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Applications of BBa_K1045003

The part BBa_K1045003 was used for in vivo and in vitro enzyme characterization. The sequence codes for an active diadenylate cyclase (DAC) from the human pathogenic bacterium Listeria monocytogenes EGD-e. The enzyme has a high in vivo activity and a moderate in vitro activity. The reaction and the composition of the reaction mixture are described below (see Fig. 3 and 4). The native DNA sequence does not contain a Strep-tag coding sequence. However, in our experiments the Strep-tag coding sequence was fused to the 5' end of the DAC-encoding sequence. The eight amino acids long Strep-tag allows the rapid purification of the DAC by Strep-tag:Streptactin affinity purification. The DAC was shown to be active and the concentrated protein was used for crystallization screenings. The obtained diffraction pattern of the protein crystal was used to determine the 3D structure of the DAC (Fig. 6)

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UNIQdb305e484de27983-partinfo-00000000-QINU UNIQdb305e484de27983-partinfo-00000001-QINU Diadenylate cyclase domain of Listeria monocytogenes DacA (Lmo2120)

The essential signaling molecule bis-(3‘,5‘)-cyclic dimeric adenosine monophosphate (c-di-AMP, see Fig. 1) was initially identified in a crystal structure of the DNA damage checkpoint protein DisA of Bacillus subtilius and Thermotoga maritima (Witte et al., 2008). c-di-AMP is structurally similar to the signaling molecule c-di-GMP but it has a distinct physiological function.
c-di-AMP was reported to play a crucial role in cell wall metabolism and in spore formation in B. subtilis (Oppenheimer-Shaanan et al., 2011; Mehne et al., 2013). Interestingly, both lack and excess of c-di-AMP have detrimental effects on cell growth and morphology (Yun Luo and Helmann, 2012; Mehne et al., 2013). The presence of proteins containing an DAC was confirmed for Gram-positive bacteria like B. subtilis and important pathogens like Streptococcus pneumoniae, Staphylococcus aureus and L. monocytogenes (Corrigan and Gründling, 2013). In contrast, Gram-negative bacteria like Escherichia coli does not produce c-di-AMP. This implyies that c-di-AMP is not essential in Gram-negatives.
Here, we present a BioBrick with the coding sequence of the DAC domain of the DacA diadenylate cyclase from L. monocytogenes. Cloning of the full-length membrane-bound DacA protein failed in E. coli. Therefore, we decided to clone a truncated version of the DacA protein that does not contain the trans-membrane domain. The resulting DNA sequence codes for 174 amino acids long protein that comprises the amino acid residues 100 – 273 of the DacA enzyme. The non-desired SpeI restriction site within the DNA sequence was removed without changing the amino acid sequence. The protein was synthesized from the BioBrick that contains a Strep-tag coding sequence at the 5‘-end of the open reading frame. The soluble protein localizes to the cytoplasm and can easily be isolated in a highly pure manner by Strep-tag:Streptactin affinity purification, which is a standard protein purification method.

To analyze the cyclase activity of the truncated DacA protein, we introduced BBa_K1045003 into the E. coli strain BL21. Expression of the gene is driven by an Isopropyl-β-D-thiogalactopyranosid (IPTG)-dependent T7 promoter that is recognized by the RNA polymerase of the T7 bacteriophage. The protein contains an eight amino acid-long Strep-Tag (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) at its N terminus. To express the protein, an overnight culture was used to inoculate LB medium containing the appropriate antibiotic. The culture was incubated at 37°C until it reached an OD₆₀₀ of 0.5 – 0.7. Having induced the expression of the T7 polymerase by adding IPTG, the culture was further incubated for 3 h. The expression of the genome encoded T7 polymerase led to the transcription of the DAC encoding gene which is driven by the T7 promoter. In order to verify the in vivo activity of the enzyme, we have extracted c-di-AMP from the cells. For this purpose, a defined volume of the culture was taken, the cells were spun down and lysed by snap freezing and heating.
The expression of BBa_K1045003 was confirmed by SDS PAGE. The analysis of the total cell protein revealed that the DAC protein was synthesized. The protein has a relative molecular weight of about 20 kDa (see Fig. 2A). The determination of c-di-AMP concentration by LC-MS/MS revealed that c-di-AMP was present in the supernatant of lysed bacterial cells. The concentration was shown to be about 60 µg per mg total cell protein (Fig. 2B). As expected the control E. coli strain harbouring the empty vector did not produce any c-di-AMP. The heterologous expression of a truncated L. monocytogenes dacA gene encoding the DAC domain in E. coli BL21 yielded an active enzyme. In conclusion, BBa_K1045003 codes for an active DAC domain, which catalyzes the condensation reaction of two molecules ATP to a single molecule c-di-AMP in vivo.

