Genetic dissection of a motility‐associated c‐di‐GMP signalling protein of Pseudomonas putida

Abstract

Lack of the Pseudomonas putida PP2258 protein or its overexpression results in defective motility on solid media. The PP2258 protein is tripartite, possessing a PAS domain linked to two domains associated with turnover of c-di-GMP - a cyclic nucleotide that controls the switch between motile and sessile lifestyles. The second messenger c-di-GMP is produced by diguanylate cyclases and degraded by phosphodiesterases containing GGDEF and EAL or HD-GYP domains respectively. It is common for enzymes involved in c-di-GMP signalling to contain two domains with potentially opposing c-di-GMP turnover activities; however, usually one is degenerate and has been adopted to serve regulatory functions. Only a few proteins have previously been found to have dual enzymatic activities - being capable of both synthesizing and hydrolysing c-di-GMP. Here, using truncated and mutant derivatives of PP2258, we show that despite a lack of complete consensus in either the GGDEF or EAL motifs, the two c-di-GMP turnover domains can function independently of each other, and that the diguanylate cyclase activity is regulated by an inhibitory I-site within its GGDEF domain. Thus, motility-associated PP2258 can be added to the short list of bifunctional c-di-GMP signalling proteins.

New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling

“…When incubated with α-32 P-GTP, His 6 -Lpl1559 produced both c-di-GMP and pGpG, demonstrating its bifunctional activity. It is noteworthy that while bifunctional DGC/PDE enzymes are still rarely reported (Liu et al 2010;Österberg et al 2013;Römling et al 2013), it is the third example with Lpl0329 and Lpl0922 of such an enzyme in L. pneumophila (Levet-Paulo et al 2011;Allombert et al 2014).…”

The c‐di‐GMP phosphodiesterase BifA regulates biofilm development in Pseudomonas putida

“…The genome of P. putida KT2440 encodes 43 polypeptides potentially involved in c‐di‐GMP turnover (Ulrich and Zhulin, ). However, it was only recently that Matilla and colleagues () characterized the product of locus PP4959, a DGC whose synthesis is induced in response to stationary phase, microaerobiosis and the presence of root exudates, and Österberg and colleagues () characterized the product of locus PP2258, a dual activity DGC/PDE involved in the regulation of flagellar motility. To the best of our knowledge, no DGC or PDE has been unambiguously linked to the regulation of the biofilm developmental cycle in this organism.…”

Phenotypic–genotypic analysis of GGDEF/EAL/HD‐GYP domain‐encoding genes in Pseudomonas putida

“…Meanwhile, PP_2258 had no significant effect on biofilm formation but markedly promoted the motility. These findings are in line with the previous research results (Matilla et al ., ; Newell et al ., ; Österberg et al ., ; Jiménez‐Fernández et al ., ).…”

“…aeruginosa BifA, harbouring both a GGDEF and EAL domain, has been confirmed to possess PDE activity and impair biofilm formation (Kuchma et al ., ; Jiménez‐Fernández et al ., ). PP_2258 has been associated with swimming motility and confirmed to be a bifunctional enzyme involved in both the synthesis and degradation of c‐di‐GMP (Österberg et al ., ). PP_4959 behaves as a DGC and promotes biofilm formation in response to limited aeration (Matilla et al ., ).…”