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

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.

SUBMITTER: Wang H

PROVIDER: S-EPMC7901474 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Chromosome dimers occur in bacterial cells as a result of the recombinational repair of DNA. In most bacteria, chromosome dimers are resolved by XerCD site-specific recombination at the dif (deletion-induced filamentation) site located in the terminus region of the chromosome. Caulobacter crescentus, a Gram-negative oligotrophic bacterium, also possesses Xer recombinases, called CcXerC and CcXerD, which have been shown to interact with the Escherichia colidif site in vitro Previous studies on Caulobacter have suggested the presence of a dif site (referred to in this paper as dif1CC ), but no in vitro data have shown any association with this site and the CcXer proteins. Using recursive hidden Markov modeling, another group has proposed a second dif site, which we call dif2CC , which shows more similarity to the dif consensus sequence. Here, by using a combination of in vitro experiments, we compare the affinities and the cleavage abilities of CcXerCD recombinases for both dif sites. Our results show that dif2CC displays a higher affinity for CcXerC and CcXerD and is bound cooperatively by these proteins, which is not the case for the original dif1CC site. Furthermore, dif2CC nicked substrates are more efficiently cleaved by CcXerCD, and deletion of the site results in about 5 to 10% of cells showing an altered cellular morphology.IMPORTANCE Bacteria utilize site-specific recombination for a variety of purposes, including the control of gene expression, acquisition of genetic elements, and the resolution of dimeric chromosomes. Failure to resolve dimeric chromosomes can lead to cell division defects in a percentage of the cell population. The work presented here shows the existence of a chromosomal resolution system in C. crescentus Defects in this resolution system result in the formation of chains of cells. Further understanding of how these cells remain linked together will help in the understanding of how chromosome segregation and cell division are linked in C. crescentus.

Project description:BACKGROUND: Dinoroseobacter shibae, a member of the Roseobacter clade abundant in marine environments, maintains morphological heterogeneity throughout growth, with small cells dividing by binary fission and large cells dividing by budding from one or both cell poles. This morphological heterogeneity is lost if the quorum sensing (QS) system is silenced, concurrent with a decreased expression of the CtrA phosphorelay, a regulatory system conserved in Alphaproteobacteria and the master regulator of the Caulobacter crescentus cell cycle. It consists of the sensor histidine kinase CckA, the phosphotransferase ChpT and the transcriptional regulator CtrA. Here we tested if the QS induced differentiation of D. shibae is mediated by the CtrA phosphorelay. RESULTS: Mutants for ctrA, chpT and cckA showed almost homogeneous cell morphology and divided by binary fission. For ctrA and chpT, expression in trans on a plasmid caused the fraction of cells containing more than two chromosome equivalents to increase above wild-type level, indicating that gene copy number directly controls chromosome number. Transcriptome analysis revealed that CtrA is a master regulator for flagellar biosynthesis and has a great influence on the transition to stationary phase. Interestingly, the expression of the autoinducer synthase genes luxI2 and luxI3 was strongly reduced in all three mutants, resulting in loss of biosynthesis of acylated homoserine-lactones with C14 side-chain, but could be restored by expressing these genes in trans. Several phylogenetic clusters of Alphaproteobacteria revealed a CtrA binding site in the promoters of QS genes, including Roseobacters and Rhizobia. CONCLUSIONS: The CtrA phosphorelay induces differentiation of a marine Roseobacter strain that is strikingly different from that of C. crescentus. Instead of a tightly regulated cell cycle and a switch between two morphotypes, the morphology and cell division of Dinoroseobacter shibae are highly heterogeneous. We discovered for the first time that the CtrA phosphorelay controls the biosynthesis of signaling molecules. Thus cell-cell communication and differentiation are interlinked in this organism. This may be a common strategy, since we found a similar genetic set-up in other species in the ecologically relevant group of Alphaproteobacteria. D. shibae will be a valuable model organism to study bacterial differentiation into pleomorphic cells.

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

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