Dinoroseobacter shibae Outer Membrane Vesicles Are Enriched for the Chromosome Dimer Resolution Site dif.

Hui Wang,Jörn Petersen,Christian Boedeker,Irene Wagner-Döbler,Petra Henke,Helena Sztajer,Susanne Engelmann,Manfred Rohde,Johannes Mansky,Martin Kucklick,Meina Neumann-Schaal,Frank Klawonn,Nicole Beier,Jürgen Tomasch,Jörg Overmann,Janet K Jansson

doi:10.1128/msystems.00693-20

Abstract

Outer membrane vesicles (OMVs) are universally produced by prokaryotes and play important roles in symbiotic and pathogenic interactions. They often contain DNA, but a mechanism for its incorporation is lacking. Here, we show that Dinoroseobacter shibae, a dinoflagellate symbiont, constitutively secretes OMVs containing DNA. Time-lapse microscopy captured instances of multiple OMV production at the septum during cell division. DNA from the vesicle lumen was up to 22-fold enriched for the region around the terminus of replication (ter). The peak of coverage was located at dif, a conserved 28-bp palindromic sequence required for binding of the site-specific tyrosine recombinases XerC/XerD. These enzymes are activated at the last stage of cell division immediately prior to septum formation when they are bound by the divisome protein FtsK. We suggest that overreplicated regions around the terminus have been repaired by the FtsK-dif-XerC/XerD molecular machinery. The vesicle proteome was clearly dominated by outer membrane and periplasmic proteins. Some of the most abundant vesicle membrane proteins were predicted to be required for direct interaction with peptidoglycan during cell division (LysM, Tol-Pal, Spol, lytic murein transglycosylase). OMVs were 15-fold enriched for the saturated fatty acid 16:00. We hypothesize that constitutive OMV secretion in D. shibae is coupled to cell division. The footprint of the FtsK-dif-XerC/XerD molecular machinery suggests a novel potentially highly conserved route for incorporation of DNA into OMVs. Clearing the division site from small DNA fragments might be an important function of vesicles produced during exponential growth under optimal conditions.IMPORTANCE Gram-negative bacteria continually form vesicles from their outer membrane (outer membrane vesicles [OMVs]) during normal growth. OMVs frequently contain DNA, and it is unclear how DNA can be shuffled from the cytoplasm to the OMVs. We studied OMV cargo in Dinoroseobacter shibae, a symbiont of dinoflagellates, using microscopy and a multi-omics approach. We found that vesicles formed during undisturbed exponential growth contain DNA which is enriched for genes around the replication terminus, specifically, the binding site for an enzyme complex that is activated at the last stage of cell division. We suggest that the enriched genes are the result of overreplication which is repaired by their excision and excretion via membrane vesicles to clear the divisome from waste DNA.

Highlights

IMPORTANCE Gram-negative bacteria continually form vesicles from their outer membrane during normal growth
In Salmonella, the shedding of Outer membrane vesicles (OMVs) accelerates the remodeling of the lipopolysaccharide (LPS) composition of the outer membrane which is an important adaptation to environmental transitions [6]
The average number of vesicles released per cell was determined in the culture supernatant directly by NanoSight particle tracking analysis, and bacterial cells were counted by flow cytometry

Summary

Introduction

IMPORTANCE Gram-negative bacteria continually form vesicles from their outer membrane (outer membrane vesicles [OMVs]) during normal growth. OMVs were discovered to be freely suspended [8] They have approximately the same abundance as bacteria, a distinct depth distribution, and contain DNA from a variety of marine bacterial taxa [9]. OMVs from Vibrio spp. play important roles for pathogenesis and symbiosis: they act as ferries for hydrolytic enzymes and signaling molecules in the coral pathogen Vibrio shilonii [10] and the oyster pathogen Vibrio tasmaniensis [11] They can package the hydrophobic quorum sensing signal CAI-1 from the human pathogen Vibrio harveyi [12]. OMVs might play a key role in the symbiotic relationship between the flatworm Paracatenula, which has neither mouth nor gut, and its obligate chemosynthetic symbiont “Candidatus Riegeria santandreae.” They might deliver energy-rich compounds to the host tissue and protect the symbiotic bacteria from being digested [15]. In D. shibae, GTA synthesis is quorum-sensing controlled and suppressed in the wild type by the product of the autoinducer synthase LuxI2 [24]

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