The biological role of death and lysis in biofilm development

Recent studies have revealed that the regulated death of bacterial cells is important for biofilm development. Following cell death, a sub-population of the dead bacteria lyse and release genomic DNA, which then has an essential role in intercellular adhesion and biofilm stability. This Opinion focuses on the role of regulated cell death and lysis in biofilm development and provides a functional comparison between bacterial programmed cell death and apoptosis. The hypothesis that the differential regulation of these processes during biofilm development contributes to the antibiotic tolerance of biofilm cells is also explored.     

Staphylococcus aureus CidA and LrgA proteins exhibit holin-like properties

The Staphylococcus aureus cid and lrg operons are known to be involved in biofilm formation by controlling cell lysis and the release of genomic DNA, which ultimately becomes a structural component of the biofilm matrix. Although the molecular mechanisms controlling cell death and lysis are unknown, it has been hypothesized that the cidA and lrgA genes encode holin- and antiholin-like proteins and function to regulate these processes similarly to bacteriophage-induced death and lysis. In this study, we focused on the biochemical and molecular characterization of CidA and LrgA with the goal of testing the holin model. First, membrane fractionation and fluorescent protein fusion studies revealed that CidA and LrgA are membrane-associated proteins. Furthermore, similarly to holins, CidA and LrgA were found to oligomerize into high-molecular-mass complexes whose formation was dependent on disulfide bonds formed between cysteine residues. To determine the function of disulfide bond-dependent oligomerization of CidA, an S. aureus mutant in which the wild-type copy of the cidA gene was replaced with the cysteine mutant allele was generated. As determined by β-galactosidase release assays, this mutant exhibited increased cell lysis during stationary phase, suggesting that oligomerization has a negative impact on this process. When analyzed for biofilm development and maturation, this mutant displayed increased biofilm adhesion in a static assay and a greater amount of dead-cell accumulation during biofilm maturation. These studies support the model that CidA and LrgA proteins are bacterial holin-/antiholin-like proteins that function to control cell death and lysis during biofilm development.     

A Central Role for Carbon-Overflow Pathways in the Modulation of Bacterial Cell Death

Similar to developmental programs in eukaryotes, the death of a subpopulation of cells is thought to benefit bacterial biofilm development. However mechanisms that mediate a tight control over cell death are not clearly understood at the population level. Here we reveal that CidR dependent pyruvate oxidase (CidC) and α-acetolactate synthase/decarboxylase (AlsSD) overflow metabolic pathways, which are active during staphylococcal biofilm development, modulate cell death to achieve optimal biofilm biomass. Whereas acetate derived from CidC activity potentiates cell death in cells by a mechanism dependent on intracellular acidification and respiratory inhibition, AlsSD activity effectively counters CidC action by diverting carbon flux towards neutral rather than acidic byproducts and consuming intracellular protons in the process. Furthermore, the physiological features that accompany metabolic activation of cell death bears remarkable similarities to hallmarks of eukaryotic programmed cell death, including the generation of reactive oxygen species and DNA damage. Finally, we demonstrate that the metabolic modulation of cell death not only affects biofilm development but also biofilm-dependent disease outcomes. Given the ubiquity of such carbon overflow pathways in diverse bacterial species, we propose that the metabolic control of cell death may be a fundamental feature of prokaryotic development.     

Bacterial biofilms: prokaryotic adventures in multicellularity

The development of bacterial biofilms includes both the initial social behavior of undifferentiated cells, as well as cell death and differentiation in the mature biofilm, and displays several striking similarities with higher organisms. Recent advances in the field provide new insight into differentiation and cell death events in bacterial biofilm development and propose that biofilms have an unexpected level of multicellularity.     

