, T.P., Bolen, D.W., Hilser, V.J., 2009. Rational modulation of conformational fluctuations in adenylate kinase reveals a local unfolding mechanism for allostery and functional adaptation in proteins. Proceedings of the National Academy of Sciences of the United States of America 106, 16984–16989. Schulz, G., Muller, C.W., Diederichs, K., 1990. Induced-fit movements in adenylate kinases. Journal of Molecular Biology 213, 627–630. Squirrell, D.J., Murphy, M.J., 1994. Adenylate kinase as a cell marker in bioluminescent assays. In: Campbell, A.K., Kricka, L.J., Stanley, P.E. (Eds.), Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects. John Wiley, Chichester, pp. 486–489. Squirrell, D.J., Murphy, M.J., 1997. Rapid detection of very low numbers of micro-organisms using adenylate kinase as a cell marker. In: Stanley, P.E., Simpson, W.J., Smither, R. (Eds.), A Practical Guide to Industrial Uses of ATPluminescence in Rapid Microbiology. Cara Technology, Lingfield. Wills, K., 2003. ATP bioluminescence and its use in pharmaceutical microbiology. In: Easter, M.C., Raton, B. (Eds.), Rapid Microbiological Methods in the Pharmaceutical Industry. Interpharm/CRC Press, Florida.