The work of DNA gyrase in bacteria was visualized at the level of single molecules
The use of new microscopic methods made it possible to reconstruct the picture of DNA gyrase with a resolution of up to single molecules. This enzyme, essential for the replication of bacterial DNA, is an essential target for antibiotics.
DNA gyrase relaxes the supercoiled loops of the bacterial circular chromosome formed by polymerase complexes during replication and transcription. Without DNA gyrase, the polymerase complex stops, and the accumulated areas of supercoiling interfere with the separation of daughter circular genomes, which ultimately leads to the death of the bacterium. Antibiotics fluoroquinolones, effective against many bacteria, are gyrase inhibitors, however, due to the spread of antibiotic resistance, there is a need for new drugs. Visualizing the details of how DNA gyrase and the replication complex work at the molecular level can help create new highly effective antibiotics.
The authors of the article, published in Nucleic Acids Research, studied the relative position of DNA gyrase and replication complex molecules using special methods of fluorescence microscopy, allowing to achieve a resolution comparable to the size of a large protein complex. The bacteria were immobilized in low fluorescence agarose. For visualization, gyrase fused to mYPet (yellow fluorescent protein) and the replica component DnaN fused to red fluorescent protein mCherry were used. The distance between the DNA gyrase and the replication complex was taken as the distance between the brightest red and yellow pixels of the image processed by mathematical methods.
To achieve a resolution comparable to the size of the protein molecule, the PALM technology was used: the subunit of DNA gyrase, the GyrA protein in the cells was labeled with a photoactivated derivative of mCherry - PAmCherry. This protein is capable of being activated under the action of radiation and quickly "fade", that is, lose the ability to fluoresce upon repeated irradiation. At a certain level of excitatory irradiation, only a part of the molecules of such a protein is activated, and during subsequent acts of irradiation, these molecules do not give a signal. Thus, with the help of a special microscope, it is possible to record the "sequential inclusion" of molecules on a very small area so that the signals from individual protein molecules do not overlap. From such sequential frames, using computer and mathematical methods, an image is reconstructed with a resolution unattainable for a conventional fluorescence or light microscope.
An even more interesting technology based on photoactivation has been used to track the movements of DNA and gyrase complexes. Movement of molecules is recorded in the same way, sequentially removing the excited groups of fluorophores. The rate of diffusion of free molecules in an aqueous solution inside a cell is known with fairly high accuracy. All gyrase molecules that move much more slowly are likely to be bound to DNA, which means that by slowing down the diffusion of gyrase, one can monitor its activity.
An analysis of the distribution of the brightest red and yellow pixels showed that the DNA gyrase and the replication complex are separated from each other by an average of 256 nm. An additional proof of the correctness of the obtained data was the check of the frequency of colocalization of gyrase and replicas: under the experimental conditions, about 30% of bacteria did not contain replicas (division either just ended or had not started yet). Focused signals from DNA gyrase were observed in only 30% of such replicisoma-negative bacteria.
To determine the stoichiometry of DNA-gyrase "foci", a special technique was used - slimfield microscopy, which operates with polarized (slimfied) laser radiation and allows one to make millisecond images of an area several tens of square microns in size. The accuracy of linear measurements for DNA gyrase was about 40 microns, while it was possible to establish that the distance between the replicisome and "foci" containing DNA gyrase is, on average, 135 nm, which is significantly larger than the replicisome diameter (50 nm). This means that gyrase is not in direct contact with a working replication complex.
According to the data obtained during photoactivation, the total number of GyrA gyrase subunits per cell is about 1450. The authors note that, due to incomplete photoactivation, this number can be underestimated by about half, but in further calculations they use it. From this amount, more than 700 active gyrase molecules can be obtained (such a molecule includes two GyrA subunits and two more GyrB subunits). However, when treated with ciprofloxacin, which "sews" the active heterotetramer of gyrase to DNA, only about 600 gyrase complexes were immobilized, that is, about 80% of the protein is capable of forming functional molecules. In an untreated cell, about half of this amount was bound to DNA, and of these 300 complexes, only about 20 are involved in the work with the replicasome.
Since the enzyme is clearly expressed more than is necessary to ensure replication, the researchers figured out what other processes it is involved in. About 1000 transcription acts occur in a cell at a time; they introduce about 30 times more supercoils into DNA than replication, and it was natural to assume that gyrase removes them. However, inhibition of RNA polymerase by rifampicin only slightly affected the number of DNA-bound gyrase complexes. Considering that the rate of transcription of different genes is different, the authors suggested that gyrase does not directly compensate for the supercoils induced by RNA synthesis, but acts on the genome according to a more complex and not yet understood algorithm.
The mechanism of joint work of gyrase and topoisomerase IV during replication is not completely clear either. Topo IV is necessary for the separation of catenans, which are formed from the daughter and parent strands of DNA when it is duplicated. The number of gyrase and topo IV molecules in the region of the replicisome seems to be insufficient for the removal of supercoiling and separation of the chains. However, this “lack” is easier to explain if we assume that gyrase can act without dissociating with DNA, and DNA decatenation does not affect replication and is induced topologically after the passage of the replicisome. Observations of the movements of the gyrase and the replication complex suggest that positive DNA supercoils formed during replication can activate gyrase at a distance of many thousands of base pairs from the replication fork.