Impaired respiration elicits SrrAB-dependent programmed cell lysis and biofilm formation in Staphylococcus aureus.

Biofilms are communities of microorganisms attached to a surface or each other. Biofilm-associated cells are the etiologic agents of recurrent Staphylococcus aureus infections. Infected human tissues are hypoxic or anoxic. S. aureus increases biofilm formation in response to hypoxia, but how this occurs is unknown. In the current study we report that oxygen influences biofilm formation in its capacity as a terminal electron acceptor for cellular respiration. Genetic, physiological, or chemical inhibition of respiratory processes elicited increased biofilm formation. Impaired respiration led to increased cell lysis via divergent regulation of two processes: increased expression of the AtlA murein hydrolase and decreased expression of wall-teichoic acids. The AltA-dependent release of cytosolic DNA contributed to increased biofilm formation. Further, cell lysis and biofilm formation were governed by the SrrAB two-component regulatory system. Data presented support a model wherein SrrAB-dependent biofilm formation occurs in response to the accumulation of reduced menaquinone.DOI:http://dx.doi.org/10.7554/eLife.23845.001

Hyperosmolarity is widely used for food preservation by inhibiting bacterial survival and growth. Therefore, it is of great significance to reveal bacterial osmotic-response mechanism. Biofilm formation presents a significant challenge for the control and prevention of pathogenic bacteria. Our previous study showed that inactivation of the efflux protein TolC in extraintestinal pathogenic Escherichia coli (ExPEC) decreased biofilm formation by affecting curli production in a medium osmolarity-dependent manner. This study aims to explore the role of the two-component CpxA/R system in mediating TolC regulation of ExPEC biofilm formation in response to osmolarity. Various mutants derived from the parental ExPEC ΔtolC strain were constructed, and their abilities to form biofilms and produce curli fimbriae in different osmotic media were evaluated using crystal violet staining, scanning electron microscopy, Congo red assay, and real-time quantitative polymerase chain reaction. The results showed that the disruption of CpxA/R system by deleting the gene encoding histidine kinase-CpxA or response regulator-CpxR, or by introducing a point mutation at the phosphorylation site of CpxA, significantly compromised the effect of TolC inactivation on ExPEC biofilm formation and curli biosynthesis under both NaCl- and sucrose-induced osmotic stresses. Our study firstly demonstrate that the CpxAR system mediated the regulation of TolC inactivation on ExPEC biofilm formation and curli production in response to both NaCl- and sucrose-induced osmotic stresses. These findings expand the regulatory network of bacterial biofilm formation and osmotic-responsiveness, contributing to exploring potential targets for preventing and controlling pathogenic bacteria.

Excessive nitrogen fertilizer use contributes to environmental pollution and undermines agricultural sustainability. Enhancing symbiotic interactions between rice and nitrogen-fixing microorganisms offers a promising strategy to potentially improve nitrogen use efficiency (NUE). This study investigates the role of rice root exudates in promoting biofilm formation by nitrogen-fixing microbes to enhance nitrogen fixation. Nine nitrogen-fixing microbial strains were evaluated for biofilm formation in response to flavone and apigenin treatments, with Gluconacetobacter diazotrophicus KACC 12358 serving as the reference strain. The most responsive strain was selected, and a library of 1597 natural compounds was screened to identify those that promote biofilm formation in both the selected and reference strains. A. indigens KACC 11682 exhibited the highest biofilm-forming capacity, with apigenin treatment showing an OD595 value approximately 1.4 times higher than the DMSO control. Screening identified 68 compounds that enhanced biofilm formation by more than 500% compared to the control. Among them, eight compounds induced strong biofilm formation (O.D. > 2.0) in A. indigens. Cardamomin, a chalconoid flavonoid, emerged as one of the most effective compounds, showing a 245% increase in biofilm formation. Growth promotion assays showed that A. indigens increased rice fresh weight by approximately 128% compared to untreated controls. This study demonstrates the potential of rice root exudate-derived compounds to promote beneficial symbiosis with nitrogen-fixing microbes. These findings offer a novel approach that may contribute to enhancing rice NUE. Future research will focus on evaluating the long-term effects of these compounds and microorganisms, assessing their applicability in real agricultural settings, and conducting further validation across various rice cultivars.