Fig. 1. c-di-AMP production and degradation. c-di-AMP is produced from two molecules of ATP by DACs and degraded to pApA by phosphodiasterases. (Edited from Corrigan and Gründling, 2013)

Fig. 2. Confirmation of the ectopic expression and in vivo activity of BBa_K1045003. (A) SDS PAGE confirmed synthesis of DAC using BBa_K1045003 in three independent biological replicates. The DAC protein was highly abundant and had a molecular weight of about 20 kDa. Lane 1: Thermo Scientific PageRuler Plus Prestained Protein Ladder. Lane 2-4: Crude extract of of BBa_K1045003 after 3 h induction. (B) Determination of c-di-AMP by HPLC-MS/MS in samples shown on the left. The concentration is given in c-di-AMP per total cell protein.

The BioBrick is an active adenylate cyclase in vitro

DACs catalyze the condensation reaction of molecules 2 ATP to one molecule c-di-AMP (Fig. 2). The reaction releases 2 pyrophosphate (PP) molecules consisting of the β-γ-phosphates of each ATP. To visualize the activity of the DAC domain and the c-di-AMP production in vitro, we used free phosphate (P) molecules as an indirect marker. Each PP that is released during c-di-AMP synthesis was cleaved by the PP phosphatase to yield 2 P molecules. Consequently, 4 P molecules indicate the production of one molecule c-di-AMP (Fig. 3). In order to determine the P concentration, we used malachite-green that forms a complex with P and molybdate. The malachite-green-P complex absorbs light at a wavelength of 630 nm. A P standard curve with known NaP concentrations was prepared to calculate the P concentration. The measured absorbance values at defined P concentrations show a linear relation between 0 and 0.8. Thus, malachite-green is suitable to determine P concentrations in a range between 0 and 100 µM (Fig. 3A).

In order to stop the enzymatic reaction and to enable the malachite-green-complex formation a low pH is necessary. Therefore the pH of the reaction mixture has to be decreased to 1! Unfortunately, ATP, which was abundant in high concentrations since it is used as a substrate for DAC, was subject to hydrolysis caused by very low pH values. In order to analyze the hydrolysis rates of ATP, PP and the mixture of both, a solution containing 1 mM of each molecule was incubated for 2 hours after the addition of HCl (Fig. 3B). When analyzed separately, ATP and PP showed a moderate hydrolysis rate with a low amount of P complexes were formed after 20 min of incubation. The mixture of ATP and PP (1 mM each) indicated a significantly lower release of free P molecules compared to ATP and PP alone.
To circumvent any influences of the ATP hydrolysis on the measurements of P release by the DAC, a standard curve containing the same concentration of ATP was treated in the same manner as the samples. After the reaction was stopped with HCl and a following 20 min incubation to enable complete complex formation, all samples were measured at the same time. The background caused by ATP hydrolysis can be subtracted. Thus, ATP hydrolysis is neglectable.
The characterization of the DAC domain demonstrates the activity and the c-di-AMP production also in vitro. The condensation reaction of 2 ATP molecules to 1 molecule c-di-AMP results in the release of PP. Malachite-green only stains free P molecules and is not able to form a complex with PP. Therefore, it is essential to incubate the DAC with a PP to yield free P in order to visualize c-di-AMP production. In the absence of 1 U/ml PP phosphatase no P was detected. The PP phosphatase was adjusted to a concentration that it theoretically converts the complete amount of ATP in the reaction within 1 min. Thus, it is very unlikely that the enzyme is a bottleneck in our enzyme assay to monitor DAC activity. No P release was detected in the absence of ATP (Fig. 3C).
To analyze the conversion rate of ATP to c-di-AMP, the malachite-green staining was used to determine the concentration of free P molecules. In the presence of 1 mM ATP, the incubation of DAC resulted in the formation of a high amount of malachite-green-P complexes indicating a high PP release during the ATP condensation reaction (Figure 3B). Thus, BBa_K1045003 was confirmed to act as an active enzyme in the presence of a divalent cation, ATP and a buffer system at pH 8. In conclusion, the DAC domain was able to synthesize c-di-AMP in vitro, however, the catalysis rate in vivo seems to be much more efficient. The higher reaction rate in vivo may indicate an influence of different cytosolic co-factors on DAC activity. Since DACs are naturally not present in E. coli, a co-factor increasing the catalysis rate seems to be universally present in bacteria. On one hand one might envision that yet unknown divalent cations are required for DAC activity. On the other hand unknown compounds might be important for high-level DAC activity.
To examine the influence of different pH values on the reaction rate of DAC activity, we incubated the purified enzyme at pH 8.0 and pH 9.5 in a time course from 0 to 240 min. Our results suggest a slightly higher cyclase activity at pH 9.5. This is in good agreement with previous observations showing that the activity of the DisA enzyme, a DAC from Bacillus thuringiensis, is affected by the pH (Zheng et al., 2013).

