Microbiological applications for bacterial metabolism on a microplate reader

Bacterial metabolism applications

Bacterial metabolism spans the complex interconnected set of chemical reactions that support life in bacteria, ancient single-celled organisms that represent one of the most diverse forms of life on Earth. Bacterial metabolism is the foundation for bacterial growth and is inextricably linked to other organisms in the ecosystem.

Many applications in microbiology hinge on the monitoring of enzymatic reactions or the measurement of specific metabolites, enzyme concentrations, and chemical reactions involved in the molecular processes of life. They may also address crucial steps in the control of metabolism at the levels of proteins or genes. These applications are incredibly useful for basic research, studying the environment, probing different aspects of biotechnology, or contributing to undertakings like drug discovery.^1-3

In this blog we examine bacterial metabolism: its scope, its interest to researchers, and how diverse applications, including those linked to metabolic control, measurement of metabolites and bacterial growth, can be determined using microplate readers. 

What is bacterial metabolism?

Cells extract energy from their environment and convert smaller molecules into cell components by a highly integrated network of chemical reactions and interactions referred to as metabolism. The underlying strategy of metabolism is to form ATP, NADPH, which are used as energy currency, and large molecule precursors and this is true for most organisms. ⁴ In the world of bacteria, bacterial metabolism refers to the complex set of reactions that occur within these organisms to sustain life. However, bacteria are inexorably intertwined with their environment and the impact of bacterial metabolism goes beyond bacterial populations.

Anabolic and catabolic pathways

Bacterial metabolism allows bacteria to grow, reproduce, maintain their structures and respond to environmental changes. There are at least a thousand chemical reactions in a bacterium like Escherichia coli.⁴ The number of reactions is large but the number of kinds of reactions is relatively small. Overall, these reactions can be divided into two broad types referred to as anabolic and catabolic pathways (Fig. 1). Anabolic processes involve the synthesis of complex molecules from simpler ones. These reactions require energy, in many cases the ATP generated from within the bacterial cell. Catabolic processes and catabolic pathways involve the breakdown of complex molecules like carbohydrates, fats and proteins into simpler ones (like glucose and amino acids) releasing energy in the process which can be used to drive the processes of anabolic metabolism. These anabolic and catabolic pathways are essential to life.

Key metabolic pathways

Bacteria are incredibly diverse and have evolved various metabolic pathways to allow them to adapt to many environments. Some can switch between different metabolic pathways and different biochemical reactions depending on environmental conditions. Others are highly specialized for specific and often very fastidious environments.

Some of the most commonly found and important metabolic pathways in bacteria (Fig. 2) include the biochemical reactions of glycolysis (the conversion of glucose to pyruvate), the pentose phosphate pathway, the citric acid cycle (the citric acid cycle comprises oxidation of acetyl-CoA from carbohydrates, fatty acids and amino acids), different types of fermentation, the Calvin cycle (part of photosynthesis to generate glucose from CO₂), and nitrogen fixation (conversion of molecular nitrogen into ammonia).⁵

Types of microbial metabolism

Some of the capabilities of bacteria include the metabolic pathways and metabolic processes linked to aerobic respiration, anaerobic respiration, fermentation, photosynthesis or the generation of energy from the oxidation of inorganic molecules like ammonia or hydrogen sulfide. This adaptability has allowed them to adjust to environments as diverse as deep-sea vents or the human gut. Depending on their capabilities, microbes can be categorised into different metabolic types (Table 1) with different metabolic pathways. A heterotrophic microbe cannot produce its own food and instead produces energy from other sources of organic carbon like sugar. In contrast, autotrophic microbes can convert abiotic sources of energy into stored energy like sugars from CO2. Photolithotrophs oxidize inorganic substrate molecules and use the sunlight as energy source like photoorganotrophs but depend on different hydrogen sources.

Table 1: Types of microorganisms and their nutritional sources. N/A, not applicable.

Bacterial metabolic control

Metabolism and metabolic pathways are controlled in multiple ways. The amounts of critical enzyme molecules and enzyme concentrations for example may be controlled by the regulation of the rate of protein synthesis and degradation as well as the regulation of gene expression. In addition, the catalytic activity of some enzyme molecules is regulated by allosteric interactions (for example feedback inhibition) or by covalent modifications. Distinct pathways for the synthesis and degradation of metabolites also contribute to metabolic control.

Bacterial metabolism and bacterial growth

Bacterial metabolism and metabolic pathways are inextricably linked to bacterial growth.⁶ It provides the energy needed to support cell division, synthesize necessary macromolecules, make use of nutrients, manage waste products, and adapt to environmental conditions. Efficient metabolism supports robust growth and promotes survival in various environments, many of which are challenging to life.

Why measure bacterial metabolism?

