demonstration-membrane-transport-histidine-using-goat-intestinal

Demonstration of Membrane Transport of Histidine using Goat Intestinal Inverted Sacs: An Experiential Pedagogical Tool for Undergraduates

Histidine is an essential amino acid that is also a precursor for metabolites implicated in the immune system, pulmonary ventilation, and vascular ...

University of delhi

Mycobacterium tuberculosis genes Rv0844c/Rv0845 encoding the NarL response regulator and NarS histidine kinase are hypothesized to constitute a two-component system involved in the regulation of nitrate metabolism. However, there is no experimental evidence to support this. In this study, we established M. tuberculosis NarL/NarS as a functional two-component system and identified His(241) and Asp(61) as conserved phosphorylation sites in NarS and NarL, respectively. Transcriptional profiling between M. tuberculosis H37Rv and a ΔnarL mutant strain during exponential growth in broth cultures with or without nitrate defined an ∼30-gene NarL regulon that exhibited significant overlap with DevR-regulated genes, thereby implicating a role for the DevR response regulator in the regulation of nitrate metabolism. Notably, expression analysis of a subset of genes common to NarL and DevR regulons in M. tuberculosis ΔdevR, ΔdevSΔdosT, and ΔnarL mutant strains revealed that in response to nitrite produced during aerobic nitrate metabolism, the DevRS/DosT regulatory system plays a primary role that is augmented by NarL. Specifically, NarL itself was unable to bind to the narK2, acg, and Rv3130c promoters in phosphorylated or unphosphorylated form; however, its interaction with DevR∼P resulted in cooperative binding, thereby enabling co-regulation of these genes. These findings support the role of physiologically derived nitrite as a metabolic signal in mycobacteria. We propose NarL-DevR binding, possibly as a heterodimer, as a novel mechanism for co-regulation of gene expression by the DevRS/DosT and NarL/NarS regulatory systems.

Mycobacterium tuberculosis response regulators, DevR and NarL, interact in vivo and co-regulate gene expression during aerobic nitrate metabolism.

Erratum to: The Mycobacterium tuberculosis relBE toxin: antitoxin genes are stress-responsive modules that regulate growth through translation inhibition.

The DevR response regulator of  is an established regulator of the dormancy response in mycobacteria and can also be activated during aerobic growth conditions in avirulent strains, suggesting a complex regulatory system. Previously, we reported culture medium-specific aerobic induction of the DevR regulon genes in avirulent  H37Ra that was absent in the virulent H37Rv strain. To understand the underlying basis of this differential response, we have investigated aerobic expression of the  operon using  H37Ra and H37Rv  overexpression strains, designated as LIX48 and LIX50, respectively. Overexpression of DevR led to the up-regulation of a large number of DevR regulon genes in aerobic cultures of LIX48, but not in LIX50. To ascertain the involvement of PhoP response regulator, also known to co-regulate a subset of DevR regulon genes, we complemented the naturally occurring mutant  gene of LIX48 with the WT  gene. PhoP dampened the induced expression of the DevR regulon by >70-80%, implicating PhoP in the negative regulation of  expression. Electrophoretic mobility shift assays confirmed phosphorylation-independent binding of PhoP to the  promoter and further revealed that DevR and PhoP proteins exhibit differential DNA binding properties to the target DNA. Through co-incubations with DNA, ELISA, and protein complementation assays, we demonstrate that DevR forms a heterodimer with PhoP but not with the mutant PhoP protein. The study puts forward a new possible mechanism for coordinated expression of the dormancy regulon, having implications in growth adaptations critical for development of latency.

Interplay of PhoP and DevR response regulators defines expression of the dormancy regulon in virulent .

The genome sequencing of , the causative organism of tuberculosis, has significantly improved our understanding of the mechanisms that drive the establishment of infection and disease progression. Several clinical strains of  exhibit single-nucleotide polymorphisms (SNPs), the implications of which are only beginning to be understood. Here, we examined the impact of a specific polymorphism in PhoR, the sensor kinase of the PhoPR two-component system. Biochemical analysis revealed reduced autophosphatase/ATPase activity, which led to enhanced downstream gene expression. We complemented  H37Ra with the wild-type and mutant  genes and characterized the strains in a cell line infection model. We provide an explanation for the low prevalence of the SNP in clinical strains (∼1%), as the mutation causes a survival disadvantage in the host cells. The study provides a rare example of selection of a signaling node under competing evolutionary forces, wherein a biochemically superior mutation aids bacterial adaptation within-host but has low fitness for infection and hence is not selected. Our study highlights the importance of accounting for such SNPs to test therapeutic and co-therapeutic methods to combat TB.

