Bacillus subtilis?de yvfI geninin genom ölçeğinde analizi

Tez (Doktora) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2012Transkripsiyonel düzenleyici proteinleriden GntR ailesi yaygın olarak kanatlı sarmal-dönüş-sarmal motifi ile karakterize edilmişlerdir. GntR ailesine mensup, FadR alt grubunun üyesi olan proteinler aminoasit metabolizmasının regülasyonuyla ve aspartat, pirüvat, glikolat gibi metabolik yol izleriyle ilişkilidir. Bu çalışmanın da hedef aldığı, basilisin üretiminde gerekli olduğu da ortaya konmuş olan B. subtilis yvfI geninin GntR ailesi transkripsiyonel düzenleyici proteinleriyle benzerlik gösterdiği bilinmektedir. Bu sunulan çalışmada, YvfI transkripsiyonel faktörü tarafından etkilenmesi mümkün genleri tanımlayabilmek için YvfI kodlayamayan TEK1 mutantı ve kontrol suşu PY79 arasında, logaritmik faz sırasında ve durağan fazda detaylı karşılaştırmalı transkriptom analizi gerçekleştirilmiştir. Büyümenin durağan evresinde YvfI proteini tarafından düzenlenmiş bir çok yolizi tanımlanmasının keşfi sağlanmıştır. Ek olarak, logaritmik fazda YvfI proteinin birçok önemli metabolik prosesde görevli genleri düzenlemede rol aldığı gösterilmiştir. Bunların yanı sıra, karşılaştırmalı transkriptom çalışmaları IPTG ile indüklenen B. subtilis PY79 amyE::Pspac::yvfI, yvfI::Tn10 mutant suşu ve aynı suşun indüklenmeyen hali arasında yürütülmüştür. Bu çalışma YvfI'nın olağandan fazla düzeyde ifade edilmiş olmasının farklı elementlerin ve metabolizmaların uyarılmasına ya da baskılanmasına sebebiyet verdiği ortaya konmuştur. Ayrıca, bu araştırma 5'-RACE-PCR analizi ile yvfI geninin promotör bölgesinin tanımlanması ve yerinin saptanmasını amaçlanmaştır. Sonuç olarak yvfI transkripsiyonunun ? A-tipi promotör tarafından kontrol edildiği, yvfI gen dizisi ve promotör bölgesi üzerinde global düzenleyici proteinlerin bağlanma motifleri olduğu gösterilerek, bu genin quorum-sensing yol izi üzerinden düzenlendiği belirlenmiştir. Sonuç olarak, YvfI'nın muhtelif düzenleyici davranışları YvfI'nın, bir çok değişik tipte regulona baskı ve indüksiyon uygulayarak düzenleyici aktivite gösteren GntR ailesine ait olduğu önerisi ile uyum göstermektedir.Most characteristic trait of GntR family transcriptional repressors is their winged helix-turn-helix motif functioning as DNA binding domain. One of the subclasses of GntR-type proteins was identified to be FadR and FadR-like proteins function in regulation of amino acid metabolism and many pathways including aspartate, pyruvate, glycolate. This study focuses on a novel gene, yvfI, required for bacilysin biosynthesis in Bacillus subtilis and YvfI shows homology with GNTR regulatory proteins. The present research aimed to identify possible genes that are affected by YvfI transcriptional factor. Therefore comparative transcriptome analysis of control strain PY79 and its yvfI disrupted mutant, TEK1 has revealed that many pathways are regulated by YvfI during stationary phase of growth. YvfI was also found to participate in regulation of genes involved in a diverse types of metabolic processes during exponential phase of growth. Furthermore, comparative studies were performed between IPTG induced and non-induced version of ?B. subtilis PY79 amyE::Pspac::yvfI, yvfI::Tn10? mutant strain. This study clarified that when overexpression achieved, YvfI gets related with the regulation of many elements and metabolic pathways. Moreover, identification and localization of promoter region of yvfI gene by 5?-RACE-PCR analysis showed that yvfI transcription was controlled from a ? A-type promoter and consisted of Spo0A, three putative AbrB and one CodY binding consensus sequences. This result indicated that this gene is regulated by global regulatory proteins through quorum-sensing pathway. Hence, as a conclusion, varied regulatory behavior of YvfI further strengtened the proposition that YvfI belongs to GntR family regulatory proteins.DoktoraPh.D

In Bacillus subtilis LutR is part of the global complex regulatory network governing the adaptation to the transition from exponential growth to stationary phase