Many applications

Measurements of bacterial metabolism give researchers a handle on many processes and applications. They can provide industrial uses like biotechnologies for optimizing fermentation processes such as those geared to the production of antibiotics, enzymes and fuels. Antimicrobial resistance alone is a large driver for new approaches to develop novel drugs informed by understanding of bacterial metabolism and bacterial growth. 

Improved understanding

Understanding metabolism is also crucial for drug discovery and investigating disease mechanisms. Knowledge of metabolic control in bacteria is not only important for practical applications in biotechnology, medicine, and the environmental sciences but is also a foundation for fundamental research in laboratories across the globe.

Measurements of bacterial metabolism on microplate readers 

At first glance, there is a bewildering array of diversity to the approaches used to study bacterial metabolism. However, whatever approach is selected, researchers are interested in technologies that deliver efficiencies to their work.

What are some examples of applications relevant to bacterial metabolism? Here we look at some practical examples that can either be used directly or adapted to measure parameters like metabolic control, the amounts of specific metabolites, or which act as surrogate measurements like bacterial growth and OD600. 

Central elements of every metabolic pathway are active enzymes. No substrate can be converted to a metabolic product if no active enzyme is available to metabolize that substrate. Enzyme activity is therefore a good indicator of bacterial metabolism that can be used for detection purposes. In the application note Lysine deacetylase activity monitored by a fluorogenic assay using the CLARIOstar the kinetic properties of the Escherichia coli lysine deacetylase CobB enzymatic reaction were measured using CycLex SIRT1 assay kits (Fig. 3).

Enzymatic deacetylation of a lysine residue within the peptide in the assay kit generates a functional peptidase cleavage site. The fast subsequent cleavage of the deacetylated peptide by the peptidase separates the fluorophore from the quencher and allows fluorescent readout of the reaction. This method was used to determine the kinetic properties of the E. coli enzyme (Fig. 4). Lysine deacetylation is linked to control of gene expression, protein function and stability as well as the control of metabolism. Developments in epigenetics relevant to drug discovery have driven interest in measuring deacetylating enzymes and this type of approach has wide applications. You can read more about deacetylation reactions and microplate readers in the blog Histone deacetylases (HDACs): erasers of epigenetic marks.

Another important control point for bacterial metabolism is at the level of redox enzymes. NADH/NAD+ and NADPH/NADP+ are cofactors used by numerous redox enzymes involved in metabolism and being able to monitor the reduction of these cofactors provides a reliable way of measuring the activities of these enzymes. In the application note Overview of ELISA assays and NADH/NADPH conversion detection the capability of the UV visible spectrometer in BMG LABTECH readers is highlighted for the rapid determination of oxidized and reduced nicotinamide cofactors (Fig. 5). The reduction of NAD⁺ to NADH and NADP⁺ to NADPH can be monitored at 340 nm because the oxidized forms do not absorb light at this wavelength. By using the spectrometer feature, researchers can select either a full spectrum (220-1000 nm) or part of a spectrum (220-400 nm) with the same measuring time to achieve rapid and accurate measurements of redox states. This type of absorbance assay has wide utility for measuring different reactions in bacterial metabolism. In addition, modern microplate readers come equipped with data management and analysis software which facilitates the discovery of findings and the reporting of results. Reactive oxygen species are products of oxidative metabolism in cells. The ability to measure reactive oxygen species can provide important information on, for example, cell signaling events including those that take place in bacteria and other cells. Such events may impact the control of gene expression and enzyme activities and lead to changes in metabolism in bacteria. In the application note Fluorescence analysis of reactive oxygen species (ROS) generated by six isolates of Aspergillus fumigatus a team of researchers describes a fluorescence-based assay that can be used to measure reactive oxygen species in bacterial cells. The assay uses 2’,7’-dichlorfluoresceindiacetate, a chemical dye whose fluorescence changes in the presence of peroxide. In this case, intracellular H₂O₂ was readily detected with a lower limit of detection of 5 nM in 200 µl samples.

In many cases when studying bacterial metabolism, it is necessary to quantify the levels of specific metabolites quickly and accurately. Glucose plays a central role in energy consumption and serves as a primary metabolic fuel for bacteria and other cells. Glucose can also be metabolized to produce lactate by fermentation. Measurement of glucose and lactate levels can therefore provide researchers with insight into the metabolic activity of specific bacterial cells and how they respond to different environmental conditions. These types of assays benefit from miniaturization and multiplex-type approaches. In the application note Glucose assay and lactate assay allow to monitor cellular glucose metabolism precisely commercially available luminescence-based assays were successfully miniaturized and read on the VANTAstar^® microplate reader. The assays make use of glucose and lactate dehydrogenase enzymes coupled to the activity of a luciferase enzyme (Fig. 6). The enhanced dynamic range of the VANTAstar microplate reader makes these assays easy to perform and eliminates the need for manual intervention from the start of the reactions to the luminescent readout.

