A matter of hierarchy: activation of orfamide production by the post-transcriptional Gac-Rsm cascade of Pseudomonas protegens CHA0 through expression upregulation of the two dedicated transcriptional regulators.

In this work, we surveyed the genome of P. protegens CHA0 in order to identify novel mRNAs possibly under the control of the Gac-Rsm cascade that might, for their part, serve to elucidate as-yet-unknown functions involved in the biocontrol of plant pathogens and/or in cellular processes required for fitness in natural environments. In view of the experimental evidence from former studies on the Gac-Rsm cascade, we developed a computational screen supported by a combination of sequence, structural and evolutionary constraints that led to a dataset of 43 potential novel mRNA targets. We then confirmed several mRNA targets experimentally and next focused on two of the respective genes that are physically linked to the orfamide biosynthetic gene cluster and whose predicted open-reading frames resembled cognate LuxR-type transcriptional regulators of cyclic lipopeptide clusters in related pseudomonads. In this report, we demonstrate that in strain CHA0, orfamide production is stringently dependent on a functional Gac-Rsm cascade and that both mRNAs encoding transcriptional regulatory proteins are under direct translational control of the RsmA/E proteins. Our results have thus revealed a hierarchical control over the expression of orfamide biosynthetic genes with the final transcriptional control subordinated to the global Gac-Rsm post-transcriptional regulatory system.

Sobrero PM ，Muzlera A ，Frescura J ，Jofré E ，Valverde C ... -  《Environmental Microbiology Reports》

Coregulation of the cyclic lipopeptides orfamide and sessilin in the biocontrol strain Pseudomonas sp. CMR12a.

Cyclic lipopeptides (CLPs) are synthesized by nonribosomal peptide synthetases (NRPS), which are often flanked by LuxR-type transcriptional regulators. Pseudomonas sp. CMR12a, an effective biocontrol strain, produces two different classes of CLPs namely sessilins and orfamides. The orfamide biosynthesis gene cluster is flanked up- and downstream by LuxR-type regulatory genes designated ofaR1 and ofaR2, respectively, whereas the sessilin biosynthesis gene cluster has one LuxR-type regulatory gene which is situated upstream of the cluster and is designated sesR. Our study investigated the role of these three regulators in the biosynthesis of orfamides and sessilins. Phylogenetic analyses positioned OfaR1 and OfaR2 with LuxR regulatory proteins of similar orfamide-producing Pseudomonas strains and the SesR with that of the tolaasin producer, Pseudomonas tolaasii. LC-ESI-MS analyses revealed that sessilins and orfamides are coproduced and that production starts in the late exponential phase. However, sessilins are secreted earlier and in large amounts, while orfamides are predominantly retained in the cell. Deletion mutants in ofaR1 and ofaR2 lost the capacity to produce both orfamides and sessilins, whereas the sesR mutant showed no clear phenotype. Additionally, RT-PCR analysis showed that in the sessilin cluster, a mutation in either ofaR1 or ofaR2 led to weaker transcripts of the biosynthesis genes, sesABC, and putative transporter genes, macA1B1. In the orfamide cluster, mainly the biosynthesis genes ofaBC were affected, while the first biosynthesis gene ofaA and putative macA2B2 transport genes were still transcribed. A mutation in either ofaR1, ofaR2, or sesR genes did not abolish the transcription of any of the other two.

Olorunleke FE ，Kieu NP ，De Waele E ，Timmerman M ，Ongena M ，Höfte M ... -  《MicrobiologyOpen》

A single mutation in the oprF mRNA leader confers strict translational control by the Gac/Rsm system in Pseudomonas fluorescens CHA0.

Crespo MC ，Valverde C 《-》

GacS/GacA activates pyoluteorin biosynthesis through Gac/Rsm-RsmE cascade and RsmA/RsmE-driven feedback loop in Pseudomonas protegens H78.

The Gac/Rsm regulatory pathway in Pseudomonas spp. activates the production of various secondary metabolites, such as antibiotics, siderophores and exoenzymes. However, the biosynthesis of antifungal compound pyoluteorin (Plt) in Pseudomonas protegens H78 is almost entirely inhibited by double deletion of two csrA/rsmA family genes, namely, rsmA and rsmE. Here, we investigated the complicated regulatory mechanism of RsmA and RsmE in Plt biosynthesis in P. protegens H78. RsmE negatively regulated Plt biosynthesis and pltLABCDEFG expression by directly interacting with the mRNA leaders of pltR and pltAB. Conversely, the transcription of pltL-G and pltR was positively influenced by RsmA through an uncharacterized mechanism. Further analyses demonstrated that pltL-G expression was diminished in the rsmA/E mutant. The deficiency of pltL-G expression in the gacA mutant was not reversed by any of the rsmA/E single or double mutations. The double deletion of rsmA/E reduced gacA expression by approximately 50% and almost completely inhibited the promoter activities of rsmXYZ sRNAs. The rsmY mutation significantly inhibited Plt biosynthesis. Taken together, GacS/GacA modulates Plt biosynthesis through two distinct pathways: Gac/Rsm-RsmE traditional positive regulatory cascade and RsmA-mediated positive transcriptional regulation. Moreover, a new positive feedback loop between RsmA/E and GacS/A-RsmXYZ is essential for activating Plt biosynthesis.

Wang Z ，Huang X ，Liu Y ，Yang G ，Liu Y ，Zhang X ... -  《-》

Distinctive features of the Gac-Rsm pathway in plant-associated Pseudomonas.

Productive plant-bacteria interactions, either beneficial or pathogenic, require that bacteria successfully sense, integrate and respond to continuously changing environmental and plant stimuli. They use complex signal transduction systems that control a vast array of genes and functions. The Gac-Rsm global regulatory pathway plays a key role in controlling fundamental aspects of the apparently different lifestyles of plant beneficial and phytopathogenic Pseudomonas as it coordinates adaptation and survival while either promoting plant health (biocontrol strains) or causing disease (pathogenic strains). Plant-interacting Pseudomonas stand out for possessing multiple Rsm proteins and Rsm RNAs, but the physiological significance of this redundancy is not yet clear. Strikingly, the components of the Gac-Rsm pathway and the controlled genes/pathways are similar, but the outcome of its regulation may be opposite. Therefore, identifying the target mRNAs bound by the Rsm proteins and their mode of action (repression or activation) is essential to explain the resulting phenotype. Some technical considerations to approach the study of this system are also given. Overall, several important features of the Gac-Rsm cascade are now understood in molecular detail, particularly in Pseudomonas protegens CHA0, but further questions remain to be solved in other plant-interacting Pseudomonas.

Ferreiro MD ，Gallegos MT 《-》