The exponential increase in the number of conducted studies combined with the development of sequencing methods have led to an enormous accumulation of partially processed experimental data in the past two decades. Here, we present an approach using literature-mined data complemented with gene expression kinetic modeling and promoter sequence analysis. This approach allowed us to identify the regulon of Bacillus subtilis sigma factor SigB of RNA polymerase (RNAP) specifically expressed during germination and outgrowth. SigB is critical for the cell’s response to general stress but is also expressed during spore germination and outgrowth, and this specific regulon is not known. This approach allowed us to (i) define a subset of the known SigB regulon controlled by SigB specifically during spore germination and outgrowth, (ii) identify the influence of the promoter sequence binding motif organization on the expression of the SigB-regulated genes, and (iii) suggest additional sigma factors co-controlling other SigB-dependent genes. Experiments then validated promoter sequence characteristics necessary for direct RNAP–SigB binding. In summary, this work documents the potential of computational approaches to unravel new information even for a well-studied system; moreover, the study specifically identifies the subset of the SigB regulon, which is activated during germination and outgrowth.

Chemical modifications of RNA affect essential properties of transcripts, such as their translation, localization and stability. 5’-end RNA capping with the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD ⁺ ) has been discovered in organisms ranging from bacteria to mammals. However, the hypothesis that NAD ⁺ capping might be universal in all domains of life has not been proven yet, as information on this RNA modification is missing for Archaea. Likewise, this RNA modification has not been studied in the clinically important Mycobacterium genus. Here, we demonstrate that NAD ⁺ capping occurs in the archaeal and mycobacterial model organisms Methanosarcina barkeri and Mycobacterium smegmatis. Moreover, we identify the NAD ⁺ -capped transcripts in M. smegmatis, showing that this modification is more prevalent in stationary phase, and revealing that mycobacterial NAD ⁺ -capped transcripts include non-coding small RNAs, such as Ms1. Furthermore, we show that mycobacterial RNA polymerase incorporates NAD ⁺ into RNA, and that the genes of NAD ⁺ -capped transcripts are preceded by promoter elements compatible with σ ^A /σ ^F dependent expression. Taken together, our findings demonstrate that NAD ⁺ capping exists in the archaeal domain of life, suggesting that it is universal to all living organisms, and define the NAD ⁺ -capped RNA landscape in mycobacteria, providing a basis for its future exploration.