The present study reports the two-Betamethasone Valerate domain architecture of M. xanthus CdnL and the NMR structures of each of these domains and of the full-length protein. We also describe our structure-based analysis of mutations that result in loss of the essential CdnL function and impair cell viability. These include mutations that disrupt the interaction with RNAP-b as well as those that leave this interaction intact. We present data that CdnL stabilizes open complex formation and stimulates transcription at an rRNA promoter by RNAP holoenzyme containing the major M. xanthus housekeeping sA, and that the loss-of-function CdnL mutants lack this activity. Our results are discussed in the context of our data of the RNAP recognition domain of TtCdnL, the T. thermophilus CdnL homolog and those from other groups on full-length TtCdnL and MtCdnL, both of which exist in bacteria lacking CarD as well as DksA. The involvement of CdnL in GNE-7915 sA-dependent rRNA promoter activity and of CarD in the action of several ECF-s factors thus illustrates the evolution of two related members of an important bacterial protein family to regulate promoter activity dependent on different s factors. Mutations of corresponding basic residues in MtCdnL have been reported to reduce its DNA binding in vitro, with that equivalent to R90A/R91A/R93A producing the greatest effect, but their consequences in vivo have not been described. It is thus noteworthy that mutating the basic R90/R91/R93, but not R128/K129, is detrimental to function in vivo despite no apparent DNA binding by M. xanthus CdnL. Additionally, the lack of an effect on mutating the highly conserved W88 in M. xanthus CdnL contrasts with the observation that the equivalent mutation in MtCdnL or TtCdnL impairs function. The CdnL NMR solution structure and its structure-based mutational analysis in this study provide molecular insights into the cellular roles and modes of action of this RNAP-binding protein that is essential for growth and viability but has unknown functions in M. xanthus. CdnL has a two-domain architecture in solution.