Materials and methods for identifying gyrase inhibitors

A screening assay to identify one or more compounds from a plurality of compounds, wherein the screening assay comprises the steps of:

• a) providing a cell or a culture of a cell wherein the cell comprises a linear polynucleotide comprising:
• i) a first inducible promoter under the control of an inducer and operably linked to a first gene and at least one terminator, and
• ii) a second promoter operably linked to a second gene;
• wherein the first inducible promoter is PT7lac, Ptrc, Plac, PT7Al/O4, PlacUV5, PrhaBAD, Para, PtetA, PrecA, PphoA, Ptrp, Pnar, PPL, PcspA, PproU, or Pcst-4 and the second promoter is PgyrA or PgyrB;
• wherein the second promoter is divergently coupled to the first inducible promoter such that the first inducible promoter is located between the second promoter and the first gene and the second promoter is located between the first inducible promoter and the second gene, wherein transcription of the first gene under the control of the first inducible promoter causes negative supercoiling of the second promoter, and said negative supercoiling of the second promoter inhibits transcription of the second gene from the second promoter;
• b) optionally, culturing the cell or the culture of the cell;
• c) incubating a plurality of test portions of the culture, provided in step a) or cultured in step b), each test portion in the plurality of test portions in the presence of one compound from the plurality of compounds and incubating a control portion of the culture, provided in step a) or cultured in step b), in the absence of any compound from the plurality of compounds;
• d) measuring the expression of the first gene and/or the second gene in each of the plurality of test portions and the control portion after the incubation of step c);
• and
• e) identifying each compound in the plurality of compounds as:
• i) a gyrase inhibitor:
• A) if the expression of the second gene is higher in the test portion compared to the expression of the second gene in the control portion, or
• B) if the expression of the first gene is higher in the control portion compared to the expression of the first gene in the test portion;
• or
• ii) not a gyrase inhibitor:
• A) if the expression of the second gene is not higher in the test portion compared to the expression of the second gene in the control portion, or
• B) if the expression of the first gene is not higher in the control portion compared to the expression of the first gene in the test portion.

A method comprising the steps of:

• a) providing a cell or a culture of a cell, wherein the cell comprises a linear polynucleotide comprising:
• i) a first inducible promoter under the control of an inducer and operably linked to a first gene and at least one terminator, and
• ii) a second promoter operably linked to a second gene;
• wherein the first inducible promoter is PT7lac, Ptrc, Plac, PT7Al/O4, PlacUV5, PrhaBAD, Para, PtetA, PrecA, PphoA, Ptrp, Pnar, PPL, PcspA, ProU, or Pest-4 and the second promoter is PgyrA or PgyrB;
• wherein the second promoter is divergently coupled to the first inducible promoter such that the first inducible promoter is located between the second promoter and the first gene and the second promoter is located between the first inducible promoter and the second gene, wherein transcription of the first gene under the control of the first inducible promoter causes negative supercoiling of the second promoter, and said negative supercoiling of the second promoter inhibits transcription of the second gene from the second promoter;
• b) optionally, culturing the cell or the culture of the cell;
• c) incubating a first portion of the culture, provided in step a) or cultured in step b), in the presence of the compound and incubating a second portion of the culture, provided in step a) or cultured in step b), in the absence of the compound;
• and
• d) measuring the expression of the first gene and/or the second gene in the first portion and/or the second portion after the incubation of step c).