The ability to sense extracellular stimuli is a fundamental property of living cells. However, little is known about the diversity of extracellular sensor elements of biological receptors. By using sensitive database searches combined with detailed protein sequence analysis, we have identified several novel extracellular sensory domains that are found in various bacterial transmembrane receptors. All of the domains are found in sensor histidine kinases and nucleotide (adenylate and diguanylate) cyclases, similar to the distribution of two previously described extracellular sensory domains, Cache (3
) and CHASE (4
). CHASE2 was recently described as being an exclusively cyanobacterial (CMS, cyanobacterial membrane sensor) domain (45
). Indeed, in the cyanobacterium Nostoc
sp. strain PCC 7120, the CHASE2 domain is present in 13 proteins. However, our results demonstrate that CHASE2 is found in transmembrane receptors in many species outside the cyanobacterial lineage. These include Deinococcus radiodurans
, spirochetes, and various proteobacterial species (Fig. and ). Similarly, the CHASE3 domain is present in several proteins in cyanobacteria but is also found in D. radiodurans
and various gram-positive bacteria and proteobacteria (Fig. and ).
While neither CHASE2 nor CHASE3 has been found in Archaea, CHASE4 and CHASE6 domains are encoded in archaeal species as well as in representatives of gram-positive bacteria and proteobacteria (CHASE4) and cyanobacteria (CHASE6). Thus, novel sensory domains have a wide phyletic distribution. Two-component regulatory systems are found in only few species of Archaea
and could have been acquired from Bacteria
through lateral gene transfer (34
). Thus, the prevalence of CHASE-like sensory domains in Bacteria
is not surprising.
Some of the proteins that contain newly defined domains have been studied experimentally, for example, the CHASE3-containing adenylate cyclase CyaA from the cyanobacteria Spirulina platense
sp. strain PCC 7120 (31
) and histidine kinase VsrA from Ralstonia
). The histidine kinase VsrA was identified as a critical sensor required for expression of virulence factors, both polysaccharides and proteins, in this wilt-inducing phytopathogen. CHASE3-containing sensors are also found in other important pathogens of plants and mammals, such as Pseudomonas syringae, Burkholderia fungorum, Legionella pneumophila
, and Bacillus anthracis
(unpublished genomes; data not shown). A recent study of the adenylate cyclase CyaA from Myxococcus xanthus
demonstrated that this CHASE2 domain-containing protein participates in signal transduction during osmotic stress and might function as an osmosensor (35
). Thus, it is an attractive hypothesis that the CHASE2 domain is the osmosensing module in all other types of transmembrane receptors in which it is found.
At this time, the stimuli that are recognized by other novel domains are unknown. Identification of conserved residues in CHASE-like domains presented in this work, such as the RGFLLT motif in the VsrA protein (Fig. ), may assist investigators in future studies of the ligand specificity of various transmembrane receptors.
The CHASE2 and CHASE4 domains have not been found in MCPs. However, as sensor elements of histidine kinases, they are present in proteobacterial species that have large numbers of MCPs, for example, Pseudomonas aeruginosa
), Ralstonia solanacearum
), and Vibrio cholerae
). This observation suggests that CHASE2 and CHASE4 recognize stimuli that are not important for the immediate motility response but might be critical for regulation of metabolism or cell development. It is remarkable that in archaea, CHASE4 appears to be associated exclusively with histidine kinases, while in bacteria it is associated exclusively with diguanylate cyclases (Fig. ). Uncovering the reasons for this dichotomy should help in understanding the principles of signal transduction in these two groups of prokaryotes.
It is important to note that the complex multidomain organization of transmembrane sensors seriously complicates functional annotation of these proteins. In the course of microbial genome sequencing projects, signaling proteins containing the CHASE2, CHASE3, and CHASE4 domains have been repeatedly misannotated and deposited in GenBank under obscure or erroneous names. For example, the histidine kinase (All3347, gi 17230839) and an uncharacterized protein (Alr0357, gi 17227853) from Nostoc
sp. strain PCC 7120 (Fig. ) were both annotated as adenylate cyclases (30
), although their similarity to adenylate cyclases was limited to the common N-terminal CHASE2 domain. Curiously, three virtually identical CHASE2-containing proteins were annotated as adenylate cyclase (All7310, gi 17233326), similar to adenylate cyclase (All3180, gi 17230672), and a hypothetical protein (Alr1378, gi 17228873), although none of them contained the cyclase domain (Fig. ). In fact, annotation of transmembrane sensors as unknown, hypothetical, or conserved hypothetical proteins is quite common.
As we have argued earlier, short of a systematic mistake in sequencing, a protein that is conserved across diverse phylogenetic lineages is not hypothetical anymore (20
). We hope that delineation of the novel sensor domains in this work would help simplify the recognition of transmembrane receptor proteins and eventually improve their annotation. It is clear, however, that fully consistent annotation of the CHASE-type domain-containing sensors will be impossible without experimentally identifying their ligands and understanding the full set of environmental signals sensed by these domains.
Although most of the novel domains described here are found in transmembrane receptors, they might utilize different mechanisms of signal transduction across the membrane. CHASE3 and CHASE4 are often found in proteins that also contain the HAMP domain (6
). Moreover, the CHASE3 domain shows remote (likely topological) similarity to the periplasmic-ligand binding domain of the HAMP domain-containing Tar chemoreceptor (41
). Therefore, in CHASE3-containing receptors, signal translocation across the membrane might be similar to the piston model suggested for Tar (12
). However, the CHASE2 domain is never found in combination with the HAMP domain but is always followed by three transmembrane helices. This unusual motif occurred without exception in all proteins in which CHASE2 was detected (Fig. ) and might be an attractive subject for future studies on novel mechanisms of transmembrane signaling.
In summary, our results indicate that various transmembrane receptors that transduce signals into diverse regulatory pathways may utilize similar sensory (input) domains. The pervasiveness of a particular sensor domain in receptors from diverse regulatory networks within a given organism suggests that the stimuli recognized by this domain might be important for the biology of the organism.