Subinhibitory arsenite concentrations lead to population dispersal in Thiomonas sp

Biofilms represent the most common microbial lifestyle, allowing the survival of microbial populations exposed to harsh environmental conditions. Here, we show that the biofilm development of a bacterial species belonging to the Thiomonas genus, frequently found in arsenic polluted sites and playing a key role in arsenic natural remediation, is markedly modified when exposed to subinhibitory doses of this toxic element. Indeed, arsenite [As(III)] exposure led to a considerable impact on biofilm maturation by strongly increasing the extracellular matrix synthesis and by promoting significant cell death and lysis within microcolonies. These events were followed by the development of complex 3D-biofilm structures and subsequently by the dispersal of remobilized cells observed inside the previously formed hollow voids. Our results demonstrate that this biofilm community responds to arsenite stress in a multimodal way, enhancing both survival and dispersal. Addressing this complex bacterial response to As(III) stress, which might be used by other microorganisms under various adverse conditions, may be essential to understand how Thiomonas strains persist in extreme environments.     

Cell death in Pseudomonas aeruginosa biofilm development

Bacteria growing in biofilms often develop multicellular, three-dimensional structures known as microcolonies. Complex differentiation within biofilms of Pseudomonas aeruginosa occurs, leading to the creation of voids inside microcolonies and to the dispersal of cells from within these voids. However, key developmental processes regulating these events are poorly understood. A normal component of multicellular development is cell death. Here we report that a repeatable pattern of cell death and lysis occurs in biofilms of P. aeruginosa during the normal course of development. Cell death occurred with temporal and spatial organization within biofilms, inside microcolonies, when the biofilms were allowed to develop in continuous-culture flow cells. A subpopulation of viable cells was always observed in these regions. During the onset of biofilm killing and during biofilm development thereafter, a bacteriophage capable of superinfecting and lysing the P. aeruginosa parent strain was detected in the fluid effluent from the biofilm. The bacteriophage implicated in biofilm killing was closely related to the filamentous phage Pf1 and existed as a prophage within the genome of P. aeruginosa. We propose that prophage-mediated cell death is an important mechanism of differentiation inside microcolonies that facilitates dispersal of a subpopulation of surviving cells.     

Prophage spontaneous activation promotes DNA release enhancing biofilm formation in Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) is able to form biofilms in vivo and previous studies propose that pneumococcal biofilms play a relevant role both in colonization and infection. Additionally, pneumococci recovered from human infections are characterized by a high prevalence of lysogenic bacteriophages (phages) residing quiescently in their host chromosome. We investigated a possible link between lysogeny and biofilm formation. Considering that extracellular DNA (eDNA) is a key factor in the biofilm matrix, we reasoned that prophage spontaneous activation with the consequent bacterial host lysis could provide a source of eDNA, enhancing pneumococcal biofilm development. Monitoring biofilm growth of lysogenic and non-lysogenic pneumococcal strains indicated that phage-infected bacteria are more proficient at forming biofilms, that is their biofilms are characterized by a higher biomass and cell viability. The presence of phage particles throughout the lysogenic strains biofilm development implicated prophage spontaneous induction in this effect. Analysis of lysogens deficient for phage lysin and the bacterial major autolysin revealed that the absence of either lytic activity impaired biofilm development and the addition of DNA restored the ability of mutant strains to form robust biofilms. These findings establish that limited phage-mediated host lysis of a fraction of the bacterial population, due to spontaneous phage induction, constitutes an important source of eDNA for the S. pneumoniae biofilm matrix and that this localized release of eDNA favors biofilm formation by the remaining bacterial population.     

Modulation of eDNA Release and Degradation Affects Staphylococcus aureus Biofilm Maturation

Recent studies have demonstrated a role for Staphylococcus aureus cidA-mediated cell lysis and genomic DNA release in biofilm adherence. The current study extends these findings by examining both temporal and additional genetic factors involved in the control of genomic DNA release and degradation during biofilm maturation. Cell lysis and DNA release were found to be critical for biofilm attachment during the initial stages of development and the released DNA (eDNA) remained an important matrix component during biofilm maturation. This study also revealed that an lrgAB mutant exhibits increased biofilm adherence and matrix-associated eDNA consistent with its proposed role as an inhibitor of cidA-mediated lysis. In flow-cell assays, both cid and lrg mutations had dramatic effects on biofilm maturation and tower formation. Finally, staphylococcal thermonuclease was shown to be involved in biofilm development as a nuc mutant formed a thicker biofilm containing increased levels of matrix-associated eDNA. Together, these findings suggest a model in which the opposing activities of the cid and lrg gene products control cell lysis and genomic DNA release during biofilm development, while staphylococcal thermonuclease functions to degrade the eDNA, possibly as a means to promote biofilm dispersal.     