Knowledge of bacterial metabolism can also translate to innovation that improves the performance of other methods used by scientists. Mycoplasma are bacteria that are unusual in that they lack a cell wall. They are often an unwanted contaminant in the cell culture systems used in the laboratory and scientists need quick and accurate ways to make sure their cultures are free from contamination with these organisms. The application note entitled Lonza's MycoAlert assay on a BMG LABTECH plate reader describes an assay that detects mycoplasma in less than 20 minutes. The method detects a metabolic enzyme that is specific to the metabolism of mycoplasma. In the MycoAlert™ assay, a specific substrate catalyses the formation of ATP from adenosine diphosphate which can then be detected in a highly sensitive luminescence-based assay (Fig. 7). An increase of ATP levels over the background levels indicates the presence of mycoplasma thanks to a rapid, simple and robust bioluminescence assay.

Oxygen levels and pH are important parameters for many measurements pertinent to bacterial metabolism. They directly influence metabolic pathways and the activities of specific enzymes, affect the efficiency of these steps, and may impact the overall health and growth of bacterial populations. Measuring and understanding the significance of oxygen levels and pH allows researchers to better control bacterial processes in natural and artificial environments. Both parameters may affect for example cellular bioenergetics, the way in which bacteria and other cells generate and use energy. Open-flow respirometry has been used as a physiologically relevant approach that allows for the measurement of respiration in cells. In the application note Measuring changes in cellular metabolism by monitoring extracellular acidification and oxygen consumption in real-time this system was translated to a 96-well microplate format that readily allows oxygen and pH levels to be measured in real-time under physiologically relevant atmospheric conditions. The system was developed on a CLARIOstar^® microplate reader equipped with an atmospheric control unit.

The Atmospheric Control Unit from BMG LABTECH provides researchers with a system that uniquely enables control of both the oxygen and carbon dioxide concentrations in an independent manner. This can be useful to achieve the optimal growth conditions for bacteria that may be challenging to grow for experimental study. Oxygen consumption under defined atmospheric conditions was measured efficiently and accurately with this system using a phosphorescent oxygen and pH probe from Cayman Chemical (Fig. 8). While the experiments were performed for human cells the system can be easily adapted for use with bacterial cells and serves as a “push button” method to measure oxygen and pH levels for bacterial cells.

Modern microplate readers offer features that have special relevance to the study of bacterial metabolism. These range from atmospheric control units and shaking options that keep bacteria under optimal growth conditions to enhanced dynamic range features that allow reliable detection of samples at a large range of concentrations and signal intensities with no manual intervention. A high-quality microplate reader will offer different shaking modes with adjustable speeds over a large range to provide optimum aeration settings for different bacterial strains.

Microplate readers offer high throughput, quantitative, and time-saving analysis for many assays that support research into bacterial metabolism. The use of different detection technologies means that researchers can deploy different techniques with varying sensitivities of measurement depending on the needs of their experimental systems.

The use of 96-, 384- or 1536-well microplates allows the processing of many samples simultaneously or in quick succession. This allows large numbers of samples to be measured in a single run which reduces the time for the collection of data. Real-time or time-resolved measurements are suitable to map the dynamic changes that may take place in bacterial metabolism. This applies to the kinetics of bacterial growth and enzyme activities for example, two frequently encountered applications relevant to bacterial metabolism.

Bacterial growth is only possible with an active bacterial metabolism. The measurement of bacterial growth is therefore an effective surrogate measurement for some studies on bacterial metabolism. Measurement of bacterial growth based on optical density (OD₆₀₀) is frequently used in many laboratories. The method comprises taking readings of bacterial samples over time that correlate with the number of organisms in a sample. OD₆₀₀ measures the amount of light scattering by a bacterial suspension and not its absorbance and you can learn more about the options available in the BMG LABTECH blog Bacterial growth measurements on a microplate reader.

Microplate readers also offer the capacity to make parallel measurements under a wide range of conditions using different detection modes. Multi-mode microplate readers therefore offer benefits in speed, reproducibility and scale for routine and advanced measurements of bacterial metabolism. For example, fluorescence and luminescence measurements offer ways to measure bacterial growth as a surrogate for bacterial metabolism. Both detection methods are highly sensitive and can detect low numbers of bacterial cells. They include the use of fluorophores like green fluorescent protein (GFP) that can be expressed in the bacterial organism of interest. In the application note Expression of a stable green fluorescent protein mutant in group B Streptococcus: Growth, detection and monitoring with the CLARIOstar the use of a GFP biomarker was used to track the growth of bacteria in liquid media. The CLARIOstar was used to measure fluorescence, absorbance and fluorescence polarization simultaneously. These measurements provide a highly sensitive alternative to conventional OD₆₀₀ and fluorescence measurements which in this case were measured at the same time.

