Microbiology

Expanding our understanding of drug-gut bacteria interactions requires high-throughput drug measurements in complex bacterial cultures. Quantification of drugs in the cultures, media, and cell pellets is prone to strong matrix effects. We have developed a liquid chromatography–high resolution mass spectrometry (LC–HRMS) method for quantifying duloxetine from high-throughput gut-drug interaction experiments. The method is partially validated for its reproducibility, sensitivity, and accuracy, which makes it suitable for largescale drug screens. We extensively used this method to study biotransformation and bioaccumulation of duloxetine and other drugs in several species of gut bacteria.

Strategies to control the levels of key enzymes of bacterial metabolism are commonly based on the manipulation of gene of interest within the target pathway. The development of new protocols towards the manipulation of biochemical processes is still a major challenge in the field of metabolic engineering. On this background, the FENIX (functional engineering of SsrA/NIa-based flux control) system allows for the post-translational regulation of protein levels, providing both independent control of the steady-state protein amounts and inducible accumulation of target proteins. This strategy enables an extra layer of control over metabolic fluxes in bacterial cell factories (see Graphical abstract below). The protocol detailed here describes the steps needed to design FENIX-tagged proteins and to adapt the system to virtually any pathway for fine-tuning of metabolic fluxes.

Graphical abstract

Sulfatase activity is often used as a measure of the activity of soil microorganisms. It is thus a suitable tool to investigate the response of microbes to plants. Here we present a method to determine the influence of various Arabidopsis genotypes on the function of soil microbiota using the sulfatase as a quantitative measure. We grew the plants in soil/sand mix under control conditions and measured the sulfatase activity in soil using a spectrophotometric determination of the product. This protocol can be used to test the contribution of individual genes to control of microbiome assembly through analysis of mutants as well as the influence of environment on plant-microbe interactions.

Trehalose (and glycogen) is a major storage carbohydrate in many cells, including S. cerevisiae. Typically, trehalose (a disaccharide of glucose) is synthesized and stored through gluconeogenesis. However, trehalose can also be made directly from glucose, if glucose-6-phosphate is channeled away from glycolysis or pentose phosphate pathway. Therefore, analyzing trehalose synthesis, utilization or its accumulation, can be used as a sentinel read-out for either gluconeogenesis or rewired glucose utilization. However, the steady-state measurements alone of trehalose cannot unambiguously distinguish the nature of carbon flux in a system. Here, we first summarize simple steady-state enzymatic assays to measure trehalose (and glycogen), that will have very wide uses. Subsequently, we describe methods of highly sensitive, quantitative LC-MS/MS based to measure trehalose. We include methods of ¹³C stable-isotope based pulse-labeling experiments (using different carbon sources) with which to measure rates of trehalose synthesis, from different carbon metabolism pathways. This approach can be used to unambiguously determine the extent of carbon flux into trehalose coming from gluconeogenesis, or directly from glucose/glycolysis. These protocols collectively enable comprehensive steady-state as well as carbon flux based measurements of trehalose. This permits a dissection of carbon flux to distinguish between cells in a gluconeogenic state (conventionally leading to trehalose synthesis), or cells with rewired glucose metabolism (also leading to trehalose synthesis). While the methods presented are optimized for yeast, these methods can be easily adapted to several types of cells, including many microbes.

Biogenic volatile compounds (VCs) mediate various types of crucial intra- and inter-species interactions in plants, animals, and microorganisms owing to their ability to travel through air, liquid, and porous soils. To study how VCs produced by Verticillium dahliae, a soilborne fungal pathogen, affect plant growth and development, we slightly modified a method previously used to study the effect of bacterial VCs on plant growth. The method involves culturing microbial cells and plants in I plate to allow only VC-mediated interaction. The modified protocol is simple to set up and produces reproducible results, facilitating studies on this poorly explored form of plant-fungal interactions. We also optimized conditions for extracting and identifying fungal VCs using solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS).

There is a pressing need to develop sustainable and efficient methods to protect and stabilize iron objects. To develop a conservation-restoration method for corroded iron objects, this bio-protocol presents the steps to investigate reductive dissolution of ferric iron and biogenic production of stabilizing ferrous iron minerals in the strict anaerobe Desulfitobacterium hafniense (strains TCE1 and LBE). We investigated iron reduction using three different Fe(III) sources: Fe(III)-citrate (a soluble phase), akaganeite (solid iron phase), and corroded coupons. This protocol describes a method that combines spectrophotometric quantification of the complex Fe(II)-Ferrozine^® with mineral characterization by scanning electron microscopy and Raman spectroscopy. These three methods allow assessing reductive dissolution of ferric iron and biogenic mineral production as a promising alternative for the development of an innovative sustainable method for the stabilization of corroded iron.

Invertase can catalyze the hydrolysis of sucrose, and is widely distributed in cells of cyanobacteria and plants. Being responsible for the first step for sucrose metabolism, invertase plays important physiological roles and its enzymatic activity is frequently needed to be determined. All the methods for determination of the invertase activity are dependent on detection of the glucose product generated by the invertase. Here we describe an ion chromatography based protocol of our laboratory for determination of cyanobacterial intracellular invertase activity.

Bacteria release cysteine to moderate the size of their intracellular pools. They can also evolve hydrogen sulfide, either through dissimilatory reduction of oxidized forms of sulfur or through the deliberate or inadvertent degradation of intracellular cysteine. These processes can have important consequences upon microbial communities, because excreted cysteine autoxidizes to generate hydrogen peroxide, and hydrogen sulfide is a potentially toxic species that can block aerobic respiration by inhibiting cytochrome oxidases. Lead acetate strips can be used to obtain semiquantitative data of sulfide evolution (Oguri et al., 2012). Here we describe methods that allow more-quantitative and discriminatory measures of cysteine and hydrogen sulfide release from bacterial cells. An illustrative example is provided in which Escherichia coli rapidly evolves both cysteine and sulfide upon exposure to exogenous cystine (Chonoles Imlay et al., 2015; Korshunov et al., 2016).

Most of the cyanobacteria accumulate osmolytes including sucrose, glucosylglycerol, in their cells in response to salt stress. Here we describe a protocol of our laboratory for extraction and quantification of cyanobacterial intracellular sucrose and glucosylglycerol. We have confirmed this protocol was applicable to at least four kinds of cyanobacteria, filamentous cyanobacterium Anabaena sp. PCC 7120, unicellular cyanobacterium Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942 and halotolerant unicellular cyanobacterium Synechococcus sp. PCC 7002.

Mycobacterium tuberculosis (Mtb) has evolved to assimilate fatty acids from its host. However, until recently, there was no reliable way to quantify fatty acid uptake by the bacteria during host cell infection. Here we describe a new method to quantify fatty acid uptake by intracellular bacilli. We infect macrophages with Mtb constitutively expressing mCherry and then metabolically label them with Bodipy-palmitate. Following the labeling procedure, we isolate Mtb-containing phagosomes on a sucrose cushion and disrupt the phagosomes with detergent. After extensive washes, the isolated bacteria are analyzed by flow cytometry to determine the level of Bodipy-palmitate signal associated with the bacteria. Using a Mtb mutant strain defective in fatty acid uptake in liquid culture we determined that this mutant assimilated 10-fold less Bodipy-palmitate than the wild type strain during infection in macrophages. This quantitative method of fatty acid uptake can be used to further identify pathways involved in lipid uptake by intracellular Mtb and possibly other bacteria.