Biofilm development and cell death in the marine bacterium Pseudoalteromonas tunicata

The newly described green-pigmented bacterium Pseudoalteromonas tunicata (D2) produces target-specific inhibitory compounds against bacteria, algae, fungi, and invertebrate larvae and is frequently found in association with living surfaces in the marine environment. As part of our studies on the ecology of P. tunicata and its interaction with marine surfaces, we examined the ability of P. tunicata to form biofilms under continuous culture conditions within the laboratory. P. tunicata biofilms exhibited a characteristic architecture consisting of differentiated microcolonies surrounded by water channels. Remarkably, we observed a repeatable pattern of cell death during biofilm development of P. tunicata, similar to that recently reported for biofilms of Pseudomonas aeruginosa (J. S. Webb et al., J. Bacteriol. 185:4585-4595, 2003). Killing and lysis occurred inside microcolonies, apparently resulting in the formation of voids within these structures. A subpopulation of viable cells was always observed within the regions of killing in the biofilm. Moreover, extensive killing in mature biofilms appeared to result in detachment of the biofilm from the substratum. A novel 190-kDa autotoxic protein produced by P. tunicata, designated AlpP, was found to be involved in this biofilm killing and detachment. A Delta alpP mutant derivative of P. tunicata was generated, and this mutant did not show cell death during biofilm development. We propose that AlpP-mediated cell death plays an important role in the multicellular biofilm development of P. tunicata and subsequent dispersal of surviving cells within the marine environment.     

Holins in Bacteria,Eukaryotes, and Archaea: Multifunctional Xenologues with Potential Biotechnological and Biomedical Applications

Holins form pores in the cytoplasmic membranes of bacteria for the primary purpose of releasing endolysins that hydrolyze the cell wall and induce cell death. Holins are encoded within bacteriophage genomes, where they promote cell lysis for virion release, and within bacterial genomes, where they serve a diversity of potential or established functions. These include (i) release of gene transfer agents, (ii) facilitation of programs of differentiation such as those that allow sporulation and spore germination, (iii) contribution to biofilm formation, (iv) promotion of responses to stress conditions, and (v) release of toxins and other proteins. There are currently 58 recognized families of holins and putative holins with members exhibiting between 1 and 4 transmembrane α-helical spanners, but many more families have yet to be discovered. Programmed cell death in animals involves holin-like proteins such as Bax and Bak that may have evolved from bacterial holins. Holin homologues have also been identified in archaea, suggesting that these proteins are ubiquitous throughout the three domains of life. Phage-mediated cell lysis of dual-membrane Gram-negative bacteria also depends on outer membrane-disrupting “spanins” that function independently of, but in conjunction with, holins and endolysins. In this minireview, we provide an overview of their modes of action and the first comprehensive summary of the many currently recognized and postulated functions and uses of these cell lysis systems. It is anticipated that future studies will result in the elucidation of many more such functions and the development of additional applications.     

A Differential Effect of E. coli Toxin-Antitoxin Systems on Cell Death in Liquid Media and Biofilm Formation

Toxin-antitoxin (TA) modules are gene pairs specifying for a toxin and its antitoxin and are found on the chromosomes of many bacteria including pathogens. Here we report how each of five such TA systems in E. coli affect bacterial cell death differently in liquid media and during biofilm formation. Of all these systems, only the TA system mazEF mediated cell death both in liquid media and during biofilm formation. At the other extreme, as our results have revealed here, the TA system dinJ-YafQ is unique in that it is involved only in the death process during biofilm formation. Cell death governed by mazEF and dinJ-YafQ seems to participate in biofilm formation through a novel mechanism.     

