Microbiology

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.

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).

Manganese (Mn) is an essential micronutrient for all photoautotrophic organisms. Two distinct pools of Mn have been identified in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), with 80% of the Mn residing in the periplasm and 20% in cytoplasm and thylakoid lumen (Keren et al., 2002). In this protocol, we describe a method to quantify the periplasmic and intracellular pools of Mn in Synechocystis accurately, using inductively coupled plasma mass spectrometry (ICP-MS).

AmTracs are the first example of “activity sensors”, since they report the activity of ammonium transporters by means of fluorescence readout in vivo (De Michele et al., 2013). AmTracs are based on a single fluorescent protein, a circularly permuted GFP (cpGFP), inserted into the cytosolic loop connecting the two pseudosymmetrical halves of the Arabidopsis and yeast plasma membrane ammonium transporters AtAMTs and ScMEP (Figure 1). Recently, FRET-based activity sensors for nitrate and peptide transporters have also been developed (Ho et al., 2014). Since transporter activity directly depends on the availability of substrate, AmTracs measure extracellular ammonium concentrations. Several versions of AmTrac exist, with different fluorescence intensity (FI) responses and affinities for ammonium, and based on different ammonium transporters (AmTrac: AtAMT1;3; AmTrac1;2: At AMT1;2; MepTrac: ScMEP2). Currently, the most useful AmTrac versions are probably AmTrac-GS (bright, with Km of 50 µM) and AmTrac-100 (a high capacity version with Km of 100 µM). The protocol for measuring ammonium concentrations in yeast cells at the fluorimeter is the same for all versions.


Figure 1. Model of AmTrac/MepTrac sensors and AMT transport mechanism. We propose that AMT switches between at least two distinct states during transport of ammonium: An outward, open state A and an inward, open state B. The movement of TMH-V (red) and TMH-VI (blue) is transmitted to the connecting loop, affecting the inserted cpGFP (green) and resulting in a change in fluorescence emission.

Secondary active transport of substrates across the inner membrane is vital to the bacterial cell. Of the secondary active transporter families, the ubiquitous major facilitator superfamily (MFS) is the largest and most functionally diverse (Reddy et al., 2012). Recently, it was reported that the MFS multidrug efflux protein MdtM from Escherichia coli (E. coli) functions physiologically in protection of bacterial cells against bile salts (Paul et al., 2014). The MdtM transporter imparts bile salt resistance to the bacterial cell by coupling the exchange of external protons (H⁺) to the efflux of bile salts from the cell interior via an antiport reaction. This protocol describes, using fluorometry, how to detect the bile salt/H⁺ antiport activity of MdtM in inverted membrane vesicles of an antiporter-deficient strain of E. coli TO114 cells by measuring transmembrane ∆pH. This method exploits the changes that occur in the intensity of the fluorescence signal (quenching and dequenching) of the pH-sensitive dye acridine orange in response to changes in [H⁺] in the vesicular lumen. Due to low levels of endogenous transporter expression that would normally make the contribution of individual transporters such as MdtM to proton-driven antiport difficult to detect, the method typically necessitates that the transporter of interest be overexpressed from a multicopy plasmid. Although the first section of the protocol described here is very specific to the overexpression of MdtM from the pBAD/Myc-His A expression vector, the protocol describing the subsequent measurement of bile salt efflux by MdtM can be readily adapted for measurement of antiport of other substrates by any other antiporter that exchanges protons for countersubstrate.

