Extracellular DNA, there's more than one way to lyse a cell
2 recent papers have highlighted the importance of eDNA in biofilm formation and found different mechanisms for externalisation
Two papers have recently come out highlighting the importance of extracellular DNA (eDNA) in two important Gram-positive bacteria, Streptococcus mutans and Staphylococcus aureus.
eDNA is an important constituent of many biofilms, yet the mechanism for it's externalisation is not 100% clear. One of the predominant hypotheses is that a subset of cells in the biofilm lyse and release their DNA which is then trapped by proteins in the extracellular matrix. There may be other transport mechanism as well.
The two studies give support to the hypothesis that bacteria produce eDNA by lysing, but they report completely different mechanisms.
Mechanism 1: Streptococcus mutans autolysin
Jung et al showed that knockouts in the AltA autolysin couldn't produce eDNA and form biofilms. The phenotype was very similar to wildtype cells with a lysis inhibitor added.
Mechanism 2: Staphylococcus aureus membrane destabilisation
DeFrancesco et al hunted for S. aureus genes that were involved in eDNA released. Notably, they found that autolysins did not seem to be involved but mutants that had a more stable cell membrane (i.e. were resistant to Congo Red) produced less eDNA. This suggests that a certain level of membrane instability is important for the production of eDNA in S. aureus biofilms.
Read both of the papers below.
AtlA mediates extracellular DNA release that contributes to Streptococcus mutans biofilm formation in an experimental infective endocarditis rat model
Host factors, like platelets, have been shown to enhance biofilm formation of oral commensal streptococci, inducing infective endocarditis (IE), but how bacterial components contribute to biofilm formation in vivo is still not clear. We previously demonstrated that an isogenic mutant strain of Streptococcus mutans deficient in autolysin AtlA (ΔatlA) showed reduced ability to cause vegetation in a rat model of bacterial endocarditis. However, the role of AtlA in bacterial biofilm formation is unclear. Herein, confocal laser scanning microscopy analysis showed the extracellular DNA (eDNA) embedded inside S. mutans GS5 floes during biofilm formation on damaged heart valves, but ΔatlA strain could not form bacterial aggregates. Semiquantification of eDNA by polymerase chain reaction with bacterial 16S ribosomal RNA primers demonstrated ΔatlA mutant strain produced dramatically less eDNA compared to wild type. Similar results were also observed with in vitro biofilm models. Adding polyanethole sulfonate, a chemical lysis inhibitor, revealed eDNA release mediated by bacterial cell lysis is required for biofilm initiation and maturation in wild type strain. Supplementation of cultures with calcium ions reduced wild type growth but increased eDNA release and biofilm mass. The effect of calcium ions on biofilm formation was abolished in ΔatlA cultures and by addition of polyanethole sulfonate. The VicK sensor, and not CiaH, was found to be required for induction of eDNA release or biofilm formation stimulated by calcium ions. These data suggest that calcium ion-regulated AtlA maturation mediates S. mutans eDNA release that contributes to biofilm formation in infective endocarditis.
Jung CJ, Hsu RB, Shun CT, Hsu CC, Chia JS. Infect Immun. 2017 Jul 3. pii: IAI.00252-17. doi: 10.1128/IAI.00252-17.
Genome-wide screen for genes involved in eDNA release during biofilm formation by Staphylococcus aureus
Staphylococcus aureus is a leading cause of both nosocomial and community-acquired infection. Biofilm formation at the site of infection reduces antimicrobial susceptibility and can lead to chronic infection. During biofilm formation, a subset of cells liberate cytoplasmic proteins and DNA, which are repurposed to form the extracellular matrix that binds the remaining cells together in large clusters. Using a strain that forms robust biofilms in vitro during growth under glucose supplementation, we carried out a genome-wide screen for genes involved in the release of extracellular DNA (eDNA). A high-density transposon insertion library was grown under biofilm-inducing conditions, and the relative frequency of insertions was compared between genomic DNA (gDNA) collected from cells in the biofilm and eDNA from the matrix. Transposon insertions into genes encoding functions necessary for eDNA release were identified by reduced representation in the eDNA. On direct testing, mutants of some of these genes exhibited markedly reduced levels of eDNA and a concomitant reduction in cell clustering. Among the genes with robust mutant phenotypes were gdpP, which encodes a phosphodiesterase that degrades the second messenger cyclic-di-AMP, and xdrA, the gene for a transcription factor that, as revealed by RNA-sequencing analysis, influences the expression of multiple genes, including many involved in cell wall homeostasis. Finally, we report that growth in biofilm-inducing medium lowers cyclic-di-AMP levels and does so in a manner that depends on the gdpP phosphodiesterase gene.
Alicia S. DeFrancesco, Nadezda Masloboeva, Adnan K. Syed, Aaron DeLoughery, Niels Bradshaw, Gene-Wei Li, Michael S. Gilmore, Suzanne Walker, and Richard Losick
Genome-wide screen for genes involved in eDNA release during biofilm formation by Staphylococcus aureus. PNAS 2017; published ahead of print July 3, 2017, doi:10.1073/pnas.1704544114