The lutR gene, encoding a product resembling a GntR-family transcriptional regulator, has previously been identified as a gene required for the production of the dipeptide antibiotic bacilysin in Bacillus subtilis. To understand the broader regulatory roles of LutR in B. subtilis, we studied the genome-wide effects of a lutR null mutation by combining transcriptional profiling studies using DNA microarrays, reverse transcription quantitative PCR, lacZ fusion analyses and gel mobility shift assays. We report that 65 transcriptional units corresponding to 23 monocistronic units and 42 operons show altered expression levels in lutR mutant cells, as compared with lutR+ wild-type cells in early stationary phase. Among these, 11 single genes and 25 operons are likely to be under direct control of LutR. The products of these genes are involved in a variety of physiological processes associated with the onset of stationary phase in B. subtilis, including degradative enzyme production, antibiotic production and resistance, carbohydrate utilization and transport, nitrogen metabolism, phosphate uptake, fatty acid and phospholipid biosynthesis, protein synthesis and translocation, cell-wall metabolism, energy production, transfer of mobile genetic elements, induction of phage-related genes, sporulation, delay of sporulation and cannibalism, and biofilm formation. Furthermore, an electrophoretic mobility shift assay performed in the presence of both SinR and LutR revealed a close overlap between the LutR and SinR targets. Our data also revealed a significant overlap with the AbrB regulon. Together, these findings reveal that LutR is part of the global complex, interconnected regulatory systems governing adaptation of bacteria to the transition from exponential growth to stationary phase

Detailed polymorphism study on cytomegalovirus DNA polymerase gene to reveal the most suitable genomic targets for quantitative Real-time PCR

The human cytomegalovirus (HCMV) is an important human pathogen primarily affecting immunocompromised patients, like transplant recipients or HIV- infected individuals. Early diagnosis of cytomegalovirus (CMV) infection in high-risk patients is essential in order to start preemptive treatments. pol (UL54) gene encoding for HCMV viral DNA polymerase is a well-defined target for HCMV detection in clinical samples and identifying most highly conserved regions for primer design remains crucial. Though real-time polymerase chain reaction (qPCR) is a rapid and sensitive method for HCMV detection, failure to detect some HCMV strains due to primer and target mismatches have led the researchers to explore more sensitive and reliable methods. Hence, to understand the broader diversity of the pol mutations in HCMV and to specify the most suitable region for primer-probe design to be used in qPCR assay, we studied both nucleotide and amino acid heterogeneities in 60 HCMV positive samples that were collected to represent national mutational prevalence of pol gene of HCMV in Turkey. The test was designed with a new set of primers- probe for HCMV detection and quantification based on the sequencing data which revealed the most conserved region on the pol gene. Statistical probit analysis was applied on qPCR studies which revealed a 95% detection limit of 100 copies/mL. In addition, linearity, reproducibility, and precision of the new test were assessed for diagnostic purposes

In Bacillus subtilis LutR is part of the global complex regulatory network governing the adaptation to the transition from exponential growth to stationary phase

The lutR gene, encoding a product resembling a GntR-family transcriptional regulator, has previously been identified as a gene required for the production of the dipeptide antibiotic bacilysin in Bacillus subtilis. To understand the broader regulatory roles of LutR in B. subtilis, we studied the genome-wide effects of a lutR null mutation by combining transcriptional profiling studies using DNA microarrays, reverse transcription quantitative PCR, lacZ fusion analyses and gel mobility shift assays. We report that 65 transcriptional units corresponding to 23 monocistronic units and 42 operons show altered expression levels in lutR mutant cells, as compared with lutR+ wild-type cells in early stationary phase. Among these, 11 single genes and 25 operons are likely to be under direct control of LutR. The products of these genes are involved in a variety of physiological processes associated with the onset of stationary phase in B. subtilis, including degradative enzyme production, antibiotic production and resistance, carbohydrate utilization and transport, nitrogen metabolism, phosphate uptake, fatty acid and phospholipid biosynthesis, protein synthesis and translocation, cell-wall metabolism, energy production, transfer of mobile genetic elements, induction of phage-related genes, sporulation, delay of sporulation and cannibalism, and biofilm formation. Furthermore, an electrophoretic mobility shift assay performed in the presence of both SinR and LutR revealed a close overlap between the LutR and SinR targets. Our data also revealed a significant overlap with the AbrB regulon. Together, these findings reveal that LutR is part of the global complex, interconnected regulatory systems governing adaptation of bacteria to the transition from exponential growth to stationary phase.