Bacteria form dense surface-associated communities known as biofilms that are central to their persistence and how they affect us. Biofilm formation is commonly viewed as a cooperative enterprise, where strains and species work together for a common goal. Here we explore an alternative model: biofilm formation is a response to ecological competition. We co-cultured a diverse collection of natural isolates of the opportunistic pathogen Pseudomonas aeruginosa and studied the effect on biofilm formation. We show that strain mixing reliably increases biofilm formation compared to unmixed conditions. Importantly, strain mixing leads to strong competition: one strain dominates and largely excludes the other from the biofilm. Furthermore, we show that pyocins, narrow-spectrum antibiotics made by other P. aeruginosa strains, can stimulate biofilm formation by increasing the attachment of cells. Side-by-side comparisons using microfluidic assays suggest that the increase in biofilm occurs due to a general response to cellular damage: a comparable biofilm response occurs for pyocins that disrupt membranes as for commercial antibiotics that damage DNA, inhibit protein synthesis or transcription. Our data show that bacteria increase biofilm formation in response to ecological competition that is detected by antibiotic stress. This is inconsistent with the idea that sub-lethal concentrations of antibiotics are cooperative signals that coordinate microbial communities, as is often concluded. Instead, our work is consistent with competition sensing where low-levels of antibiotics are used to detect and respond to the competing genotypes that produce them.

Staphylococcus epidermidis: metabolic adaptation and biofilm formation in response to different oxygen concentrations.

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract In their natural environment, most bacteria preferentially live as complex surface-attached multicellular colonies called biofilms. Biofilms begin with a few cells adhering to a surface, where they multiply to form a mature colony. When conditions deteriorate, cells can leave the biofilm. This dispersion is thought to be an important process that modifies the overall biofilm architecture and that promotes colonization of new environments. In Caulobacter crescentus biofilms, extracellular DNA (eDNA) is released upon cell death and prevents newborn cells from joining the established biofilm. Thus, eDNA promotes the dispersal of newborn cells and the subsequent colonization of new environments. These observations suggest that eDNA is a cue for sensing detrimental environmental conditions in the biofilm. Here, we show that the toxin–antitoxin system (TAS) ParDE4 stimulates cell death in areas of a biofilm with decreased O2 availability. In conditions where O2 availability is low, eDNA concentration is correlated with cell death. Cell dispersal away from biofilms is decreased when parDE4 is deleted, probably due to the lower local eDNA concentration. Expression of parDE4 is positively regulated by O2 and the expression of this operon is decreased in biofilms where O2 availability is low. Thus, a programmed cell death mechanism using an O2-regulated TAS stimulates dispersal away from areas of a biofilm with decreased O2 availability and favors colonization of a new, more hospitable environment. Editor's evaluation In this work, the authors present compelling evidence that a toxin-antitoxin system contributes to biofilm dispersal under oxygen limited conditions. This work makes important contributions to two areas of microbial physiology; functional understanding of toxin-antitoxin systems, which have remained largely elusive, and mechanistic regulation or biofilm dispersal, is a critical, but less understood aspect of biofilm physiology. https://doi.org/10.7554/eLife.80808.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Bacteria are more social than what had long been expected. While they can swim around on their own, most of them in fact settle down as part of a surface-bound community. The plaque on our teeth and the gooey deposit in our bathroom pipes are the visible results of this communal lifestyle. Inside this slimy ‘biofilm’, cells share resources and are protected from toxic substances such as antibiotics. However, being tied to one spot is not always a good thing: it may be advantageous for a cell in a biofilm to strike out on its own and resume ‘single life’ if local conditions deteriorate. Caulobacter crescentus bacteria do not always have this choice, as adult cells in this species become permanently glued into place upon joining a biofilm. When these divide, however, their daughters have a choice: swim away, or stick with the group. Previous research has shown that this