Determination of genes under the direct control of GntR-type transcriptional factor LutR in Bacillus subtilis PY79 by CHIP and EMSA methods

LutR transcription factor, one of the members of GntR family, is a global regulatory protein encoded by lutR gene in B. subtilis. When lutR gene was first discovered, it was named as "yvfI". But it was re-named as "lutR" (Lactate utilisation gene), when lutR was found to be involved in lactate utilization. B. subtilis LutR protein is a homolog of E. coli FadR regulatory protein. The sequence of LutR protein has also a significant homology with orthologs of the different species in FadR subfamily of GntR superfamily. Additionally, FadR-like proteins are involved in the regulation of many metabolic pathways. GntR family is the HTH-type protein superfamily consisting of regulatory proteins which contain a Helix-Turn-Helix (HTH) motif to bind to DNA. CDD analysis in different studies revealed that "FadR C-terminal ligand-binding region", which is quite common in GntR family, also has been found in LutR C-terminal (PFAM 07729). Furthermore, it was determined that there is a significant similarity between the 44 amino acids at the LutR N-terminus and the GntR wHTH domain (PF00392). A member of GntR family contains a DNA-binding domain (DBD) with HTH motif at N-terminus and an effector-binding or oligomerization (EBD) domain at C-terminus. The DNA-binding region including a small β-strand and 3-helix core is highly conserved in all GntR family, while the other regions out of the DNA-binding site may be highly variable. The main antibiotic producer member of Bacillus genus is B. subtilis. Approximately 30 different antibiotics are known to be produced by B. subtilis and most of them act against a wide range of bacteria. Approximately 350-kb region in B. subtilis genome includes antibiotic encoding genes and this region constitutes 4-5% of total genome. The most important feature of B. subtilis antibiotics they cannot only act as antimicrobial agent, but also effect the regulation of cell morphology and physiology. Bacilysin [L-alanine-(2.3-epoxycyclohexanone-4)-L-alanine] is a dipeptide antibiotic produced by non-ribosomal pathway. A monocistronic gene ywfH and a polycistronic bacABCDEF (formerly ywfHBCDEFG) are responsible for bacilysin biosynthesis in B. subtilis. Bacilysin is composed of two amino acids; L-alanine at N-terminus and non-proteinogenic L-anticapsin at C-terminus. The antimicrobial activity of bacilysin depends on its proteolytic cleavage into L-alanine and L-anticapsin by a peptidase after its transport into the cells. Since L-anticapsin causes to lysis of bacterial or fungal cells by inhibiting activity of glucosamine synthase. Together with global regulators ComA, Spo0A, AbrB and CodY, LutR was found to participate to the regulation of bacilysin biosynthesis. On the other hand, a very recent study performed by our group indicated that LutR is a pleiotropic regulator that is involved in the regulation of a wide variety of cellular processes associated with the onset of stationary phase, such as degradative enzyme production, antibiotic production and resistance, carbohydrate utilization and transport, transfer of mobile genetic elements, induction of phage related genes, sporulation, sporulation delay and cannibalism, and biofilm formation. The other protein including helix-turn-helix at N-terminal is SinR. SinR protein exhibits dual regulatory role because it activates the competence and motility during late-growth periods in B. subtilis, but inhibits sporulation and exoprotease production. SinR acts as a pleiotropic regulatory key molecule such as Spo0A and prevent the other unnecessary potential responses by providing the development of adaptive responses. lutABC operon was also found to be under the control of both LutR and SinR. These proteins repress lutABC operon acting together. It is known that 18 gene is also repressed by SinR. These genes are belonging to epsA-O and yqxM-sipW-tasA operons responsible for the extracellular matrix holding together the long cell chains known as biofilm. Environmental conditions in microbial growth media change quickly and continuously. These sudden alterations threat to the survival of the cells. Therefore, it is an extremely critical necessity for each cell to be adapted to the environmental contiditions in the fastest and most convenient way. B. subtilis cells regulate its metabolism according to the environmental conditions by genome-wide controlling the gen expression via quorum sensing mechanism. Quorum sensing is a mechanism, which microorganisms communicate each-other using some chemical signal molecules in the environment in order to create an adaptational behavior depending on the increasing cell population. Bacteria synthesize these molecules in cytoplasma, then secrete them out of cell and sense these small, signalling molecules that accumulate in the growth medium as cells grow to high density. Quorum sensing signal molecules can be N-acyl-homoserine lactones (AHLs) for Gram-negative bacteria, or small peptides for Gram-positive bacteria or furan derivatives for some gram positive and negative bacteria. Bacillus subtilis is a model organism for scientific research because of it is one of the best-studied microorganisms, its genome sequencing is completed, its non-pathogenicity, easy handling, easy transformability, high growth rate, adaptive metabolism. B. subtilis cells are a rod-shaped, aerobic or facultative aerobic, endospore-forming bacteria. Bacillus subtilis is able to respond to the changes in its environment in order to survive. B. subtilis regulates its genes by quorum sensing mechanism to manage the optimal growth conditions. All phenotypes such as motility, chemotaxis, competence, degradative enzyme and antibiotic production, competition with the competitors, secretion of enzymes such as proteases and sporulation are controlled strigently by using quroum sensing. In this thesis, the main aim is to determine the genes under the direct control of LutR in B. subtilis. For this purpose, we applied in vitro chromatin immunoprecipitation (ChIP) method modified according to our purpose. The LutR protein produced and purified as a recombinant protein in E. coli pQE60::lutR strain. Electrophoretic mobility shift assay (EMSA) was also used to verify that LutR protein binds directly to the candidate genes determined by our modified-ChIP method. Additionally, RT-qPCR methods were used to determine the effects of LutR on the expression of those genes. Furthermore, in the doctoral thesis of Öykü İrigül-Sönmez (2012), it was determined the lrpB, yodT, rpsP, yutK, nasA, yvsG, ytsD and yvnB genes could be under the direct control of LutR. These results obtained from microarray analysis applied with B. subtilis PY79 (amyE::Pspac::lutR lutR::Tn10::spc) strain which overexpresses lutR gene. In the present study, we used EMSA methods to gain certainty about which of those genes expressions (lrpB, yodT, rpsP, yutK, nasA, yvsG, ytsD and yvnB) are affected directly by LutR protein. Previous study of our group indicated that SinR is capable of interacting with the regulatory regions of all of the LutR-target genes tested. According to this data, it was aimed to highlight the possible new genes regulated by both LutR and SinR, EMSA analysis were performed in the presence of both regulators. The results of these EMSA analysis indicated that LutR and SinR proteins regulate the gene expressions of yuxO, lrpB, yodT, rpsP, yutK, nasA, yvsG, ytsD and yvnB genes together. Finally, besides to experimental analysis, it was aimed to obtain bioinformatic data about LutR protein by comparative in silico analysis and charachterization studies. For this purpose, we performed two different approaches: In first analysis, the physichochemical, structural, and phylogenetic features of LutR proteins belonging to different species were studied by several bioinformatic tools and determined the similarities and differentiations among various species. In the second analysis, structural comparative analysis of only one LutR protein belonging to B. subtilis 168 with well-known GntR HTH type transcription factors FadR and YvoA was accomplished by PyMOL. As the last, the procedure developed for ChIP method in this thesis provides a methodological innovation because it is not only useful for procaryotic systems but also it can be applied without antibody usage.