Eukaryotic-like signaling and gene regulation in a prokaryote that undergoes multicellular development.

H ow cell–cell signaling coordinates cell movement, gene expression, and differentiation during the development of multicellular organisms is a fundamental question. At the molecular level, the answer is complex, even in the simplest cases. Myxococcus xanthus is one of the simplest organisms that undergo development to produce a multicellular structure of uniform size and shape. Cells of this bacterium sense nutrient limitation and alter their gliding movements to produce mounds (Fig. 1). Within these nascent fruiting bodies, cells differentiate into spores. Driving this developmental process is a program of gene expression that is temporally and spatially regulated by intracellular and extracellular signals. Are the typical prokaryotic signaling and gene regulatory mechanisms sufficient for multicellular development of M. xanthus? Previous work has indicated that eukaryotic-like mechanisms play a role. The work of Jelsbak et al. (1) in this issue of PNAS extends this observation by describing a group of 12 M. xanthus genes that seem to encode enhancer-binding proteins (EBPs) with a forkhead-associated (FHA) domain. The authors show that the FHA domain of one of the EBPs is required for normal development. Because FHA domains interact with phosphothreonine residues, the results suggest a crucial link to eukaryotic-like Ser Thr protein kinases (STPKs), which are abundant in M. xanthus. Bacterial EBPs derive their name from the fact that they activate transcription by 54-RNA polymerase ( 54-RNAP) in much the same way some EBPs activate transcription in eukaryotes. 54-RNAP requires interaction with an EBP to form the transcriptionally competent open promoter complex. Bacterial EBPs bind to DNA enhancer elements typically located 70–150 bp upstream of the transcription start site (reviewed in ref. 2). Bending of DNA allows the EBP to contact 54RNAP, forming a DNA loop. ATP hydrolysis by the conserved central domain of the EBP enables it to convert the 54RNAP closed promoter complex to the open complex. Although eukaryotic EBPs also bind DNA and in some cases cause a DNA loop to form by contacting RNAP or a general transcription factor, they do not perform the ATP hydrolysis necessary for open complex formation [e.g., transcription factor IIH (TFIIH) does this in the case of RNAP II transcription]. The work of Jelsbak et al. (1) builds on previous efforts to identify and characterize EBPs of M. xanthus (3–5). By using the conserved central ATPase domain to search the recently completed M. xanthus genome sequence, Jelsbak et al. (1) found 52 putative EBPs. This is the largest number so far found in a bacterial genome. At least 16 of these EBPs have been shown to be required for normal fruiting body development or motility (refs. 3–5 and references therein). Clearly, this form of regulation is critical for M. xanthus development. Moreover, 54 is essential for growth of this bacterium (6), unlike other bacteria so far tested. The situation in M. xanthus is strikingly different from that in Streptomyces coelicolor, another prokaryotic model for development, which is devoid of 54 and EBPs (7). Jelsbak et al. (1) recognize FHA domains in 12 of the M. xanthus EBPs. The FHA domain mediates phosphorylationdependent, protein–protein interactions by recognizing a phosphothreoninecontaining epitope in a protein partner (reviewed in ref. 8). Originally recognized in a subset of eukaryotic forkheadtype transcription factors, the FHA domain has been found in eukaryotic proteins with diverse functions, including signal transduction, protein transport, and DNA repair. Likewise, FHA domains are found in a variety of prokaryotic proteins, implicating them in many bacterial processes (9). However, until now, only two putative FHA domain-containing EBPs (FHA-EBPs) have been described in bacteria (10). The finding of 12 such proteins in M. xanthus (1) suggests abundant connections between EBP-dependent transcription by 54-RNAP and phosphorylation of Thr residues in proteins. There is ample opportunity for phosphothreonine formation in proteins of M. xanthus. The first eukaryotic-like STPK found in bacteria was discovered in M. xanthus (11), and subsequent work revealed a large family of STPKs, many required for normal development (12). Half of the 12 FHA-EBP genes are next to or near STPK genes in the M. xanthus genome (1). This proximity suggests that the FHA domain of these EBPs might interact with the STPK encoded nearby. According to this model (Fig. 2), Thr autophosphorylation of the STPK in response to a signal would promote interaction with the FHA-EBP. Transfer of phosphate from the STPK to the FHAEBP would allow it to activate transcription by 54-RNAP. A precedent for parts of this model has been described recently (13). PknH, an STPK, and EmbR, an FHA domaincontaining transcription factor, are encoded by adjacent genes in Mycobacterium tuberculosis. PknH can autophosphorylate on Ser and Thr residues in vitro, then interact with the FHA domain of EmbR and transfer phosphate to one or more of its Thr residues. Presumably, phosphorylation of EmbR enables it to activate transcription. Although EmbR lacks the conserved central ATPase domain of EBPs, the N-terminal two-thirds of EmbR is similar to AfsR, a transcriptional activator of S. coelicolor. AfsR does not have a recognizable FHA domain, but it is phosphorylated by an STPK, and this phosphorylation enhances its ability to bind to the 35 region of its target promoter, where its ATPase activity is proposed to isomerize the RNAP closed promoter