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

Abstract

The CarD-CarG complex controls various cellular processes in the bacterium Myxococcus xanthus including fruiting body development and light-induced carotenogenesis. The CarD N-terminal domain, which defines the large CarD_CdnL_TRCF protein family, binds to CarG, a zinc-associated protein that does not bind DNA. The CarD C-terminal domain resembles eukaryotic high-mobility-group A (HMGA) proteins, and its DNA binding AT hooks specifically recognize the minor groove of appropriately spaced AT-rich tracts. Here, we investigate the determinants of the only known CarD binding site, the one crucial in CarDCarG regulation of the promoter of the carQRS operon (P QRS ), a light-inducible promoter dependent on the extracytoplasmic function (ECF) factor CarQ. In vitro, mutating either of the 3-bp AT tracts of this CarD recognition site (TTTCCAGAGCTTT) impaired DNA binding, shifting the AT tracts relative to P QRS had no effect or marginally lowered DNA binding, and replacing the native site by the HMGA1a binding one at the human beta interferon promoter (with longer AT tracts) markedly enhanced DNA binding. In vivo, however, all of these changes deterred P QRS activation in wild-type M. xanthus, as well as in a strain with the CarD-CarG pair replaced by the Anaeromyxobacter dehalogenans CarD-CarG (CarD Ad -CarG Ad ). CarD Ad -CarG Ad is functionally equivalent to CarD-CarG despite the lower DNA binding affinity in vitro of CarD Ad , whose C-terminal domain resembles histone H1 rather than HMGA. We show that CarD physically associates with RNA polymerase (RNAP) specifically via interactions with the RNAP ␤ subunit. Our findings suggest that CarD regulates a light-inducible, ECF -dependent promoter by coupling RNAP recruitment and binding to a specific DNA site optimized for affinity and position.

The CarD/CarG regulatory complex is required for the action of several members of the large set of Myxococcus xanthus extracytoplasmic function σ factors

“…This site, spanning positions −77 to −65, has two 3 bp AT‐rich tracts with a 7 bp spacer, and the AT‐tract length and position are strictly optimized for P QRS activation in vivo (García‐Heras et al ., ). Nonetheless, CarD can also recognize other AT‐rich tracts (4–8 bp long) that are 6–8 bp apart, like mammalian HMGA in vitro (Solomon et al ., ; Maher and Nathans, ; Padmanabhan et al ., ; García‐Heras et al ., 2009; 2013). A search using Virtual Footprint (http://prodoric.tu-bs.de/vfp; Munch et al ., ) for DNA stretches conforming to the pattern [AT][AT][AT].{4,10}.…”

“…As mentioned earlier, our previous work established that CarG binds to CarD but not to DNA or RNAP, whereas CarD interacts via its N‐terminal domain with CarG and the β subunit of RNAP and via its C‐terminal domain with DNA. A DNA site with two 3 bp AT tracts and a 7 bp spacer recognized by the CarD/CarG complex and located 30 bp upstream of the light‐inducible P QRS promoter is crucial for activation by CarQ (Peñalver‐Mellado et al ., ; García‐Heras et al ., ). CarD also binds to a probe containing P ddvSA , while in silico analysis reveals that several of the CarD/CarG‐dependent P ecf identified in this work contain possible CarD‐binding sites.…”

“…A notable feature of myxobacteria evident from available genome data is the presence of large numbers of ECF‐σ factors (from 19 to as many as 118, as listed in the MiST2 database), and out of 11 myxobacterial species sequenced, orthologous CarD/CarG pairs exist in eight, all within the Myxococcales order that includes M. xanthus . Furthermore, we have shown that the M. xanthus CarD/CarG pair is functionally equivalent to the ones in Stigmatella aurantiaca and Anaeromyxobacter dehalogenans , with 32 and 23 putative ECF‐σ factors respectively (Cayuela et al ., ; García‐Heras et al ., 2009; 2013). Hence, at least in myxobacteria, with their large pool of ECF‐σs, CarD/CarG‐mediated regulation of ECF‐σ function presents a new facet among the control mechanisms exploited to selectively activate and control ECF‐σ activity in response to specific extracytoplasmic stresses.…”

“…However, diverse sites are compatible with the observed plasticity of the CarD DNA‐binding module, a basic, intrinsically unfolded domain functionally replaceable by other basic, unstructured domains such as that in histone H1 (García‐Heras et al ., ). Even so, AT tract length and position relative to the promoter of the CarD‐binding site required for P QRS activation in vivo has been found to be strictly optimized (García‐Heras et al ., ). This suggests that the CarD‐binding site, even if divergent for different P ecf , may be specific for a given ECF‐σ to act at its P ecf .…”

“…−65, has two 3 bp AT-rich tracts with a 7 bp spacer, and the AT-tract length and position are strictly optimized for PQRS activation in vivo (García-Heras et al, 2013). Nonetheless, CarD can also recognize other AT-rich tracts (4-8 bp long) that are 6-8 bp apart, like mammalian HMGA in vitro (Solomon et al, 1986;Maher and Nathans, 1996;Padmanabhan et al, 2001;García-Heras et al, 2009;2013).…”