Genetic Blueprint for Stringent Response in Betaproteobacterial Aromatoleum/Azoarcus/Thauera Cluster

The Aromatoleum/Azoarcus/Thauera (AAT) cluster comprises anaerobic degradation specialists (Aromatoleum, Thauera) and N2-fixing endophytes (Azoarcus). Omics-based and genetic studies with associated model strains implicate stringent response (SR) in adaptation to nutrient limitation and plant colonization. SR is well-studied in standard bacteria such as Escherichia coli and known as adaptive strategy to nutrient limitations by adjusting e.g., transcription and stress response. SR involves the alarmone (p)ppGpp, whose cellular level is controlled by the synthetases/hydrolases RelA/SpoT and the noncanonical transcription factor DksA, whose interaction with RNA polymerase (RNAP) binding of (p)ppGpp enhances.

DksA-mediated SR occurs across Proteobacteria and other phylogenetic groups such as Myxococcia and Spirochaetia, mostly applying to pathogens. Furthermore, all three DksA variants (four, two or one cysteine residue(s) for Zn2+-binding) were found. Genes encoding SR components are present in all studied 37 genomes representing 31 species from the AAT cluster. Each genome encodes a synthesizing RelA, a hydrolyzing SpoT, a four cysteine-containing DksA, and mostly also a one cysteine-containing DksA. Opposing functions of RelA and SpoT in Aromatoleum aromaticum EbN1T, Aromatoleum sp. strain CIB, Azoarcus olearius BH72, and Thauera aromatica K172T (also entire AAT cluster) are implicated by full conservation of amino acids (E and D vs. 2H2D motif and ED diad) essential for catalysis by their synthetase versus hydrolase domains. Likewise, functionality of the predicted C4-type DksAs from these four model strains was visually assessed by structural modeling and comparison of key features (binding sites for Zn2+/(p)ppGpp; CC tip for RNAP interaction) to those of the available E. coli DksA cryo-EM structure.

SR as a global adaptation strategy should contribute to the success of the AAT cluster in its distinct habitats: complex and highly variable soils/sediments (high molecular and microbial diversity, fluctuating nutrient availabilities and redox states) of the free-living degradation specialists versus the defined endorhizosphere (more stable conditions, less complex community) of the endophytes. A noteworthy exception is Aromatoleum sp. strain CIB by combining degradation and endophytic features. Thus, future investigations into the role of SR in the habitat success of such bacteria reflecting their divergent environmental niches are needed as well as promising.