Delprince, A. | bioRxiv Science

bioRxiv2026

Functional impact of a type VI secretion system on RsmE-mediated spatial structure formation in Pseudomonas fluorescens Pf0-1

Bacteria naturally form densely structured populations wherein both space and nutrients become locally limited. In densely populated Pseudomonas fluorescens Pf0-1 colonies, spatiogenetic patches with drastically reduced cellular density naturally emerge through spontaneous mutations in rsmE. RsmE is a posttranscriptional regulator that binds to specific mRNAs, including those that consequently repress the production of extracellular secretions. We have previously shown that {Delta}rsmE produces an extracellular polysaccharide (EPS) and biosurfactant that collectively function to define the spatiogenetic structure and locally outcompete the WT. Here, we identify additional RsmE-regulated secretions through a combination of RNA-sequencing and LC-MS/MS. In particular, a type VI secretion system (T6SS) was exclusively produced by {Delta}rsmE. Confocal microscopy imaging of WT and genetically modified {Delta}rsmE cocultures showed that the T6SS kills WT cells that invade the low- density spatiogenetic structure. However, its impact on competition with the WT was both quantifiably and spatially limited in the absence of the normally co-produced EPS and biosurfactant, largely due to the consequently altered spatial structure. These results highlight the importance of understanding both the individual and collective spatial functions of extracellular secretions, especially in the ecological context of densely structured microbial populations. IMPORTANCEIn crowded wild type (WT) Pseudomonas fluorescens Pf0-1 colonies, rsmE mutants spontaneously emerge by forming a low cellular density spatial structure that is devoid of WT cells. RsmE functions primarily to repress the production of various extracellular secretions, including an extracellular polysaccharide and biosurfactant, that collectively form the unique spatial structure. Here, we describe an RsmE-regulated type VI secretion system that kills the invading WT cells and protect the low-density structure. Importantly, individual RsmE-regulated secretions carry out unique functions, but their efficacies largely depend on other co-regulated products. Extracellular secretions that are mechanistically similar to those described here are co- produced across diverse biofilm-forming and virulent bacterial species, where they also likely play complex molecular and ecological roles.

microbiology↗

bioRxiv2022

Spatial structure formation by RsmE-regulated extracellular secretions in Pseudomonas fluorescens Pf0-1

Cells in microbial communities on surfaces live and divide in close proximity, which greatly enhances the potential for social interactions. Spatiogenetic structures manifest through competitive and cooperative interactions among the same and different genotypes within a shared space, and extracellular secretions appear to function dynamically at the forefront. A previous experimental evolution study utilizing Pseudomonas fluorescens Pf0-1 colonies demonstrated that diverse mutations in the rsmE gene are repeatedly and exclusively selected through the formation of a dominant spatial structure. RsmEs primary molecular function is translation repression, and its homologs regulate various social and virulence phenotypes. Pseudomonas spp. possess multiple paralogs of Rsm proteins, and RsmA, RsmE, and RsmI are the most prevalent. Here, we demonstrate that the production of a mucoid polymer and a biosurfactant are exclusively regulated through RsmE, contradicting the generalized notion of functional redundancy among the Rsm paralogs. Furthermore, we identify the biosurfactant as the cyclic lipopeptide gacamide A. Competition and microscopy analyses show that the mucoid polymer is solely responsible for creating a space of low cellular density, which is shared exclusively by the same genotype. Gacamide A and other RsmE-regulated products appear to establish a physical boundary that prevents the encroachment of the competing genotype into the newly created space. Although cyclic lipopeptides and other biosurfactants are best known for their antimicrobial properties and reducing surface tension to promote the spreading of cells on various surfaces, they also appear to help define spatial structure formation within a dense community. IMPORTANCEIn densely populated colonies of the bacterium Pseudomonas fluorescens Pf0-1, diverse mutations in the rsmE gene are naturally selected by solving the problem of overcrowding. Here, we show that RsmE-regulated secretions function together to create and protect space of low cell density. A biosurfactant generally promotes the spreading of bacterial cells on abiotic surfaces, however, it appears to function atypically within a crowded population by physically defining genotypic boundaries. Another significant finding is that these secretions are not regulated by RsmEs paralogs that share high sequence similarity. The experimental pipeline described in this study is highly tractable and should facilitate future studies to explore additional RsmE-regulated products and address why RsmE is functionally unique from its paralogs.

microbiology↗