A Low-Prevalence Single-Nucleotide Polymorphism in the Sensor Kinase PhoR in  Suppresses Its Autophosphatase Activity and Reduces Pathogenic Fitness: Implications in Evolutionary Selection.

The DNA polymorphisms found in clinical strains of Mycobacterium tuberculosis drive altered physiology, virulence, and pathogenesis in them. Although the lineages of these clinical strains can be traced back to common ancestor/s, there exists a plethora of difference between them, compared to those that have evolved in the laboratory. We identify a mutation present in ~80% of clinical strains, which maps in the HATPase domain of the sensor kinase MtrB and alters kinase and phosphatase activities, and affects its physiological role. The changes conferred by the mutation were probed by in-vitro biochemical assays which revealed changes in signaling properties of the sensor kinase. These changes also affect bacterial cell division rates, size and membrane properties. The study highlights the impact of DNA polymorphisms on the pathophysiology of clinical strains and provides insights into underlying mechanisms that drive signal transduction in pathogenic bacteria.

A high-frequency single nucleotide polymorphism in the MtrB sensor kinase in clinical strains of Mycobacterium tuberculosis alters its biochemical and physiological properties.

The Mycobacterium tuberculosis protein kinase K regulates growth adaptation by facilitating mycobacterial survival in response to a variety of  and  stress conditions. Here, we further add that  transcription is responsive to carbon and nitrogen starvation signals. The increased survival of an M. tuberculosis Δ mutant strain under carbon- and nitrogen-limiting growth conditions compared to the wild-type (WT) H37Rv suggests an integral role of PknK in regulating growth during metabolic stress. To identify the downstream targets of PknK-mediated signaling, we compared phosphoproteomic and transcription profiles of mycobacterial strains overexpressing WT and phosphorylation-defective PknK. Results implicate PknK as a signaling protein that can regulate several enzymes involved in central metabolism, transcription regulation, and signal transduction. A key finding of this study was the identification of two essential two-component response regulator (RR) proteins, PrrA and MtrA, and Rho transcription terminator, as unique targets for PknK. We confirm that PknK interacts with and phosphorylates PrrA, MtrA, and Rho . PknK-mediated phosphorylation of MtrA appears to increase binding of the RR to the cognate probe DNA. However, dual phosphorylation of MtrA and PrrA response regulators by PknK and their respective cognate sensor kinases  showed nominal additive effect on the mobility of the protein-DNA complex, suggesting the presence of a potential fine-tuning of the signal transduction pathway which might respond to multiple cues.  Networks of gene regulation and signaling cascades are fundamental to the pathogenesis of Mycobacterium tuberculosis in adapting to the continuously changing intracellular environment in the host. M. tuberculosis protein kinase K is a transcription regulator that responds to diverse environmental signals and facilitates stress-induced growth adaptation in culture and during infection. This study identifies multiple signaling interactions of PknK and provides evidence that PknK can change the transcriptional landscape during growth transitions by connecting distinctly different signal transduction and regulatory pathways essential for mycobacterial survival.

Mycobacterium tuberculosis PknK Substrate Profiling Reveals Essential Transcription Terminator Protein Rho and Two-Component Response Regulators PrrA and MtrA as Novel Targets for Phosphorylation.

Article	Year
Mycobacterium tuberculosis response regulators, DevR and NarL, interact in vivo and co-regulate gene expression during aerobic nitrate metabolism. The Journal of biological chemistry\| PubMed ID: 25659431	2015
Erratum to: The Mycobacterium tuberculosis relBE toxin: antitoxin genes are stress-responsive modules that regulate growth through translation inhibition. Journal of microbiology (Seoul, Korea)\| PubMed ID: 26626358	2015
Interplay of PhoP and DevR response regulators defines expression of the dormancy regulon in virulent . The Journal of biological chemistry\| PubMed ID: 30181216	2018
A Low-Prevalence Single-Nucleotide Polymorphism in the Sensor Kinase PhoR in Suppresses Its Autophosphatase Activity and Reduces Pathogenic Fitness: Implications in Evolutionary Selection. Frontiers in microbiology\| PubMed ID: 34512602	2021
A high-frequency single nucleotide polymorphism in the MtrB sensor kinase in clinical strains of Mycobacterium tuberculosis alters its biochemical and physiological properties. PloS one\| PubMed ID: 34529706	2021
Mycobacterium tuberculosis PknK Substrate Profiling Reveals Essential Transcription Terminator Protein Rho and Two-Component Response Regulators PrrA and MtrA as Novel Targets for Phosphoryl... Microbiology spectrum\| PubMed ID: 35404097	2022

Vandana Malhotra

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