INTRODUCTION
Bacteria sense the fluctuations in their external environment and respond by expressing genes required for adapting to the altered environmental conditions. Expression of new sets of genes is initiated at promoter sequences recognized explicitly by RNA polymerase with specific sigma (σ) factors. While a primary house-keeping σ factor initiates gene expression in exponentially growing cells, alternative σ factors are activated under specific conditions to control the expression of a specific set of genes by recognizing alternative promoter sequences 1,2. Based on their sequence, domain architecture, and function, σ factors of the σ70 family are divided into four groups3,4. The primary σ factor belongs to Group 1 and contains four highly conserved domains (designated σ1 through σ4) along with a non-conserved region 3. Group 2 σ factors are closely related to Group 1 but are not essential for growth. However, the Group 3 σ factors lack the σ1 domain and control cellular processes such as sporulation, flagella biosynthesis, or heat shock response. Group 4 constitutes the largest and most diverse group of σ factors that regulate the cellular response to extracellular stimuli, hence known as extra-cytoplasmic function (ECF) σ factors5-7. In contrast to the other σ70 family members, the ECF σ factors contain only two of the four conserved domains, σ2 and σ4, which are enough for promoter recognition and interaction with core enzyme.
Based on sequence similarity and conservation of genomic context, ECF σ factors have been subdivided into 40 phylogenetically distinct groups8. An ECF σ factor is usually co-transcribed with a gene encoding its cognate anti-σ factor, regulating the σ factor activity 6,8,9. ECF σ factors are also characterized by auto-regulation of their promoter. Genes encoding ECF σ factor and anti- σ factor are often organized as part of the same operon6,8-10. After their expression, anti-σ factors sequester their cognate ECF σ factors to occlude their binding to the core enzyme and their cognate promoters. Specific intracellular or extracellular stimuli inactivate the anti-σ factor either by changing its conformation or by proteolytical degradation 9-11. This sets the ECF σ factor free to associate with the core enzyme to initiate transcription from its target promoters. Many ECF σ factors, however, are not associated with anti-σ factors. Instead, they harbor a C-terminal extension fused to the σ4 domain with the help of a flexible linker 8,9,12. A conserved SnoaL_2 like domain (Pfam: PF12680) in the C-terminal extension of the ECF41 family of σ factors was thought to play a dual role as an activator and inhibitor of the ECF σ activity 13 by interacting with the core regions of the ECF41 σ factor 14.
Azospirillumbrasilense is a plant growth-promoting rhizobacterium, which colonizes many grasses’ roots and promotes their growth by producing phytohormones and fixing atmospheric nitrogen. The genome of A. brasilense encodes a primary σ factor and 22 alternative σ factors. Out of its 10 ECF σ factors, two are co-transcribed with and translationally coupled to their cognate Zinc-binding anti-σ factors 15,16. These two pairs of ECF σ- and anti-σ factors constitute two different regulatory cascades, which control the biosynthesis of carotenoids in A. brasilense17. One of the ECF σ factors encoded in the A. brasilense genome was not accompanied with an anti-σ factor and contained an extension of 119 amino acids at its C-terminus, suggesting its similarity to the ECF41 type of σ factors. Crystal structure of SigJ of Mycobacterium tuberculosis (Mtb-SigJ), which belongs to the ECF41 family of σ factors, sheds some light on the role of the C-terminal SnoaL_2 domain on the structure and function of ECF41 type of σ factors 18. Direct coupling analysis combined with mutational analysis of the conserved residues of the C-terminal region of the ECF41 σ factor of Bacillus licheniformis identified the contact interface between the C-terminal extension and the core σ factor regions required for controlling ECF activity14.
In this study, we investigated the role of the two conserved motifs in the SnoaL_2 -like an extension of the C-terminal domain of ECF41 σ factor of A. brasilense Sp245: a proximal DGGGR motif and a distal NPDKV motif. Despite the increasing attention to the role of SnoaL_2 domain in modulating ECF41 σ activity and function13,14,18, the physiological role of ECF41 family σ factors is not known yet 13,14. We have recently shown the ECF41 σ factor (RpoE10) role in controlling the motility and biogenesis of lateral flagella in A. brasilense Sp24519. Here, we describe the role of the two conserved motifs in the SnoaL_2 domain of RpoE10 in the inhibition and activation of its activity, respectively. An all-atom Molecular Dynamics (MD) simulations and principal component analysis (PCA) of RpoE10 within silico mutations at conserved motifs structural study was carried out along with the experimental validation of the consequence.