The Stigmatella aurantiaca Homolog of Myxococcus xanthus High-Mobility-Group A-Type Transcription Factor CarD: Insights into the Functional Modules of CarD and Their Distribution in Bacteria

Abstract

Transcriptional factor CarD is the only reported prokaryotic analog of eukaryotic high-mobility-group A (HMGA) proteins, in that it has contiguous acidic and AT hook DNA-binding segments and multifunctional roles in Myxococcus xanthus carotenogenesis and fruiting body formation. HMGA proteins are small, randomly structured, nonhistone, nuclear architectural factors that remodel DNA and chromatin structure. Here we report on a second AT hook protein, CarD Sa , that is very similar to CarD and that occurs in the bacterium Stigmatella aurantiaca. CarD Sa has a C-terminal HMGA-like domain with three AT hooks and a highly acidic adjacent region with one predicted casein kinase II (CKII) phosphorylation site, compared to the four AT hooks and five CKII sites in CarD. Both proteins have a nearly identical 180-residue N-terminal segment that is absent in HMGA proteins. In vitro, CarD Sa exhibits the specific minor-groove binding to appropriately spaced AT-rich DNA that is characteristic of CarD or HMGA proteins, and it is also phosphorylated by CKII. In vivo, CarD Sa or a variant without the single CKII phosphorylation site can replace CarD in M. xanthus carotenogenesis and fruiting body formation. These two cellular processes absolutely require that the highly conserved N-terminal domain be present. Thus, three AT hooks are sufficient, the N-terminal domain is essential, and phosphorylation in the acidic region by a CKII-type kinase can be dispensed with for CarD function in M. xanthus carotenogenesis and fruiting body development. Whereas a number of hypothetical proteins homologous to the N-terminal region occur in a diverse array of bacterial species, eukaryotic HMGA-type domains appear to be confined primarily to myxobacteria.

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

“…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 .…”

“…1B). All probes were generated by PCR (except for Mut5b and Mut10b, which were synthesized by GenScript USA, Inc.), and 32 P 5= end labeled at the coding strand as described elsewhere (5). Electrophoretic mobility shift assays (EMSA) were carried out in 20-l total reaction volumes of binding buffer (50 mM NaCl, 15 mM HEPES, 4 mM Tris, pH 7.9, 1 mM DTT, 10% glycerol, 1 mg/ml bovine serum albumin [BSA], and 0.1% Nonidet P-40) containing 1 to 3 pM of end-labeled probe (ϳ13,000 cpm), the amount of protein indicated in the corresponding figures and legends, and 1 g of double-stranded poly(dGdC) as nonspecific competitor DNA.…”

“…CarD and CarG are found only in a number of other myxobacteria. One striking feature of CarD and its orthologs in the closely related myxobacteria Stigmatella aurantiaca, Myxococcus fulvus, and Corallococcus coralloides is that these bacterial proteins have a DNA binding domain resembling eukaryotic high-mobility-group A (HMGA) proteins (5,6). The latter are small, relatively abundant, minor-groove DNA binding factors that remodel chromatin in the assembly of specific nucleoprotein complexes essential in transcription, replication, recombination, and repair (7,8).…”

“…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).…”

Operator Design and Mechanism for CarA Repressor-mediated Down-regulation of the Photoinducible carB Operon in Myxococcus xanthus

“…Once liberated, CarQ up-regulates expression of car-QRS and crtI (18). In this, CarQ is aided by the high mobility group A-type protein CarD (23)(24)(25)(26) and the histone-like protein IhfA (integration host factor ␣ subunit) (27). The photo-induced expression of the carB operon is regulated by CarA, which is produced independently of light from an adjacent operon, and by CarS (28,29).…”