Compounds that inhibit bacterial metabolism may also inhibit bacterial growth. The minimum inhibitory concentration or minimum bactericidal concentration may therefore be useful parameters in the initial screens for new compounds affecting specific steps in bacterial metabolism.

Quorum sensing is a phenomenon allowing bacteria to adapt to environmental conditions. Specifically, it is a type of cell-to-cell communication that depends on secreted chemical signaling molecules, bacterial cell density, and changes in gene expression. Researchers study quorum sensing since it can directly influence how certain bacteria regulate their metabolic activities. In the application note Monitoring bacterial cell-to-cell communication quorum sensing using a BMG LABTECH microplate reader experiments are described whereby a BMG LABTECH microplate reader was able to measure microbial growth (Fig. 9) and the bioluminescence arising from quorum sensing in parallel. This type of measurement has applications not only for studies of bacterial metabolism but is also useful in looking for new ways to combat biofilm formation and antimicrobial resistance. You can read more about quorum sensing in the BMG LABTECH blog Quorum sensing: how bacteria stay in touch.

Future developments for bacterial metabolism applications

Our knowledge of bacterial metabolism continues to grow at pace and scientists have only scratched the surface of the bacterial world on Earth. The demand for measurements of bacterial metabolism will therefore continue to grow in the years ahead as new discoveries, technologies and applications emerge from laboratories worldwide. Further advances in detection technologies and innovation in the specific assays available for microplate readers should serve as a formidable catalyst for further developments.

BMG LABTECH solutions for bacterial metabolism assays

What is the preferred BMG LABTECH microplate reader for specific needs and applications related to bacterial metabolism? Absorbance detection for the measurement of OD₆₀₀ is available on BMG LABTECH’s complete portfolio of microplate readers with the ultrafast spectrometer. The exception is the NEPHELOstar^® Plus which is a dedicated laser-based nephelometer for light scattering and turbidity measurements. However, this does not mean that it cannot be used to examine bacterial growth. The NEPHELOstar Plus offers turbidimetric measurements for the determination of bacterial growth at very high sensitivity. It can be used for example to study the early stages of bacterial growth.

Most of BMG LABTECH’s readers are available as multi-mode detection devices for sensitive fluorescence and luminescence measurements. Both the VANTAstar® and CLARIOstar Plus further allow for wavelength flexibility and include Enhanced Dynamic Range technology for superior performance in a single luminescence or fluorescence run. They also offer increased light transmission and sensitivity courtesy of Linear Variable Filter Monochromators^TM and different filter options.

Bacteria require specific temperatures and aeration for maximum growth rates. To ensure optimal growth conditions, all BMG LABTECH readers offer accurate temperature regulation up to 45°C (some devices even offer the option of temperature regulation up to 65°C). Three shaking modes with adjustable speed up to 700 rpm (optionally to 1100 rpm) provide optimum aeration settings for your strain. Additionally, the VANTAstar, CLARIOstar Plus, the Omega series and the SPECTROstar Nano can be equipped with an extraordinary robust transport system for shaking 24/7 where required.

The VANTAstar, the CLARIOstar Plus, the Omega series and NEPHELOstar Plus can be combined with the Atmospheric Control Unit making them the preferred choice for different kinds of live cell assays including bacterial growth assays with specific requirements for the surrounding atmosphere.

All BMG LABTECH microplate readers have exceptionally fast reading capabilities. In addition, the Omega series, NEPHELOstar Plus, CLARIOstar Plus,  and PHERAstar^® FSX microplate readers come with on-board injectors that can offer the very best options for detection at the time of injection. The VANTAstar can be equipped with a modular injection unit. The SPECTROstar Nano comes with a dedicated cuvette-port which can also be used to study bacterial growth over time in a cuvette-based approach.

Collectively, BMG LABTECH multi-mode readers combine high-quality measurements with miniaturised assays, short measurement times, and offer considerable savings on materials and other resources.

References

1. Gerhard Gottschalk (1985) Bacterial metabolism. Second edition, Springer Verlag.
2. Wood WB (1974) The molecular basis of metabolism. McGraw-Hill.
3. Atkinson DE (1977) Cellular energy metabolism and its regulation. Academic Press.
4. Stryer L (1975) Biochemistry. WH Freeman and Company, San Francisco.
5. Richardson AR, Somerville GA, Sonenshein AL. (2015) Regulating the intersection of metabolism and pathogenesis in Gram-positive bacteria. Microbiology Spectrum 3(3):MBP-0004-2014. doi: 10.1128/microbiolspec.MBP-0004-2014. Fig.2 in this blog adapted from this reference under license CC-BY 4.0.
6. Dean, A. C. R., and Hinshelwood, C. (1966) Growth, Function and Regulation in Bacterial Cells. London: Oxford University Press.