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Applications of BBa_K1045003

The part BBa_K1045003 was used for in vivo and in vitro analysis of protein functionality. The sequence codes for an active adenylate cyclase with a high activity in vivo and a moderate activity in vitro in a reaction solution containing 10 mM MgCl2 at a pH between 8 and 9.5. The coding sequence was equipped with a 5'-Strep-tag for purification, crystallized and the defraction pattern determined.

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UNIQ4cd30db28b3cd9f1-partinfo-00000000-QINU UNIQ4cd30db28b3cd9f1-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) 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 seems to have a distinct physiological function.
c-di-AMP was reported to play a crucial role in cell wall synthesis and spore formation in B. subtilis (Oppenheimer-Shaanan et al., 2011; Mehne et al., 2013). Interestingly, both absence 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 adenylate cyclase domain (DAC) was confirmed for Gram-positive bacteria like Bacillus subtilis and important pathogens like Streptococcus pneumoniae, Staphylococcus aureus and Listeria monocytogenes (Corrigan and Gründling, 2013). In striking contrast to the severe effects that alterations in c-di-AMP homeostasis have on Gram-positive bacteria, Escherichia coli as a Gram-negative representative does not produce c-di-AMP, implying that this molecule is not essential in Gram-negatives.
Here, we introduce a BioBrick with the coding sequence of the DacA cyclase domain of Listeria monocytogenes. Cloning of the full-length membrane-bound DacA protein failed in E. coli. Therefore, we decided to exclude the trans-membrane domains ending up with a coding sequence of 100 – 273 amino acids of DacA. Unsuitable restriction sites within the gene were removed without changing the amino acid sequence. The protein was expressed from the BioBrick equipped with a Strep-tag on the 5‘-end of the gene. The soluble protein localizes to the cytoplasm and can easily be extracted with standard protein purification methods.

To analyze the cyclase activity of the truncated DacA protein, we introduced BBa_K1045003 into the E. coli strain BL21 with an N-terminal Strep-Tag and under the control of a T7-promoter. 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 Isopropyl-β-D-thiogalactopyranosid (IPTG), the culture was then incubated for 3 h. The expression of the genome encoded T7-polymerase led to the transcription of the DAC which was driven by a T7-promotor. In order to extract c-di-AMP from the cell, a defined volume of the culture was taken and the cells lysed by snap freezing and heating.
The expression of BBa_K1045003 was confirmed by SDS gel electrophoresis showing a thick ectopic expression band with a weight of about 20 kDa (Fig. 1A). The c-di-AMP determination using LC-MS/MS revealed the presence of c-di-AMP in the supernatant of lysed bacterial cells. The measured concentration was determined as ca. 60 µg/mg of the total protein extract (Fig. 1B). The empty vector control did not show a measurable amount of c-di-AMP. The ectopic expression of the L. monocytogenes DAC-domain in E. coli BL21 yielded an active enzyme. Thus, BBa_K1045003 codes for an active adenylate cyclase domain which catalyzes the condensation reaction of two molecules ATP to a single molecule c-di-AMP in vivo.

Fig. 1. Cyclic di-AMP production and degradation. Cyclic di-AMP is produced from two molecules of ATP by diadenylyl cyclase (DAC) enzymes 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 gel electrophoresis confirmed the high expression of BBa_K1045003 in 3 biological replicates showing a thick band at 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) The measured content of c-di-AMP in relation to the total protein amount.

Diadenylate-cyclases catalyze the condensation reaction of 2 ATP-molecules yielding one molecule c-di-AMP (Fig. 2). The reaction releases 2 pyrophosphate 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 molecules as an indirect marker. Each pyrophosphate that is released during c-di-AMP synthesis was cleaved by the pyrophosphatase to yield 2 phosphate molecules. Consequently, 4 phosphate molecules indicate the production of one molecule c-di-AMP (Figure 3). In order to determine the phosphate concentration, we used malachite-green that forms a complex with phosphate and molybdate. The malachite-green-phosphate complex absorbs light at a wavelength of 630 nm. A phosphate standard curve with known sodium phosphate concentrations was prepared to calculate the phosphate concentration. The measured absorbance values with the corresponding phosphate concentrations show a linear relation between 0 and 0.8. Thus, malachite-green is suitable to determine phosphate concentrations between 0 and 100 µM (Figure 3A).

