Abstract
Most bacteria grow in nature forming multicellular structures named biofilms. The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key player in the regulation of the transition from planktonic to sessile lifestyles and this regulation is crucial in the development of biofilms. In Pseudomonas putida KT2440, Rup4959, a multidomain response regulator with diguanylate cyclase activity, when overexpressed causes an increment in the intracellular levels of c-di-GMP that gives rise to a pleiotropic phenotype consisting of increased biofilm formation and crinkly colony morphology. In a broad genomic screen we have isolated mutant derivatives that lose the crinkly morphology, designed as cfc (crinkle free colony). A total of 19 different genes have been identified as being related with the emergence of the cfc phenotype either because the expression or functionality of Rup4959 is compromised, or due to a lack of transduction of the c-di-GMP signal to downstream elements involved in the acquisition of the phenotype. Discernment between these possibilities was investigated by using a c-di-GMP biosensor and by HPLC-MS quantification of the second messenger. Interestingly five of the identified genes encode proteins with AAA+ ATPase domain. Among the bacterial determinants found in this screen are the global transcriptional regulators GacA, AlgU and FleQ and two enzymes involved in the arginine biosynthesis pathway. We present evidences that this pathway seems to be an important element to both the availability of the free pool of the second messenger c-di-GMP and to its further transduction as a signal for biosynthesis of biopolimers. In addition we have identified an uncharacterized hybrid sensor histidine kinase whose phosphoaceptor conserved histidine residue has been shown in this work to be required for in vivo activation of the orphan response regulator Rup4959, which suggests these two elements constitute a two-component phosphorelay system.
Highlights
In recent years cyclic diguanosine monophosphate (c-di-GMP) has emerged as an important second messenger in prokaryotic cells, where its role in defining the bacterial mode of life has been established
An independent transposon insertional inactivation located in the core of the algU gene did not cause any mutant phenotype. This result was consistent in all the clones and c-di-GMP Regulatory Network indicates that the loss of functionality of algU was probably not the reason for the crinkle-free phenotype of the variant cfcK-16
Unlike what was observed with the wild type, we have shown above that the global levels of c-di-GMP were undetectable in the mutant cfcK-54 carrying pMAMV1 and that this mutant is hampered in a histidine kinase (HK)
Summary
In recent years cyclic diguanosine monophosphate (c-di-GMP) has emerged as an important second messenger in prokaryotic cells, where its role in defining the bacterial mode of life has been established. High intracellular levels of c-di-GMP favor the settlement of bacteria in the form of sessile populations (surface-attached cells and biofilms), whilst low levels of this molecule promote motility and the planktonic lifestyle. C-di-GMP is considered one of the key elements controlling biofilm formation and dispersal (Boyd and O’Toole, 2012). The turnover of this second messenger is carried out by proteins containing the domains GGDEF or EAL/HD-GYP, which are responsible for the synthesis and hydrolysis of c-di-GMP through their diguanylate cyclase (DGC) and phosphodiesterase activities (PDE), respectively. Often multiple of these proteins are found in the same organism and there appears to be some redundancy (Römling et al, 2005; Ulrich and Zhulin, 2010)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
