CdnL, a member of the large CarD-like family of bacterial proteins, is vital for Myxococcus xanthus and differs functionally from the global transcriptional regulator CarD

Abstract

CarD, a global transcriptional regulator in Myxococcus xanthus, interacts with CarG via CarDNter, its N-terminal domain, and with DNA via a eukaryotic HMGA-type C-terminal domain. Genomic analysis reveals a large number of standalone proteins resembling CarDNter. These constitute, together with the RNA polymerase (RNAP) interacting domain, RID, of transcription–repair coupling factors, the CarD_TRCF protein family. We show that one such CarDNter-like protein, M. xanthus CdnL, cannot functionally substitute CarDNter (or vice versa) nor interact with CarG. Unlike CarD, CdnL is vital for growth, and lethality due to its absence is not rescued by homologs from various other bacteria. In mycobacteria, with no endogenous DksA, the function of the CdnL homolog mirrors that of Escherichia coli DksA. Our finding that CdnL, like DksA, is indispensable in M. xanthus implies that they are not functionally redundant. Cells are normal on CdnL overexpression, but divide aberrantly on CdnL depletion. CdnL localizes to the nucleoid, suggesting piggyback recruitment by factors such as RNAP, which we show interacts with CdnL, CarDNter and RID. Our study highlights a complex network of interactions involving these factors and RNAP, and points to a vital role for M. xanthus CdnL in an essential DNA transaction that affects cell division.

High-Mobility-Group A-Like CarD Binds to a DNA Site Optimized for Affinity and Position and to RNA Polymerase To Regulate a Light-Inducible Promoter in Myxococcus xanthus

“…7). The only interaction observed by the two-hybrid analysis was that of CarD with the RNAP ␤ subunit, concordant with our earlier data using specific domains of either protein (16). Thus, CarD physically associates with RNAP, and this appears to be achieved via the ␤ subunit of RNAP, without involving the other RNAP core subunits or CarQ.…”

“…If this were so, stable or transient interactions could occur between RNAP and CarD. Two-hybrid analysis had actually indicated that the CarD N-terminal domain can interact with a fragment from the N-terminal region of the M. xanthus RNAP ␤ subunit (16). However, it remained to be validated if this physical interaction persisted for the full-length forms of CarD and RNAP ␤.…”

“…Thus, availability of CarQ for its association with the core RNAP and the selective recognition of P QRS by RNAP-CarQ holoenzyme are fundamental in activating transcription from P QRS . Previous studies have established two more proteins, CarD and CarG, as indispensable for P QRS activation in vivo (5,6,(13)(14)(15)(16). Both factors are coexpressed from the same operon and act as a single, physically linked functional unit, but only CarD recognizes and directly binds to a DNA site upstream of P QRS .…”

“…The E. coli two-hybrid system employed is based on functional complementation of the T25 and T18 fragments of the Bordetella pertussis adenylate cyclase catalytic domain (CyaA) when two test proteins interact (23). pKT25-carD and pUT18-carG have been described before (13,15,16). Coding sequences and flanking regions for each M. xanthus RNAP subunit were obtained from genome data (NCBI accession numbers YP_631525, YP_631280, YP_631281, and YP_633047, respectively, for the ␣, ␤, ␤=, and subunits of RNAP).…”

“…1A) (13)(14)(15). In contrast to mammalian HMGA, CarD also has a structurally well-defined Nterminal domain of ϳ180 residues which is essential for function and defines the widely distributed CarD_CdnL_TRCF family of bacterial proteins or protein modules that have been implicated in interactions with RNA polymerase (RNAP) (5,(13)(14)(15)(16)(17). Through this domain CarD forms a stable complex with CarG, which is indispensable in every known CarD-dependent process (13,15).…”