Biofilm Development and Cell Death in the Marine Bacterium Pseudoalteromonas tunicata

The newly described green-pigmented bacterium Pseudoalteromonas tunicata (D2) produces target-specific inhibitory compounds against bacteria, algae, fungi, and invertebrate larvae and is frequently found in association with living surfaces in the marine environment. As part of our studies on the ecology of P. tunicata and its interaction with marine surfaces, we examined the ability of P. tunicata to form biofilms under continuous culture conditions within the laboratory. P. tunicata biofilms exhibited a characteristic architecture consisting of differentiated microcolonies surrounded by water channels. Remarkably, we observed a repeatable pattern of cell death during biofilm development of P. tunicata, similar to that recently reported for biofilms of Pseudomonas aeruginosa (J. S. Webb et al., J. Bacteriol. 185:4585-4595, 2003). Killing and lysis occurred inside microcolonies, apparently resulting in the formation of voids within these structures. A subpopulation of viable cells was always observed within the regions of killing in the biofilm. Moreover, extensive killing in mature biofilms appeared to result in detachment of the biofilm from the substratum. A novel 190-kDa autotoxic protein produced by P. tunicata, designated AlpP, was found to be involved in this biofilm killing and detachment. A ΔalpP mutant derivative of P. tunicata was generated, and this mutant did not show cell death during biofilm development. We propose that AlpP-mediated cell death plays an important role in the multicellular biofilm development of P. tunicata and subsequent dispersal of surviving cells within the marine environment.     

Hydrogen peroxide linked to lysine oxidase activity facilitates biofilm differentiation and dispersal in several gram-negative bacteria

The marine bacterium Pseudoalteromonas tunicata produces an antibacterial and autolytic protein, AlpP, which causes death of a subpopulation of cells during biofilm formation and mediates differentiation, dispersal, and phenotypic variation among dispersal cells. The AlpP homologue (LodA) in the marine bacterium Marinomonas mediterranea was recently identified as a lysine oxidase which mediates cell death through the production of hydrogen peroxide. Here we show that AlpP in P. tunicata also acts as a lysine oxidase and that the hydrogen peroxide generated is responsible for cell death within microcolonies during biofilm development in both M. mediterranea and P. tunicata. LodA-mediated biofilm cell death is shown to be linked to the generation of phenotypic variation in growth and biofilm formation among M. mediterranea biofilm dispersal cells. Moreover, AlpP homologues also occur in several other gram-negative bacteria from diverse environments. Our results show that subpopulations of cells in microcolonies also die during biofilm formation in two of these organisms, Chromobacterium violaceum and Caulobacter crescentus. In all organisms, hydrogen peroxide was implicated in biofilm cell death, because it could be detected at the same time as the killing occurred, and the addition of catalase significantly reduced biofilm killing. In C. violaceum the AlpP-homologue was clearly linked to biofilm cell death events since an isogenic mutant (CVMUR1) does not undergo biofilm cell death. We propose that biofilm killing through hydrogen peroxide can be linked to AlpP homologue activity and plays an important role in dispersal and colonization across a range of gram-negative bacteria.     

Caspase-1-dependent pore formation during pyroptosis leads to osmotic lysis of infected host macrophages

Salmonella enterica serovar Typhimurium invades host macrophages and induces a unique caspase-1-dependent pathway of cell death termed pyroptosis, which is activated during bacterial infection in vivo. We demonstrate DNA cleavage during pyroptosis results from caspase-1-stimulated nuclease activity. Although poly(ADP-ribose) polymerase (PARP) activation by fragmented DNA depletes cellular ATP to cause lysis during oncosis, the rapid lysis characteristic of Salmonella-infected macrophages does not require PARP activity or DNA fragmentation. Membrane pores between 1.1 and 2.4 nm in diameter form during pyroptosis of host cells and cause swelling and osmotic lysis. Pore formation requires host cell actin cytoskeleton rearrangements and caspase-1 activity, as well as the bacterial type III secretion system (TTSS); however, insertion of functional TTSS translocons into the host membrane is not sufficient to directly evoke pore formation. Concurrent with pore formation, inflammatory cytokines are released from infected macrophages. This mechanism of caspase-1-mediated cell death provides additional experimental evidence supporting pyroptosis as a novel pathway of inflammatory programmed cell death.     

