GGDEF and EAL/HD-GYP protein domains are responsible for the synthesis and hydrolysis of the bacterial secondary messenger cyclic diguanylate (c-di-GMP) through their diguanylate cyclase and phosphodiesterase activities, respectively. Forty-three genes in Pseudomonas putida KT2440 are putatively involved in the turnover of c-di-GMP. Of them only rup4959 (locus PP4959) encodes a GGDEF/EAL response regulator, which was identified in a genome wide analysis as preferentially induced while this bacterium colonizes roots and adjacent soil areas (the rhizosphere). By using fusions to reporter genes it was confirmed that the rup4959 promoter is active in the rhizosphere and inducible by corn plant root exudates and microaerobiosis. Transcription of rup4959 was strictly dependent on the alternative transcriptional factor σ(S) . The inactivation of the rup4959-4957 operon altered the expression of 22 genes in the rhizosphere and had a negative effect upon oligopeptide utilization and biofilm formation. In multicopy or when overexpressed, rup4959 enhanced adhesin LapA-dependent biofilm formation, the development of wrinkly colony morphology, and increased Calcofluor stainable exopolysaccharides (EPS). Under these conditions the inhibition of swarming motility was total and plant root tip colonization considerably less efficient, whereas swimming was partially diminished. This pleiotropic phenotype, which correlated with an increase in the global level of c-di-GMP, was not acquired with increased levels of Rup4959 catalytic mutant at GGDEF as a proof of this response regulator exhibiting diguanylate cyclase activity. A screen for mutants in putative targets of c-di-GMP led to the identification of a surface polysaccharide specific to KT2440, which is encoded by the genes cluster PP3133-PP3141, as essential for phenotypes associated with increased c-di-GMP. Cellulose and alginate were discarded as the overproduced EPS, and lipopolysaccharide (LPS) core and O-antigen were found to be essential for the development of wrinkly colony morphology.
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.
In Pseudomonas putida KT2440, cfcR encodes an orphan multidomain response regulator with diguanylate cyclase activity, which is responsible for the synthesis of c-di-GMP, a second messenger key in the transition from planktonic to sessile bacterial lifestyles. When overexpressed, cfcR enhances biofilm formation and causes other phenotype alterations. The cfcA gene, encoding a membrane-anchored multisensory CHASE3/GAF hybrid histidine kinase (HK), is required to develop this pleiotropic phenotype.Here we show autophosphorylation of CfcA through HisKA/HATPase_c domains and then transfer of the phosphoryl group to an internal receiver (REC) domain. CfcA REC domains are nonessential for phosphotransfer from CfcA$P to the REC domain of CfcR. CfcA$P also phosphorylates the REC domain of CfcD, a second HK encoded in the same gene cluster as CfcA, which negatively regulates the CfcA/ CfcR pathway. To evaluate the impact of CfcA domains on CfcR activity, a battery of mutants with in-frame domain deletions was generated, whose CfcA protein locations were also examined. CfcA membrane anchorage contributes to protein stability and CfcR activation. Salt enhances c-di-GMP levels through CfcR, a response which is hampered by alteration of a presumed ligand-binding motif in the CHASE3 sensor domain. Thus, in P. putida, c-di-GMP is salt-regulated through the CfcA/CfcR/CfcD system.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.