In addition to transmembrane receptors, there are several well-studied histidine kinases that have no transmembrane segments, such as chemotaxis histidine kinase CheA and nitrogen regulation protein NtrB (GlnL) from E. coli
, sporulation kinase KinA from B. subtilis,
or rhizobial oxygen sensor FixL (see Hoch and Silhavy, 1995
; for reviews). Receptor census () shows that free-living bacteria typically encode a significant number of intracellular histidine kinases, adenylate cyclases, diguanylate cyclases and phosphodiesterases. In fact, their cytoplasmic signalling network may be as complex as transmembrane signal transduction system. The genome of M. loti
, for example, encodes 13 copies of the adenylate cyclase domain (). Of these, only one appears to be fused to a periplasmic sensor domain, and another one is fused to an integral membrane sensor domain. All the rest are found in predicted cytoplasmic proteins, fused to poorly characterized N-terminal or C-terminal domains, most of which are likely involved in signalling. Of the 32 copies of the GGDEF domain, encoded in M. loti
, 18 belong to transmembrane sensors and 14 are found in intracellular signal transduction proteins and response regulators ().
Intracellular signalling proteins typically combine N-terminal cytoplasmic sensor domains, usually PAS or GAF, with a variety of signal transduction or output domains (). Some of these proteins contain N-terminal CheY domains and can be considered bona fide
response regulators. Indeed, phosphorylation of the CheY domain was shown to affect adenylate cyclase activity of the C-terminal ACyc domain, just as it affects DNA-binding properties of classical response regulators (Coudart-Cavalli et al., 1997
). However, many intracellular signalling proteins lack the CheY domains. Such proteins should not be confused with response regulators, despite certain parallelism in their domain architectures (see and ). For example, in addition to four NtrC-type response regulators of the CheY-AAA-Fis domain architecture (AtoC, GlnG, HydG and YfhA, see COG2204), E. coli
K12 encodes three intracellular signalling proteins with GAF-AAA-Fis domain structure (FhlA, HyfA, and NorR, see COG3604) and one more protein (YgeV) with GAF-PAS-AAA-Fis domain structure. Whereas the exact nature of the ligands of most of these proteins remains obscure, there is little doubt that they are directly involved in monitoring levels of NO and other intracellular parameters and regulating transcription in response to changes in these parameters (Gardner et al., 2003
Fig. 2 Parallelism in structures of response regulators and intracellular signalling proteins. Transmission of environmental signals from sensory kinases to response regulators involves phosphorylation of Asp residues in their CheY-like receiver domains, which (more ...)
Several pioneering studies have provided experimental evidence of the involvement of cytoplasmic signalling proteins in intracellular signalling. An E. coli
protein with the PAS-GGDEF-EAL domain combination has been named a ‘direct oxygen sensor’ (DOS), based on the effect oxygen binding has on the conformation of its N-terminal domain (Delgado-Nixon et al., 2000
). Further, oxygen binding has been shown to activate the phosphodiesterase activity of a G. xylinum
protein with the same domain organization (Chang et al., 2001
). Likewise, cGMP binding to the GAF domain of human phosphodiesterase PDE5 was shown to stimulate the activity of its C-terminal enzymatic domain (Rybalkin et al., 2003
). Besides oxygen, the DOS protein could also bind NO and CO, indicating that PAS- or GAF-containing molecules could be used for sensing a variety of intracellular parameters and effecting a variety of cellular responses. Finally, the NorR protein of GAF-AAA-Fis domain architecture has been shown to regulate transcription in response to nitric oxide and reactive nitrogen species (Pohlmann et al., 2000
; Gardner et al., 2003
; Mukhopadhyay et al., 2004
). These results clearly demonstrate that ligand binding to the N-terminal PAS and/or GAF domains can modulate the activities of the downstream output domains. Thus, the similarity between CheY-containing response regulators and PAS- or GAF-containing signallers apparently extends to their regulation mechanisms: both phosphorylation of the CheY domain in response to the extracellular signal and ligand binding to PAS or GAF, comprising an intracellular signal, induce conformational changes in these domains. In turn, these conformational changes activate (rather, cause a relief of inhibition) the downstream output domains, allowing them to perform their functions, be that binding DNA or RNA, catalysing synthesis or hydrolysis of cAMP or c-diGMP, demethylation of MCPs, and so on.
Unfortunately, most intracellular signalling proteins are still poorly studied and remain to be recognized as legitimate members of the bacterial signalling network.