The widespread bacterial second messenger cyclic diguanylate (c-di-GMP) 
regulates a variety of processes, including protein secretion, motility, cell 
development, and biofilm formation. c-di-GMP-dependent responses are often 
mediated by its binding to the cytoplasmic receptors that contain the PilZ 
domain. Here, we present comparative structural and sequence analysis of various 
PilZ-related domains and describe three principal types of them: (i) the 
canonical PilZ domain, whose structure includes a six-stranded beta-barrel and a 
C-terminal alpha helix, (ii) an atypical PilZ domain that contains two extra 
alpha helices and forms stable tetramers, and (iii) divergent PilZ-related 
domains, which include the eponymous PilZ protein and PilZN (YcgR_N) and PilZNR 
(YcgR_2) domains. We refine the second c-di-GMP binding motif of PilZ as 
[D/N]hSXXG and show that the hydrophobic residue h of this motif interacts with 
a cluster of conserved hydrophobic residues, helping maintain the PilZ domain 
fold. We describe several novel PilZN-type domains that are fused to the 
canonical PilZ domains in specific taxa, such as spirochetes, actinobacteria, 
aquificae, cellulose-degrading clostridia, and deltaproteobacteria. We propose 
that the evolution of the three major groups of PilZ domains included (i) fusion 
of pilZ with other genes, which produced Alg44, cellulose synthase, and other 
multidomain proteins; (ii) insertion of an ∼200-bp fragment, which resulted in 
the formation of tetramer-forming PilZ proteins; and (iii) tandem duplication of 
pilZ genes, which led to the formation of PilZ dimers and YcgR-like 
proteins.IMPORTANCE c-di-GMP is a ubiquitous bacterial second messenger that 
regulates motility, biofilm formation, and virulence of many bacterial 
pathogens. The PilZ domain is a widespread c-di-GMP receptor that binds c-di-GMP 
through its RXXXR and [D/N]hSXXG motifs; some PilZ domains lack these motifs and 
are unable to bind c-di-GMP. We used structural and sequence analysis to assess 
the diversity of PilZ-related domains and define their common features. We show 
that the hydrophobic residue h in the second position of the second motif is 
highly conserved; it may serve as a readout for c-di-GMP binding. We describe 
three principal classes of PilZ-related domains, canonical, tetramer-forming, 
and divergent PilZ domains, and propose the evolutionary pathways that led to 
the emergence of these PilZ types.