Regulation of nopaline uptake in Agrobacterium tumefaciens

Dr Marincs Ferenc
Regulation of nopaline uptake in Agrobacterium tumefaciens.
Doktori értekezés, Szegedi Tudományegyetem.

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Gene expression in bacteria is regulated at different levels, of which the first one is the regulation of transcription mediated by DNA-binding regulatory proteins. These proteins bind to operator sequences in the vicinity of the regulated promoters, and repress or activate gene expression in response to small effector molecules in order to ensure the correct spatial and temporal presence of gene products for the cellular biochemical processes. The mechanism of regulation of gene expression by regulatory proteins is implemented by different means in which both DNA-protein and protein-protein interactions in and around the regulated promoter regions, and particular tertiary conformational alterations at the DNA and protein levels may all be involved. Agrobacterium tumefaciens is a plant pathogenic bacterium which incites tumours on higher plants by transferring a particular portion (the T-DNA) of its large, so-called tumour-inducing (Ti) plasmid into the plant genome. After integration of the T-DNA into the plant chromosomes, expression of genes of the integrated DNA results in unbalanced production of plant hormones, which leads to uncontrolled cell division and consequently to tumour formation. Moreover, the integrated bacterial genes direct the synthesis of specific molecules, collectively called opines, in the tumour cells. Opines are catabolised and utilised as a sole carbon, nitrogen and energy source by the tumour-inducing bacteria. The catabolism of opines is directed by genes located on the Ti plasmids, but these genes are not part of the T-DNA which is transferred into the plant genome. By this mechanism, the bacteria create a unique niche for themselves, where their survival and multiplication is ensured. In this dissertation I describe molecular studies performed on the regulation of nopaline catabolism in A. tumefaciens and present a novel type of mechanism by which gene expression is regulated in the Ti plasmid pTiT37. Genes involved in nopaline catabolism had previously been identified in our laboratory by transposon mutagenesis. Two of these genes, nocR and nocP, were found to be divergently transcribed and expression of nocP and nocR was nopaline-responsive and nopaline-unresponsive, respectively. The nocR-nocP region was sequenced and sequence analysis revealed that the nocR gene encodes a putative DNA-binding regulatory protein, which belongs to the family of LysR-type bacterial activator proteins, while the nocP gene encodes a putative ABC-type transporter protein which is very likely involved in the uptake of nopaline. In vivo analysis using reporter genes indicated that the NocR protein is a regulatory protein with dual functions, i.e. NocR auto-represses its own synthesis and both represses and activates expression of the divergently transcribed nocP gene. The promoters of the nocR and nocP genes were identified by sequence and functional analyses which revealed that they do not overlap and a 131 bp region of unknown function separates the -35 hexamers of the two promoters. Although both promoters are highly homologous to the consensus Escherichia coli σ70 promoters, the nocP promoter displays features which are characteristic to supercoiling-responsive promoters. The NocR protein was expressed in E. coli and it was proven that the molecular mass of its subunit is 31.5 kDa. It was also shown that the N-terminals of the expressed and the predicted proteins are identical. It was shown by gel retardation and DNaseI footprinting assays that the NocR protein binds to the intervening region between the coding sequences of the nocR and nocP genes either in the absence or presence of nopaline. Nopaline had no influence on the binding affinity of NocR, but changed the conformation of the NocR-noc DNA complex. The binding site of NocR was localised to a 72 bp long DNA sequence in the nocR promoter region, upstream of the nocP promoter. In the binding site of NocR a CATGN4CATG tandem palindrome was identified and it was proven both in vitro and in vivo that this sequence functions as the operator for the NocR protein. Overlapping the operator sequence, two interesting sequence features, an 18 bp alternating purine-pyrimidine sequence able to form Z-DNA, and a putative gyrase (an enzyme involved in adjusting cellular superhelicity) recognition site were found. In vivo analyses using reporter genes indicted that transcription of the nocR gene on its own and in combination with the operator sequence is able to repress expression of the nocP gene to a certain extent even in the absence of the NocR protein. This indicated that the two promoters are transcriptionally coupled and that local superhelicity has a role in the regulatory mechanism by which NocR regulates expression of the nocP gene. It was also shown that the operator sequence is able to influence the supercoiling level of a plasmid in vitro, depending on the overall level of cellular superhelicity. Putting these results together, a novel type of mechanistic model was developed to explain the means by which NocR regulates expression of the nocP gene in the nopaline catabolism region of the A. tumefaciens Ti plasmid pTiT37. According to this model, binding of the NocR regulatory protein to the noc operator located in a remote position relative to the regulated nocP promoter, in combination with the negative local supercoiling generated by the transcription of the nocR gene, represses expression of nocP, because in this situation the spatial position of the hexamers of the nocP promoter is not favourable for transcription. In the presence of nopaline, the conformation of the NocR protein-noc operator complex is altered in such a way that the operator either flips from B- to Z-form or, if it was already in Z-form in the absence of nopaline, the Z-stretch is expanded towards the nocP promoter. This conformational change results in removal of helical turns and local supercoils in the region thus bringing the hexamers of the nocP promoter into the correct spatial arrangement for transcription. This means of regulation represents a coordinated, energetically favourable adaptation and survival strategy for Agrobacterium living under suboptimal conditions.

Mű típusa: Disszertáció (Doktori értekezés)
Doktori iskola: Biológia Doktori Iskola
Tudományterület / tudományág: természettudományok > biológiai tudományok
Témavezető neveBeosztás, tudományos fokozat, intézményEmail
EPrint azonosító (ID): 1447
Publikációban használt név : Dr Marincs Ferenc
A feltöltés ideje: 2012. ápr. 11. 09:41
Utolsó módosítás: 2017. jún. 13. 14:44
Egyebek (raktári szám): B 4395
Védés állapota: védett

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