In order to stop the enzymatic reaction and to enable the malachite-green-complex formation a low pH is necessary, which the reason for decreasing the pH value of the reaction solution to 1. Unfortunately, ATP, which was abundant in high concentrations since it is used as substrate for c-di-AMP production, was subject to hydrolysis caused by very low pH-values. In order to analyze the hydrolysis rates of ATP, pyrophosphate and the mixture of both, a solution containing 1 mM of each molecule was incubated for 2 hours after the addition of HCl (Figure 3B). When analyzed separately, ATP and pyrophosphate showed a moderate hydrolysis rate with a low amount of phosphate complexes after 20 min. The mixture of ATP and pyrophosphate (1 mM each) indicated a significantly lower release of free phosphate molecules compared to ATP and pyrophosphate alone.
To circumvent any influences of the ATP hydrolysis on the measurements of phosphate 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 adenylate cyclase 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 pyrophosphate. Malachite-green only stains free phosphate molecules and is not able to form a complex with pyrophosphate. Therefore, it is essential to incubate the DAC with a pyrophosphatase to yield free phosphates in order to visualize c-di-AMP production. In the absence of 1 U/ml pyrophosphatase no phosphate was detected. The pyrophosphatase was adjusted to a concentration that it theoretically converts the complete amount of ATP in the reaction within 1 min and, in consequence, is very unlikely to form a bottleneck in this staining reaction. No phosphate release was detected in the absence of ATP (Figure 3C).
To analyze the conversion rate of ATP to cyclic-di-AMP, the malachite-green staining was used to determine the concentration of free phosphate molecules. In the presence of 1 mM ATP, the incubation of DAC resulted in the formation of a high amount of malachite-green-phosphate complexes indicating a high pyrophosphate 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 one can say that 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 the catalysis capabilities of the DAC. Since diadenylate cyclases are naturally not present in E. coli, a co-factor increasing the catalysis rate seems to be universally present in bacteria. This might indicate the involvement of different divalent cations in the catalysis reaction or universally present lower and higher chemical compounds.
To examine the influence of different pH-values on the reaction rate of the cyclase activity, we incubated the purified enzyme at pH=8 and pH=9.5 in a time course from 0 to 240 min. The results suggest a slightly higher cyclase activity at pH=9.5 as reported before for the diadenylate cylase DisA from Bacillus thuringiensis (Zheng et al., 2013).

Fig. 3. Phosphate staining using malachite-green. (A) Linear standard curve for the phosphate detection. (B) Hydrolysis of ATP, Sodium pyrophosphate, and both (1 mM each). (C) Phosphate detection in the presence presence of active enzyme, ATP, and a pyrophosphatase.

Fig. 4. SDS gel showing expression and purification of DacA (Lmo2120). (A) Lane 1: Thermo Scientific PageRuler Plus Prestained Protein Ladder; NE: Non-induced; OE: Over-expression = Clone contained the cyclase domain of which the activity was induced by addition of isopropyl-ß-D-1-thiogalactopyranoside (IPTG); L: Lysate; FT: Flow-through; After wash 1 our protein was purified. (B) In vitro cyclase activity of DacA

Protein structure of the diadenylate cyclase 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 lead to the discovery of new antibacterial substance classes by computational fitting methods. To yield a sufficient amount of protein for crystallization, we expressed BBa_K1045003 in E. coli BL21 with an N-terminal Strep-tag under the control of a 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. The expression was confirmed by SDS gel electrophoresis and yielded a thick ectopic expression band with a size of 20.0 kDa (Fig. 4A). The protein size indicates the presence of the DAC in all used cell cultures. To purify the DAC-protein, 10 l of cell cultures were lysed and the cell lysate containing a Strep-tag fusion protein was applied to a column with immobilized Strep-Tactin. Subsequently, the protein was washed and eluted from the column (Fig. 4A).
After dialyzing and concentrating the first two elutions, we obtained a total protein concentration of 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.
The protein structure of DacA was revealed and shows a globular protein with a distinct ATP-binding cleft. The ribbon model demostrates the general structure composed of α-helices and ϐ-sheets (Fig. 6).

Fig. 5. DacA protein crystals and diffraction pattern. (A) Nice crystals were received with a medium concentration of alcohol and other supplements. (B) X-ray diffraction image of DacA (Lmo2120) crystals; (C) the highlighted box is shown enlarged.

Fig. 6. Protein structure of DacA. (A, B) Ribbon representation of DacA in the ATP bound state. (C, D) Surface structure of DacA and the ATP-binding pocket. (E) Magnified view into the ATP-binding pocket