Have exploding cells blown up MV dogma?

Credit: NPG

Bacterial biofilms rely on a matrix formed by extracellular DNA (eDNA) and other macromolecules for structural integrity and self-organization. Components of the matrix may also have other 'public goods' functions; for example, extracellular membrane vesicles (MVs) have been shown to protect cells in the biofilm from antibiotics. Whitchurch and colleagues now report the surprising observation that MVs in Pseudomonas aeruginosa biofilms are produced by exploding bacterial cells, which also release eDNA and other public goods into the matrix.

P. aeruginosa is an opportunistic pathogen that can form interstitial biofilms in the extracellular space of human tissue, notably in individuals with cystic fibrosis. Live-cell imaging of both interstitial and submerged P. aeruginosa biofilms showed that a subpopulation of cells undergo a rapid (<10 s) transition from a rod-shaped cell to a round cell, which in most cases precedes, by no more than 1 minute, an explosive cell lysis event that releases eDNA into the matrix. These changes are consistent with a loss of structural integrity in the peptidoglycan of the cell wall. Moreover, they are reminiscent of the cell lysis events precipitated by lytic phages, which are induced by the activation of the RecA-mediated SOS stress response. Indeed, experiments with a recA deletion mutant showed that explosive cell lysis in P. aeruginosa biofilms is also dependent on the SOS stress response. In further agreement with a phage origin for the lysis mechanism, P. aeruginosa has a cryptic prophage that is predicted to encode a phage-like endolysin, Lys. Genetic experiments showed that eDNA release was dependent on the endolytic activity of Lys and that the loss of this activity was as detrimental to the structural organization of the biofilm as eDNA degradation by DNase I.

“super-resolution microscopy showed that MVs were formed by the reannealing of membrane fragments following explosive cell lysis”

More remarkable still was the observation that explosive cell lysis seemed to underlie the production of all MVs in P. aeruginosa biofilms. This was a surprising finding because Gram-negative bacteria such as P. aeruginosa are generally assumed to produce MVs through blebbing of the outer membrane. However, in what the authors believe to be the first in vivo observation of MV formation, super-resolution microscopy showed that MVs were formed by the reannealing of membrane fragments following explosive cell lysis, generally within 1 s of lysis. By contrast, biofilms formed by lys mutants deficient for lysis did not produce any MVs. The contents of the MVs, most of which were 150−300 nm in diameter, included mRNA, rRNA and eDNA.

Although the release of eDNA in biofilms has previously been proposed — but not directly observed — to occur through cell lysis, the unexpected finding that MVs are also produced in this way underscores the fundamental role of sacrificial autolysis as a social interaction in P. aeruginosa biofilms. By sacrifice of a subpopulation of cells, which the authors suggest is mediated by a heterogeneous SOS response, components of cell detritus, such as eDNA and MVs, can be used as public goods for the greater benefit of the biofilm community. Furthermore, the role of Lys in this process is an intriguing example of the co-option of a virulent function by the host bacterium after phage domestication. Importantly, genes homologous to lys are conserved in many bacterial genera, which hints that exploding cell lysis might be a common mechanism of MV production.