Bacillus subtilis HelD, an RNA Polymerase Interacting Helicase, Forms Amyloid-Like Fibrils

Abstract

HelD, an RNA polymerase binding protein from Bacillus subtilis, stimulates transcription and helps in timely adaptation of cells under diverse environmental conditions. At present, no structural information is available for HelD. In the current study, we performed size exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS) which suggests that HelD is predominantly monomeric and globular in solution. Using combination of size exclusion chromatography and analytical ultracentrifugation, we also show that HelD has a tendency to form higher order oligomers in solution. CD experiments suggest that HelD has both α-helical (∼35%) and β sheet (∼26%) secondary structural elements. Thermal melting experiments suggest that even at 90°C, there is only about 30% loss in secondary structural contents with Tm of 44°C. However, with the increase in temperature, there was a gain in the β-sheet content and significant irreversible loss of α-helical content. Using a combination of X-ray fiber diffraction analysis, and dye based assays including Thioflavin-T based fluorescence and Congo red binding assays, we discovered that HelD forms amyloid-like fibrils at physiologically relevant conditions in vitro. Using confocal imaging, we further show that HelD forms amyloid inclusions in Escherichia coli. Bioinformatics-based sequence analysis performed using three independent web-based servers suggests that HelD has more than 20 hot-spots spread across the sequence that may aid the formation of amyloid-like fibrils. This discovery adds one more member to the growing list of amyloid or amyloid-like fibril forming cytosolic proteins in bacteria. Future studies aimed at resolving the function of amyloid-like fibrils or amyloid inclusions may help better understand their role, if any, in the bacterial physiology.

Characterization of the Radiation Desiccation Response Regulon of the Radioresistant Bacterium Deinococcus radiodurans by Integrative Genomic Analyses

“…In agreement with these data, transcriptome analysis of cells recovering from exposure to ionizing radiation showed no upregulation of expression of these three genes in a ∆irrE mutant compared to the wild-type strain [24]. In E. coli, RecG plays an important role in DNA repair, recombination, and replication [55], whereas HelD, from Bacillus subtilis or Mycobacterium smegmatis, is associated with transcriptional pathways [56,57]. In D. radiodurans, cells devoid of RecG exhibit a delay in growth and double strand break (DSB) repair kinetics, and a decrease in resistance to γ-irradiation and H 2 O 2 [58,59], whereas the ∆dr1572 mutant exhibited a greater sensitivity to H 2 O 2 , but no change in resistance to ionizing radiation and to MMC when compared to the wild-type strain [60].…”

Two Novel Amyloid Proteins, RopA and RopB, from the Root Nodule Bacterium Rhizobium leguminosarum

“…Not only biofilm proteins but also toxins and different bacterial factors of virulence form amyloids that may be either inactivated for storage or functionally active [69]. Therefore, according to contemporary data, with the exception of several proteins regulating transcription [75,76,77], most bacterial amyloids are associated with the virulence and can be harmful for multicellular hosts promoting host–pathogen interactions.…”

Microbial functional amyloids serve diverse purposes for structure, adhesion and defence

“…Within the pathogenic M. tuberculosis, the transcription regulator CarD has been shown to form amyloid fibrils both in vitro and in vivo (Kaur et al 2018b). Similarly, the RNA polymerase-binding protein HeiD from B. subtilis can form amyloid fibrils in living cells (Kaur et al 2018a). These recent discoveries about bacterial prions indicate that prion amyloid assemblies may be Fig.…”