Fig. 3. P staining using malachite-green. (A) Linear standard curve for the P detection. (B) Hydrolysis of ATP, NaPP, and both (1 mM each). (C) P detection in the presence presence of active enzyme, ATP, and a PP phosphatase.

Fig. 4. SDS PAGE showing synthesis and purification of the DAC domain from DacA (Lmo2120). (A) Lane 1: Thermo Scientific PageRuler Plus Prestained Protein Ladder; NE: Non-induced; OE: Overexpression = bacteria the DAC domain-encoding gene whose expression was induced by adding IPTG; L: Lysate; FT: Flow-through; After 5 washing steps a highly pure DAC was present in the three elution fractions. (B) In vitro cyclase activity of DacA

Protein structure of the DAC domain

Since c-di-AMP was reported to be an essential signaling molecule in Gram-positive bacteria, it is a suitable and promising target for the search of highly specific anti-bacterial substances. Thus, the molecular structure of the DAC domain can serve as a scaffold for computational fitting methods in order to identify novel antibacterial substances. To yield sufficient amounts of the protein for crystallization screenings, we synthesized the DAC from BBa_K1045003 in E. coli BL21. The protein contains a Strep-tag at its N terminus. Expression of the gene is driven by the T7 promoter. After the induction of the T7 polymerase with IPTG the cell cultures were incubated at 16°C for 12 – 14 h overnight.
Incubation at 37°C resulted in growth inhibition (data not shown). Synthesis of the highly abundant DAC protein was confirmed by SDS PAGE. As expected the protein has a relative molecular weight of about 20.0 kDa (Fig. 4A). To purify the DAC domain, the cells of 10 l cultures were harvested by centrifugation and lysed! The cell lysate containing the Strep-tag fusion protein was applied to a column with immobilized Strep-Tactin. Subsequently, the colum was washed and the protein was eluted from the matrix by Desthiobiotin (Fig. 4A).
After dialyzing and concentrating the first two elution fractions, we obtained a highly abundant protein solution. The total protein concentration was determined to be 10 mg/ml, which was necessary to test the crystallization capabilities. The best result for the crystallization reaction was given at a medium concentration of alcohol and other supplements (Fig. 5A). The crystal yields an X-ray diffraction pattern with a resolution of 2.8 Å (Fig. 5B, C). The dataset was measured at the EMBL Hamburg Beamline P13 at the PETRA III synchrotron on the DESY campus in Germany, using a PILATUS2 6M X-ray detector, 12/24 Hz frame rate, and with a bit rate of up to 1.2 GByte/s (https://www.dectris.com/).
The protein structure of the DacA DAC domain was successfully determined and shows a globular protein with a distinct ATP-binding pocket. The ribbon model demostrates the general structure composed of α-helices and ϐ-sheets (Fig. 6). The precise crystallographic values and refinement statistics confirmed the reliability of the obtained 3D-structure (Table 1).

Fig. 5. DAC protein crystals and diffraction pattern. (A) Beautiful crystals were obtained with a medium concentration of alcohol and other supplements. (B) X-ray diffraction image of the DAC crystals; (C) the highlighted box is shown enlarged.

Fig. 6. Protein structure of the DacA DAC domain. (A) Cartoon representation of DAC in its ATP-bound state. (B) General composition of the DAC consisting of α-helices and ϐ-sheets (C) Magnified view into the ATP-binding pocket.

Table 1. Crystallographic data collection and refinement statistics

Values for the data in the highest resolution shell are shown in parentheses.
^a The CC_1/2 is the correlation coefficient between two randomly selected half data sets as described in Karplus & Diederichs (2012).
^b R_free = ∑_Test||F_obs| –|F_calc||/∑Test |F_obs|, where “Test” is a test set of about 5% of the total reflections randomly chosen and set aside prior to refinement for the complex.