The transmembrane proton gradient (ΔpH) is the primary source of energy exploited by secondary active substrate/H⁺ antiporters to drive the electroneutral transport of substrates across the Escherichia coli (E. coli) inner membrane. Such electroneutral transport results in no net movement of charges across the membrane. The charge on the transported substrate and the stoichiometry of the exchange reaction, however, can result in an electrogenic reaction which is driven by both the ΔpH and the electrical (∆Ψ) components of the proton electrochemical gradient, resulting in a net movement of electrical charges across the membrane. We have shown that the major facilitator superfamily transporter MdtM - a multidrug efflux protein from E. coli that functions physiologically in protection of bacterial cells against bile salts - imparts bile salt resistance to the bacterial cell by coupling the exchange of external protons (H⁺) to the efflux of bile salts from the cell interior via an electrogenic antiport reaction (Paul et al., 2014). This protocol describes, using fluorometry, how to detect electrogenic antiport activity of MdtM in inverted membrane vesicles of an antiporter-deficient strain of E. coli TO114 cells by measuring transmembrane ∆Ψ. The method exploits changes that occur in the intensity of the fluorescence signal (quenching and dequenching) of the probe Oxonol V in response to changes in membrane potential due to the MdtM-catalysed sodium cholate/H⁺ exchange reaction. The protocol can be adapted to detect activity of any secondary active antiporter that couples the electrogenic translocation of H⁺ across a biological membrane to that of its counter-substrate, and may be used to unmask otherwise camouflaged transport activities and physiological roles.

Streptomyces species produce spores, which, while not as robust as endospores of Bacillus or Clostridium species, are capable of surviving for months or even years (Hopwood, 2006). During this time these spores remain viable, surviving by slowly degrading internal stores of carbon compounds, such as the carbohydrate trehalose. To enable metabolism to continue they must have access to an electron acceptor that allows the removal of the reducing equivalents that accumulate through metabolic activity. The most commonly used acceptor is oxygen. We describe the quantitative measurement of oxygen respiration rates by developmentally arrested spores of the streptomycete Streptomyces coelicolor (Fischer et al., 2013).

Many microorganisms have the capacity to use nitrate as a respiratory electron acceptor. Reduction of nitrate is catalyzed by a multi-subunit nitrate reductase that is often located associated with the cytoplasmic membrane and has its active site oriented toward the cytoplasm. This means that nitrate must be transported into the cell and often this occurs concomitantly with the export of the reduced nitrite product. Often nitrate and nitrite transport are coupled through the action of a nitrate: nitrite antiporter. Microbial cells, spores and mycelium harbour intracellular storage compounds such as trehalose or glycogen that, upon metabolism, function as endogenous electron donors for nitrate reduction. It is also possible to use glucose supplied exogenously as a substrate for nitrate reduction. The method described here allows the direct analysis of nitrate reduction by whole cell material without the requirement for artificial electron donors. This method is also applicable to the study of spores, particularly those of Streptomyces species (Fischer et al., 2013). The paper by Fischer et al. 2013 provides examples of datasets for the method presented below.

Transport assays allow the direct kinetic analysis of a specific transporter by measuring apparent K_m and V_max values, and permit the characterization of substrate specificity profiles through competition assays. In this protocol, we describe a rapid and easy method for performing uptake assays in the model filamentous ascomycete Aspergillus nidulans. These assays make use of A. nidulans germinating conidiospores, thus avoiding technical difficulties associated with the use of mycelia. The ease of construction genetic null mutants in this model fungus permits the rigorous characterization of any transporter in the absence of similar transporters with overlapping specificities, a common problem in relevant studies.

Choline is a methylated nitrogen compound that is widespread in nature. It is a precursor of several metabolites that perform numerous biological functions and it is predominantly used for the synthesis of essential lipid components of the cell membranes. Since there is no evidence that prokariotes can synthesize choline de novo and because choline uptake from exogenous sources is energetically more favorable than de novo synthesis, bacteria have evolved different uptake mechanisms for choline transport across the bacterial membrane. This protocol describes an easy and high sensitive method to assess choline uptake in bacteria using as tracer [³H]-choline chloride. The protocol was originally intended for Brucella abortus but could be applied for any bacteria with the corresponding modifications depending on the bacteria growth requirements (composition of the culture medium, temperature for growth, etc.). It can be useful to determine the choline uptake ability of several bacterial species under different growth conditions.