Regulation via c-di-GMP as a second messenger 
Cyclic diguanylate (c-di-GMP) is a bacterial second messenger that activates biofilm formation while inhibiting motility, thus regulating the switch between a planktonic and a sessile lifestyle.
In addition to phenotypes that affect virulence properties indirectly, c-di-GMP can also directly regulate virulence factors [56,57].
Proteins containing a so-called GGDEF domain are responsible for the synthesis of c-di-GMP, and those with a so-called EAL domain for its degradation.
The expression and activity of those GGDEF and EAL domain containing proteins is regulated by factors with a PilZ domain that binds c-di-GMP. The PilZ domain is found as a stand-alone domain or in combination with GGDEF, EAL and other domains, thus assumed to function also as an allosteric domain to control other regulatory enzymes [58,59].
In Y. enterocolitica, we identified 22 putative proteins containing GGDEF and EAL domains.
Eleven of these proteins solely contain a GGDEF-domain and six solely an EAL-domain, and both domains are found in tandem in five proteins.
The protein AdrA (YE3010, GGDEF domain) is annotated as a putative diguanylate cyclase, YE2278 (GGDEF+EAL) as a putative phosphodiesterase, YE3818 (GGDEF) as a putative regulator, and YE3806 (GGDEF+EAL) as a putative exported protein.
All other GGDEF and EAL domain-containing proteins are of unknown function.
Furthermore, two proteins with PilZ domain exist in Y. enterocolitica, namely YE3197 and BcsA (YE4074), a putative cellulose synthase.
Cellulose synthesis in bacteria has been identified to be important for the protection from chemical or mechanical stress by forming a hydrophobic extracellular matrix [60].
The expression of two of those EAL-domain containing proteins, YE4063 and YE1324, is induced at low temperature (Tab. 1).
These two factors might therefore be important for insect colonization instead for virulence against mammals.
The presence of c-di-GMP mediated regulation in Y. enterocolitica is therefore suggested to play a central role in switching from biofilm formation to the human as well as to the insect environment.
P. luminescens contains no protein with GGDEF, EAL or PilZ domain.
This phenomenon is quite surprising, because with few exceptions such as Helicobacter pylori, nearly all pathogenic bacteria use c-di-GMP as a second messenger.
It has been reported that P. luminescens forms biofilms in vitro, and that a luxS-deficient mutant unable to synthesize the quorum-sensing inducer AI-2 showed a decreased biofilm formation [42].
The lack of these protein domains in P. luminescens reveals that c-di-GMP signalling plays a major role in pathogenic bacteria when colonizing a mammalian host, and a minor for invertebrate colonization of entomopathogenic or entomoinfecting bacteria.