decision is influenced by the health of the community. Dying cells release DNA fragments which disable the structures allowing newborn cells to adhere to the environment, and a high mortality rate in the biofilm therefore forces unattached cells to leave the colony. Berne et al. wanted to build on these results and examine how exactly cells die in the biofilm. In particular, the deaths could be sudden and random, with the bacteria succumbing to injury; or they could result from cells activating one of their built-in self-destruct programs. To investigate this question, genetically modified C. crescentus bacteria were grown in the laboratory and exposed to different environments. Combining genetic and microscopic approaches revealed that as a biofilm becomes too crowded, certain individuals self-destruct via a cell death program known as the toxin-antitoxin system. Further experiments showed that low oxygen availability was the signal that triggered self-destruction. Drops in oxygen levels can happen when the environment becomes hostile or when a colony is overpopulated. The results by Berne et al. therefore suggest that by triggering self-destruction in certain members of the community, reduced oxygen access leads to newborn cells swimming away, which in turn prevents further overcrowding and allows new, more hospitable locations to be colonized. Biofilms are of growing interest in a wide range of human industries, but they also present formidable challenges. This is particularly the case in healthcare, as they tend to infest medical devices and help disease-causing species to resist treatments. Understanding how bacteria are encouraged to join or leave their colony is necessary to better control biofilms to our advantage. Introduction Biofilms are multicellular communities attached to a surface, where complex exchanges and interactions occur between the different members. Biofilms first start with the attachment of individual bacterial cells to a surface and then grow into a more complex community when attached bacteria divide and new ones join. The biofilm lifestyle is considered beneficial for bacteria, as they usually provide protection from xenobiotic stresses and predators, and increase collective nutrient availability (Flemming et al., 2016). However, when conditions deteriorate, cells can leave the biofilm through a process referred to as dispersal, disseminate to new environments, and form new biofilms, enabling the colonization of new niches (Guilhen et al., 2017). Dispersal is triggered in response to various environment and biological cues, and understanding its regulation is important to determine how biofilms can be controlled. Many Alphaproteobacteria use a strong polar adhesin to irreversibly attach to surfaces and form biofilms (Berne et al., 2015; Berne et al., 2018b), with Caulobacter crescentus holdfast being the best characterized example. C. crescentus has a dimorphic life cycle, where each division cycle yields a sessile mother stalked cell and a motile daughter swarmer cell. Newborn motile swarmer cells bear a single flagellum and multiple pili at the new pole. After the cell cycle progresses beyond a certain point, or upon contact with a surface, the newborn cells secrete a holdfast at the same pole and differentiate into stalked cells by retracting their pili, ejecting their flagellum, and synthesizing a thin cylindrical extension of the cell envelope called the stalk, which pushes the holdfast away from the cell body. While the chemical composition of the holdfast is not entirely elucidated, it is composed of polysaccharides with four different monosaccharide constituents, as well as DNA and peptide molecules of unknown nature (Merker and Smit, 1988; Hernando-Pérez et al., 2018; Hershey et al., 2019). Holdfast is an extremely strong bioadhesin (Tsang et al., 2006; Berne et al., 2013) crucial for irreversible cell adhesion to solid surfaces (Ong et al., 1990; Bodenmiller et al., 2004), colonization of air–liquid interfaces (Fiebig, 2019), and biofilm formation (Entcheva-Dimitrov and Spormann, 2004). In some bacterial species, extracellular DNA (eDNA) plays a stabilizing role in the biofilm matrix (Okshevsky and Meyer, 2015; Campoccia et al., 2021). In contrast, we previously showed that C. crescentus eDNA produced via cell lysis negatively regulates biofilm formation and stimulates cell dispersal. eDNA binding to unattached holdfasts inhibits their adhesiveness, thereby inhibiting cell attachment to surfaces (Berne et al., 2010). In contrast, eDNA does not dislodge previously bound holdfasts. Therefore, eDNA prevents newborn swarmer cells from joining mature biofilms, but does not dissociate existing biofilms. Because inhibition by eDNA is proportional to its concentration, we