Roles of Mn-catalase and a possible heme peroxidase homologue in protection from oxidative stress in Thermus thermophilus

Ebihara, Akio; Manzoku, Miho; Fukui, Kenji; Shimada, Atsuhiro; Morita, R; Masui, Ryoji; Kuramitsu, Seiki

doi:10.1007/s00792-015-0753-2

Cited by 3 publications

(2 citation statements)

References 40 publications

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“…Although little is known about the role of manganese in stress resistance in Thermus, there is evidence that cellular Mn 2+ concentrations are sufficiently high in Tth, and several Mn 2+ -dependent enzymes have been implicated in the oxidative stress response in this bacterium (54)(55)(56). We found that although Tth Gfh1 is functional in the presence of magnesium ions, its pausestimulatory activity is also greatly enhanced by manganese.…”

Section: Discussionmentioning

confidence: 68%

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Esyunina

Agapov

Kulbachinskiy

2016

Proc. Natl. Acad. Sci. U.S.A.

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Transcriptional pausing has emerged as an essential mechanism of genetic regulation in both bacteria and eukaryotes, where it serves to coordinate transcription with other cellular processes and to activate or halt gene expression rapidly in response to external stimuli. Deinococcus radiodurans, a highly radioresistant and stressresistant bacterium, encodes three members of the Gre family of transcription factors: GreA and two Gre factor homologs, Gfh1 and Gfh2. Whereas GreA is a universal bacterial factor that stimulates RNA cleavage by RNA polymerase (RNAP), the functions of lineage-specific Gfh proteins remain unknown. Here, we demonstrate that these proteins, which bind within the RNAP secondary channel, strongly enhance site-specific transcriptional pausing and intrinsic termination. Uniquely, the pause-stimulatory activity of Gfh proteins depends on the nature of divalent ions (Mg 2+ or Mn 2+ ) present in the reaction and is also modulated by the nascent RNA structure and the trigger loop in the RNAP active site. Our data reveal remarkable plasticity of the RNAP active site in response to various regulatory stimuli and highlight functional diversity of transcription factors that bind inside the secondary channel of RNAP.RNA polymerase | transcriptional pausing | Gfh factors | Deinococcus radiodurans | stress response C ellular RNA polymerases (RNAPs) are complex molecular machines whose activity during transcription is regulated by DNA-and RNA-encoded signals, protein factors, small molecules, and inhibitors. The catalytic cycle of RNAP can be interrupted by pauses of various natures that play important roles in genetic regulation in all organisms, from the classic systems of transcription attenuation and their variations in bacteria (1) to recently discovered widespread promoter-proximal pausing in eukaryotes (2). The pausing serves to activate or repress transcription rapidly at specific genomic sites in response to regulatory stimuli and to coordinate RNA synthesis with other genetic processes (e.g., DNA replication and repair, RNA translation in bacteria) (1,(3)(4)(5)(6)(7)(8).Recent structural and biochemical studies revealed distinct RNAP conformations corresponding to different functional states of the transcription elongation complex (TEC) during nucleotide addition, RNA proofreading, and pausing (9-11). The control of structural transitions between these states likely underlies the function of multiple regulatory factors acting on RNAP. However, the roles of individual RNAP conformations in transcription and the mechanisms of their switching by transcription factors remain only partially understood. During transcription, RNAP holds the DNA template and the RNA transcript within its main channel, whose opening is controlled by the mobile clamp/shelf module and the flap domain of RNAP (9-11). Nucleotide addition and TEC translocation depend on alternating cycles of the folding of the trigger loop (TL) and kinking of the bridge helix (BH) in the RNAP active site (9-11) (Fig. 1A). Analysis of the stru...

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Section: Discussionmentioning

confidence: 68%

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Esyunina

Agapov

Kulbachinskiy

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…An extremely thermophilic bacterium, T. thermophilus , possesses a protein known as MnCAT, which functions as an H 2 O 2 scavenger [ 35 ]. Native MnCAT was purified by heat treatment, followed by several purification steps [ 36 ]. In this study, we investigated the effectiveness of additives on the production of T. thermophilus MnCAT designed to be expressed from the T7 lac promoter in E. coli cells.…”

Section: Resultsmentioning

confidence: 99%

Boosting Auto-Induction of Recombinant Proteins in Escherichia coli with Glucose and Lactose Additives

Tahara

Tachibana

Takeo

et al. 2021

PPL

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Background: Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level. Objectives: In this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium. Method: First, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7lac promoter-based expression). Results: Auto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture. Conclusion: The medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.

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Parallel Molecular Evolution of Catalases and Superoxide Dismutases—Focus on Thermophilic Fungal Genomes

et al. 2020

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Catalases (CAT) and superoxide dismutases (SOD) represent two main groups of enzymatic antioxidants that are present in almost all aerobic organisms and even in certain anaerobes. They are closely interconnected in the catabolism of reactive oxygen species because one product of SOD reaction (hydrogen peroxide) is the main substrate of CAT reaction finally leading to harmless products (i.e., molecular oxygen and water). It is therefore interesting to compare the molecular evolution of corresponding gene families. We have used a phylogenomic approach to elucidate the evolutionary relationships among these two main enzymatic antioxidants with a focus on the genomes of thermophilic fungi. Distinct gene families coding for CuZnSODs, FeMnSODs, and heme catalases are very abundant in thermophilic Ascomycota. Here, the presented results demonstrate that whereas superoxide dismutase genes remained rather constant during long-term evolution, the total count of heme catalase genes was reduced in thermophilic fungi in comparison with their mesophilic counterparts. We demonstrate here, for the newly discovered ascomycetous genes coding for thermophilic superoxide dismutases and catalases (originating from our sequencing project), the expression patterns of corresponding mRNA transcripts and further analyze translated protein sequences. Our results provide important implications for the physiology of reactive oxygen species metabolism in eukaryotic cells at elevated temperatures.

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Roles of Mn-catalase and a possible heme peroxidase homologue in protection from oxidative stress in Thermus thermophilus

Cited by 3 publications

References 40 publications

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

Boosting Auto-Induction of Recombinant Proteins in Escherichia coli with Glucose and Lactose Additives

Parallel Molecular Evolution of Catalases and Superoxide Dismutases—Focus on Thermophilic Fungal Genomes

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