Improved method for the isolation of RNA from bacteria refractory to disruption, including S. aureus producing biofilm

The development of fast, reliable and inexpensive phenol protocol is described for the isolation of RNA from bacterial biofilm producers. The method was tested on Staphylococcus aureus (S. aureus) and other biofilm-producing gram-negative microorganisms and provided the highest integrity of RNA recovery in comparison to other methods reported here. In parallel experiments, bacterial lysis with Qiagen, NucleoSpin RNAII, InnuREP RNA Mini, Trizol and MasterPure RNA extraction Kits using standard protocols consistently gave low RNA yields with an absence of integrity. The boiling method presented here yielded high concentration of RNA that was free from 16S and 23S rRNA, contained 5S RNA. Higher yields due to improved biofilm bacterial cell lysis were achieved with an added hot phenol incubation step without the need for a bead mill or the enzyme. This method when used in conjunction with the Qiagen RNeasy Mini kit, RNA isolation was a success with greater integrity and contained undegraded 16S and 23S rRNA and did not require further purification. Contaminating DNA was a problem with the RNA processing samples; we used quantitative real-time PCR (RT-qPCR) to measure the recovery of RNA from bacterial biofilm cells using the method described here.     

Spontaneous bacterial cell lysis and biofilm formation in the colon of the Cape Dune mole-rat and the laboratory rabbit

A wide range of techniques, including high-throughput DNA sequencing methods, have been applied to the evaluation of the normal intestinal flora. However, the inability to grow many of those species in culture imposes substantial constraints on the techniques used to evaluate this important community. The presence of biofilms in the normal gut adds further complexity to the issue. In this study, a flow cytometric analysis was used to separate intact bacterial cells, cell debris, and other particulate matter based on bacteria-specific staining and particle size. In addition, an analysis of biofilm formation using fluorescent light microscopy was conducted. Using these approaches, the ratio of bacterial cell debris to intact bacterial cells as a measure of spontaneous lysis of bacterial cells in the gut of the Cape dune mole-rat (Bathyergus suillus) and the laboratory rabbit (Oryctolagus cuniculus) was examined, and the degree of biofilm formation was semi-quantitatively assessed. The results suggest that the degree of spontaneous cell lysis was greater in the appendix than in the cecum in both the mole-rat and the rabbit. Further, the results point toward extensive epithelial-associated biofilm formation in the proximal mole-rat and rabbit large bowel, although the biofilms may be less structured than those found in laboratory rodents and in humans.     

Phage-induced lysis enhances biofilm formation in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is capable of forming highly structured surface-attached communities. By DNase I treatment, we demonstrated that extracellular DNA (eDNA) serves as a structural component in all stages of biofilm formation under static and hydrodynamic conditions. We determined whether eDNA is released through cell lysis mediated by the three prophages LambdaSo, MuSo1 and MuSo2 that are harbored in the genome of S. oneidensis MR-1. Mutant analyses and infection studies revealed that all three prophages may individually lead to cell lysis. However, only LambdaSo and MuSo2 form infectious phage particles. Phage release and cell lysis already occur during early stages of static incubation. A mutant devoid of the prophages was significantly less prone to lysis in pure culture. In addition, the phage-less mutant was severely impaired in biofilm formation through all stages of development, and three-dimensional growth occurred independently of eDNA as a structural component. Thus, we suggest that in S. oneidensis MR-1 prophage-mediated lysis results in the release of crucial biofilm-promoting factors, in particular eDNA.     

Life after death: the critical role of extracellular DNA in microbial biofilms

The death and lysis of microbial cells leads to the release of cytoplasmic contents, many of which are rapidly degraded by enzymes. However, some macromolecules survive intact and find new functions in the extracellular environment. There is now strong evidence that DNA released from cells during lysis, or sometimes by active secretion, becomes a key component of the macromolecular scaffold in many different biofilms. Enzymatic degradation of extracellular DNA can weaken the biofilm structure and release microbial cells from the surface. Many bacteria produce extracellular deoxyribonuclease (DNase) enzymes that are apparently tightly regulated to avoid excessive degradation of the biofilm matrix. Interfering with these control mechanisms, or adding exogenous DNases, could prove a potent strategy for controlling biofilm growth.