Extracellular vesicles secreted by Gram-negative bacteria have proven to be important in bacterial defense, communication and host–pathogen relationships. They resemble smaller versions of the bacterial mother cell, with similar contents of proteins, LPS, DNA, and RNA. Vesicles can elicit a protective immune response in a range of hosts, and as vaccine candidates, it is of interest to properly characterize their cargo. Genetic sequencing data is already available for vesicles from several bacterial strains, but it is not yet clear how the genetic makeup of vesicles differ from that of their parent cells, and which properties may characterize enriched genetic material. The present study provides evidence for DNA inside vesicles from Vibrio cholerae O395, and key characteristics of their genetic and proteomic content are compared to that of whole cells. DNA analysis reveals enrichment of fragments containing ToxR binding sites, as well as a positive correlation between AT-content and enrichment. Some mRNAs were highly enriched in the vesicle fraction, such as membrane protein genes ompV, ompK, and ompU, DNA-binding protein genes hupA, hupB, ihfB, fis, and ssb, and a negative correlation was found between mRNA enrichment and transcript length, suggesting mRNA inclusion in vesicles may be a size-dependent process. Certain non-coding and functional RNAs were found to be enriched, such as VrrA, GcvB, tmRNA, RNase P, CsrB2, and CsrB3. Mass spectrometry revealed enrichment of outer membrane proteins, known virulence factors, phage components, flagella and extracellular proteins in the vesicle fraction, and a low, negative correlation was found between transcript-, and protein enrichment. This result opposes the hypothesis that a significant degree of protein translation occurs in vesicles after budding. The abundance of viral-, and flagellar proteins in the vesicle fraction underlines the importance of purification during vesicle isolation.

Membrane vesicles (MVs) are spherical particles naturally released from the membrane of Gram-negative bacteria. Bacterial MV production is associated with a range of phenotypes including biofilm formation, horizontal gene transfer, toxin delivery, modulation of host immune responses and virulence. This study reports comparative profiling of MVs from bacterial strains isolated from three widely disperse geographical areas. Mass spectrometry identified 119, 159 and 142 proteins in MVs from three different strains of Piscirickettsia salmonis isolated from salmonids in Chile (LF-89), Norway (NVI 5692) and Canada (NVI 5892), respectively. MV comparison revealed several strain-specific differences related to higher virulence capability for LF-89 MVs, both in vivo and in vitro, and stronger similarities between the NVI 5692 and NVI 5892 MV proteome. The MVs were similar in size and appearance as analyzed by electron microscopy and dynamic light scattering. The MVs from all three strains were internalized by both commercial and primary immune cell cultures, which suggest a potential role of the MVs in the bacterium’s utilization of leukocytes. When MVs were injected into an adult zebrafish infection model, an upregulation of several pro-inflammatory genes were observed in spleen and kidney, indicating a modulating effect on the immune system. The present study is the first comparative analysis of P. salmonis derived MVs, highlighting strain-specific vesicle characteristics. The results further illustrate that the MV proteome from one bacterial strain is not representative of all bacterial strains within one species.

The macromolecules of the bacterial cell occupy 20-40% of the total cytosol volume, and crowded environments have long been known to compact and stabilize DNA. Nevertheless, investigations on DNA-protein binding are generally performed in the absence of crowding, which may yield an incomplete understanding of how nucleoid-assembling proteins work. A family of such proteins, abundant in Gram-negative bacteria, is the histone-like nucleoid structuring proteins (H-NS). Herein, the synergistic role of macromolecular crowding (mimicked using polyethylene glycol, PEG) and H-NS was investigated using fluorescence correlation spectroscopy (FCS) and enzyme protection assays. We show that crowding enhances the binding of H-NS to the AT-rich tracks of the DNA, where it preferentially binds to, protecting these tracks towards enzyme digestion, inducing some DNA condensation, and inhibiting the biological function of DNA. We further suggest that the looping of DNA chains, induced by H-NS, contributes to the synergistic effect of DNA-binding protein and crowding agents, on DNA condensation.

The endemic intracellular pathogens Francisella noatunensis and Piscirickettsia salmonis are among the most severe threats to the worldwide fish farming industry, as there are currently no satisfactory vaccines available. The bacteria induce quite similar diseases, francisellosis and rickettsiosis, which are characterized by symptoms like discoloration, reduced swimming ability, swollen kidney and spleen, formation of granulomas and occasionally high mortalities, especially in farming enclosures. The bacteria have been shown to secrete outer membrane vesicles (OMVs), which have been proposed as vaccine candidates for the pathogens, but they have shown mixed protection and extremely varying levels of virulence, even among very similar subspecies. In conjunction with these vaccine experiments, the proteomics of the OMVs have been mapped to some extent, but their possible content of RNA and DNA is largely unexplored. OMVs from other Gramnegative sources have been shown to carry DNA and transfer resistance genes between bacteria, so there is significant reason to suspect that this is the case with F. noatunensis and P. salmonis as well. Furthermore, little is known on whether specific sequences of DNA are preferentially secreted in these OMVs, or if the inclusion is merely a random fragment of the genome or any present plasmid. It is known that the genomes of some Francisella spp. are populated by transposable elements, and bacterial transposons are known for their ability to transmit antibiotic resistance. This naturally raises the concern of how the use of OMVs as vaccines could be affecting the rate of which antibiotic resistance genes are transferred within the bacterial population. With these considerations in mind, the aim of the project is 1) to confirm or disprove the presence of DNA in OMVs from F. noatunensis and P. salmonis , 2) Get quantitative sequence data on this content to assess preferential packing of DNA and 3) attempt horizontal antibiotic resistance gene transfer by OMVs.