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Regulatory role of small RNAs and RNA-binding proteins in carbon metabolism and collective behaviour of Vibrio cholerae
Regulatory role of small RNAs and RNA-binding proteins in carbon metabolism and collective behaviour of Vibrio cholerae
The importance of small regulatory RNAs (sRNAs) has been recognized across all domains of life. Originally considered “non-coding RNAs,” several bacterial sRNAs have been found to encode functional proteins that are under 50 amino acids long. This group of regulators are called dual-function regulators. To date, only five such regulators have been characterized in bacteria. In the primary study, the first dual-function RNA of Vibrio cholerae was discovered and characterized. The pathogen colonizes and infects the upper intestines by producing two key virulence determinants – toxin co-regulated pilus (TCP) and cholera toxin (CT). While all the known sRNAs of V. cholerae act directly or indirectly to regulate the production of TCP, the sRNA VqmR is the only known direct repressor of CT production to date. Therefore, a forward genetic screen was employed to score for CT repression. This screen identified another promising candidate called Vcr082. Interestingly, Vcr082 also encodes 29 amino acids long ORF and hence was re-named VcdRP, for V. cholerae dual RNA regulator and protein, eponymous to their roles. The dual regulator is controlled by the global transcription factor of carbon utilization, cAMP-CRP. The riboregulatory component is conserved at the 3’ end of the dual regulator. By employing a conserved stretch of four cytosines, VcdR base-pairs with and represses mRNAs that encode for transporters that import PTS sugars. Additionally, VcdR also downregulates the phosphor-carrier proteins PtsH and PtsI that are involved in the phospho-relay during glycolysis. The small protein, VcdP exerts its regulatory role by interacting with and accelerating the activity of citrate synthase enzyme, opening the gateway into the TCA cycle. This way, both VcdR and VcdP act to block sugar uptake and modulate the flux through the TCA cycle, thereby striking a balance to maintain overall carbon metabolism in V. cholerae. The diverse environments that V. cholerae inhabits necessitates that the organism rapidly perceives changes in its external environment and appropriately tailors its gene expression paradigm. To achieve this, the bacteria employ quorum sensing (QS) to communicate and coordinate a suitable response. While this mechanism of census taking has been well-documented early on in several marine bacteria, more recent studies have identified additional QS systems in V. cholerae. Similarly, while biofilm formation has been extensively studied, the transition into and subsequent dispersal was only documented recently. These incomplete underpinnings thereby prompted further investigation of the QS pathway. Therefore, in the second study, a forward genetic screen in a V. cholerae mutant library was employed to score for an altered QS phenotypic transition. This screen identified a novel RNA-binding protein called MbrA (membrane-bound RNA-binding protein A). This protein localizes to the membrane and contains two trans-membrane domains at the N-terminus and a conserved RNA recognition motif-type RNA-binding domain located towards the C-terminus. MbrA is activated by the global transcription factor cAMP-CRP and a subsequent transcriptome analysis revealed its role in the regulation of motility genes and flagellar assembly complex in V. cholerae.
Vibrio cholerae, Hfq, citrate synthase, dual-function RNA, small protein, small RNA, quorum sensing
Venkat, Kavyaa
2022
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Venkat, Kavyaa (2022): Regulatory role of small RNAs and RNA-binding proteins in carbon metabolism and collective behaviour of Vibrio cholerae. Dissertation, LMU München: Faculty of Biology
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Abstract

The importance of small regulatory RNAs (sRNAs) has been recognized across all domains of life. Originally considered “non-coding RNAs,” several bacterial sRNAs have been found to encode functional proteins that are under 50 amino acids long. This group of regulators are called dual-function regulators. To date, only five such regulators have been characterized in bacteria. In the primary study, the first dual-function RNA of Vibrio cholerae was discovered and characterized. The pathogen colonizes and infects the upper intestines by producing two key virulence determinants – toxin co-regulated pilus (TCP) and cholera toxin (CT). While all the known sRNAs of V. cholerae act directly or indirectly to regulate the production of TCP, the sRNA VqmR is the only known direct repressor of CT production to date. Therefore, a forward genetic screen was employed to score for CT repression. This screen identified another promising candidate called Vcr082. Interestingly, Vcr082 also encodes 29 amino acids long ORF and hence was re-named VcdRP, for V. cholerae dual RNA regulator and protein, eponymous to their roles. The dual regulator is controlled by the global transcription factor of carbon utilization, cAMP-CRP. The riboregulatory component is conserved at the 3’ end of the dual regulator. By employing a conserved stretch of four cytosines, VcdR base-pairs with and represses mRNAs that encode for transporters that import PTS sugars. Additionally, VcdR also downregulates the phosphor-carrier proteins PtsH and PtsI that are involved in the phospho-relay during glycolysis. The small protein, VcdP exerts its regulatory role by interacting with and accelerating the activity of citrate synthase enzyme, opening the gateway into the TCA cycle. This way, both VcdR and VcdP act to block sugar uptake and modulate the flux through the TCA cycle, thereby striking a balance to maintain overall carbon metabolism in V. cholerae. The diverse environments that V. cholerae inhabits necessitates that the organism rapidly perceives changes in its external environment and appropriately tailors its gene expression paradigm. To achieve this, the bacteria employ quorum sensing (QS) to communicate and coordinate a suitable response. While this mechanism of census taking has been well-documented early on in several marine bacteria, more recent studies have identified additional QS systems in V. cholerae. Similarly, while biofilm formation has been extensively studied, the transition into and subsequent dispersal was only documented recently. These incomplete underpinnings thereby prompted further investigation of the QS pathway. Therefore, in the second study, a forward genetic screen in a V. cholerae mutant library was employed to score for an altered QS phenotypic transition. This screen identified a novel RNA-binding protein called MbrA (membrane-bound RNA-binding protein A). This protein localizes to the membrane and contains two trans-membrane domains at the N-terminus and a conserved RNA recognition motif-type RNA-binding domain located towards the C-terminus. MbrA is activated by the global transcription factor cAMP-CRP and a subsequent transcriptome analysis revealed its role in the regulation of motility genes and flagellar assembly complex in V. cholerae.