proposed that eDNA serves as a rheostat-like environmental cue to trigger dispersal when conditions are detrimental and cause cell death. However, it was not known if eDNA release is the simple consequence of random cell death occurring in the biofilm as conditions worsen, or if it is the result of an active mechanism, such as programmed cell death (PCD) (Berne et al., 2010; Kirkpatrick and Viollier, 2010). In this study, we demonstrate that cell death and eDNA release in a biofilm are regulated by a PCD mechanism that responds to oxygen availability. PCD in bacteria includes all genetically encoded mechanisms that lead to cell lysis (Lewis, 2000; Bayles, 2014). Toxin-antitoxin systems (TAS) are important regulators of PCD (Rice and Bayles, 2008; Peeters and de Jonge, 2018). These systems are comprised of a stable toxin and its unstable antitoxin cognate. The antitoxin molecule usually antagonizes the toxin under ‘steady state’ growth conditions; but, in PCD-triggering conditions, the antitoxin is inactivated, leading to an excess of free toxins that target key cellular processes in response to various environmental signals (Harms et al., 2018; Wang et al., 2021). There are currently eight types of TAS described in bacteria. The classification depends on the nature of the antitoxin (RNA in types I, III, and VIII, or small protein in the other TAS types), and the toxin (small protein in all but type VIII where the toxin is a small RNA), and how the antitoxin neutralizes the toxin activity (Song and Wood, 2020a, Singh et al., 2021; Srivastava et al., 2021). TAS are widespread in bacterial and archaeal genomes, but despite their abundance, the biological relevance of most TAS is still elusive (Fraikin et al., 2020). TAS were first described as plasmid addiction modules that ensure plasmid stabilization via post-segregational killing of plasmid-free cells (Ogura and Hiraga, 1983; Gerdes et al., 1986). TAS have also been shown to promote addiction to certain chromosomally encoded elements such as integrative conjugative elements (Wozniak and Waldor, 2009) or CRISPR-cas loci (Li et al., 2021). In addition, TAS have been described as defense mechanism against phage infection where host translation is inhibited by the phage (Pecota and Wood, 1996; Fineran et al., 2009; Song and Wood, 2020b, LeRoux and Laub, 2022; Vassallo et al., 2022). In bacterial cells that lose their plasmid/chromosomal element encoding the TAS, or that are infected by phage, the amount of labile antitoxin rapidly decreases, leading to toxin activation and subsequent cell death. In addition to the well accepted role of TAS in plasmid addiction and phage exclusion, TAS have been linked to diverse physiological responses, such as biofilm formation, stress response, and persistence (Kamruzzaman et al., 2021), although this is still debated (Ronneau and Helaine, 2019; Wade and Laub, 2019; Song and Wood, 2020a, Jurėnas et al., 2022). Many TAS have been reported to be transcriptionally upregulated under environmental stress conditions (Jurėnas et al., 2022), but this increase does not necessarily trigger liberation of an active toxin (LeRoux et al., 2020). Among the 18 TAS identified in the C. crescentus genome (Ely, 2021), 13 have been studied experimentally, and belong to four different groups: (1) four paralogous RelBE (Fiebig et al., 2010) operons and one HigBA (Kirkpatrick et al., 2016) operon, belonging to the type II systems where the toxins (RelE or HigB) are known to be mRNA endonucleases; (2) four type II systems belonging to the ParDE family (Fiebig et al., 2010), where ParE toxins are usually DNA gyrase inhibitors; (3) three paralogs of HipBA, also a type II system, where the HipA toxins inhibit protein synthesis (Huang et al., 2020; Zhou et al., 2021); and (4) SocAB, the only member of the type VI TAS described so far, where the SocB toxin directly inhibits DNA replication (Aakre et al., 2013). The environmental conditions that trigger any of these TAS and their biological function are not yet fully identified. In this study, we show that the ParDE4 TAS is involved in PCD and eDNA release in C. crescentus biofilms where it stimulates cell dispersal. We show that areas of a biofilm with decreased O2 availability experience more cell death. Cell viability is improved in a ∆parDE4 mutant biofilm, especially in areas of decreased O2 availability, generating less cell lysis and less eDNA release. We also show that cell dispersal is decreased when parDE4 is deleted, probably due to the lower local eDNA concentration. Expression of parDE4 is positively regulated by O2 and the expression of this operon is decreased in biofilms where O2 availability is low. Thus, PCD by an O2-regulated TAS stimulates dispersal away from areas of