Cyclic diguanylate turnover mediated by the sole GGDEF/EAL response regulator in Pseudomonas putida: its role in the rhizosphere and an analysis of its target processes

Abstract

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.

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

“…In particular, the stress sigma factor RpoS positively or negatively regulated 15 of the 21 genes upon entry into the stationary phase. Regulation of the GGDEF/EAL domain-encoding genes by RpoS has also been observed in other bacteria (325,364) and seems to represent a common trait. While six of the RpoS-controlled genes regulate CsgD and curli gene expression, downstream targets of most of the other GGDEF/EAL domain-encoding genes remain to be determined.…”

Roles of Cyclic Di-GMP and the Gac System in Transcriptional Control of the Genes Coding for the Pseudomonas putida Adhesins LapA and LapF

“…No detectable differences were observed in planktonic growth of the strains. Cultures harboring pMAMV1 were vortexed in the presence of glass beads (diameter, 0.5 mm) to disrupt aggregates that appeared as a consequence of high levels of c-di-GMP (31). Experiments were repeated at least three times with two technical repeats per sample, and data are given in Miller units.…”

“…Plasmids pMMG1 (P lapF ::lacZ), pMMG2 (P lapF ::gfp), pMMGA (P lapA :: lacZ), pMAMV21 harboring an rpoS::lacZ translational fusion, and pMAMV1 carrying the diguanylate cyclase-encoding gene rup4959 have been described elsewhere (22,31,32). E. coli was grown at 37°C in LB medium (33).…”

Fis regulates the competitiveness of Pseudomonas putida on barley roots by inducing biofilm formation

“…Since the switch of bacterial lifestyle from planktonic to sessile growth requires alterations in bacterial physiology, the expression of a large number of genes is changed during the process of colonization (Mathesius, 2009;Matilla et al, 2007Matilla et al, , 2011Ramos-González et al, 2005). Matilla et al (2007) identified 93 P. putida genes from 11 functional classes which exhibited increased expression when the bacteria grew in the maize rhizosphere, including genes for motility, such as fliL and fliB, and several transcriptional regulators.…”