Role of c-di-GMP in determining sessile or motile lifestyle in Aeromonas caviae Sch3N

The bacterial second messenger c-di-GMP has been recently found to be implicated in controlling many cellular processes in bacteria, one of them is the switch between motility and sessility. When in a sessile lifestyle (such as in a biofilm), bacteria are known to be more resistant to immune defences and to antibiotics. Mesophilic Aeromonas species are known to constitutively express a single polar flagellum which allows the bacteria to swim in liquid environments. They as well are able to express many peritrichous lateral flagella which allow them to move as a group (swarm). The multiple lateral flagella although are needed for swarming, they are, however, also required for biofilm formation. this study investigates the relationship between different components within the c-di-GMP network and the lateral flagella (laf) genes expression. Diguanylate cyclases containing GGDEF domains are responsible for c-di-GMP synthesis and phosphodiesterases containing EAL domains are known to breakdown the second messenger. Thirteen GGDEF-EAL domains encoding genes present in A. caviae Sch3N were mutated by plasmid insertion mutagenesis after which the effects of these mutations were studied phenotypically and by transcriptional fusions. We have found differences among the mutants in their ability to form a biofilm and on the activities of the lateral flagellin promoters lafA1p and lafA2p. C-di-GMP is known to bind to the PilZ domain and to RNA domains (riboswitches) to exert its function. We have mutated the pilZ gene present in the lateral flagella gene cluster by insertion of a kanamycin cassette and also we have deleted the lafK riboswitch by Spliced Overlap Extension PCR. Performance of phenotypic and beta-galactosidase assays following these mutations allowed us to conclude that the lafK c-di-GMP type I riboswitch seems to act as a c-di-GMP modulator in A. caviae. In addition, the lafK riboswitch acts as an "OFF switch" which prevents the expression of LafK, the major regulator of the laf operon.