a biofilm with decreased O2 availability. Results The ParDE4 TAS is involved in biofilm inhibition and eDNA release of C. crescentus grown under static conditions We previously showed that, in C. crescentus, eDNA is a cue that can trigger biofilm inhibition and dispersion by binding to holdfasts and reducing their adhesiveness. This mechanism is a result of cell lysis and eDNA release in the biofilm (Berne et al., 2010). To investigate if this eDNA release is the product of a specific PCD mechanism, we tested if a TAS was involved in promoting cell death in the biofilm, as previously suggested (Kirkpatrick and Viollier, 2010). If such a TAS is inactivated, one should observe less cell death, less eDNA release, and more biofilm formation. We examined the four ParDE-like and four RelBE-like individual in-frame deletion mutants previously described (Fiebig et al., 2010), as well as mutants lacking the four ParDE (‘All parDE− ' mutant), the four RelBE (‘All relBE− ' mutant) and the eight ParDE/RelBE operons (‘All parDE− All relBE− ' mutant’), for their ability to form biofilms compared to C. crescentus CB15 wild-type (WT). For these static biofilm assays, we grew cells in two-ml plastic microfuge tubes sealed with AeraSeal breathable film, to allow for gas exchange, and incubated them statically at 30°C (Figure 1). We defined these growth conditions as ‘moderate aeration’. All the tested mutants grew similarly to WT under these conditions (Figure 1—figure supplement 1). Figure 1 with 2 supplements see all Download asset Open asset Role of the eight toxin–antitoxin systems (TAS) in cell death, extracellular DNA (eDNA) release, and biofilm formation. C. crescentus WT and the different TAS in-frame deletion mutants were grown for 48 hr under moderate aeration conditions at 30°C in M2G medium, as depicted on the left. (A) Percentage of dead cells in the planktonic phase; results are expressed as a percentage of the total cells (live + dead) in the sample, quantified using the BacLight Live/Dead kit. (B) Biofilm formation, quantified by crystal violet staining; results are expressed as a percentage of biofilm formed compared to WT. (C) Quantification of eDNA released in the planktonic phase, using PicoGreen. Results are given as the average of four independent experiments, each run in duplicate, and the error bars represent the standard error of the mean (SEM). Statistical comparisons are calculated using Student’s unpaired t-tests; only samples statistically different from WT are shown. **p < 0.01. We tested the ability of the TAS mutants to form biofilms after 48 hr, and quantified cell death and eDNA release under these growth conditions (Figure 1). Among single mutants, ∆parDE4 was the only strain that behaved differently compared to WT. The percentage of dead cells was lower in this mutant and it produced ~30% more biofilm than the other strains (Figure 1A, B). Furthermore, it released only about half of the amount of eDNA in the planktonic phase compared to WT (Figure 1C and Figure 1—figure supplement 1B). The All parDE− and the All parDE− All relBE− strains, where all four parDE operons and all parDE plus all relBE operons were deleted respectively, behaved like the ∆parDE4 single deletion mutant (Figure 1). These results suggest that ParDE4 plays a role in cell death and eDNA release under our experimental conditions and that the observed changes in eDNA concentration yield differences in biofilm regulation. To test if this phenotype was specific for C. crescentus cells that are able to form biofilms, we deleted the hfsDAB holdfast synthesis cluster in the ∆parDE4 background to generate a strain unable to produce holdfast, and therefore unable to adhere to surfaces and form biofilms. The double mutant ∆parDE4 ∆hfsDAB phenocopied the ∆parDE4 strain, with lower eDNA and lower proportion of dead cells (Figure 1—figure supplement 2). These results indicate that the function of ParDE4 does not require cells to be adhered to a surface and suggest that it might be responding to the differences in medium aeration as described in a later section. The ParDE4 TAS plays a role in cell death in mature biofilms of C. crescentus The parDE4 operon is composed of the parD4 antitoxin gene (CC2985/CCNA_03080) and the parE4 toxin gene (CC2984/CCNA_03079), overlapping by 21 bp (Nierman et al., 2001; Fiebig et al., 2010; Marks et al., 2010). To assess the role of ParDE4 in cell death, eDNA release, and biofilm formation over time, we monitored biofilm formation on sterile microscopy-grade clear polyvinyl chloride (PVC) strips grown under moderate aeration as depicted in Figure 1A. Over time, we also quantified eDNA release and cell death occurring in WT and ∆parDE4 (Figure 2 and Figure 2—figure supplement 1). Cell death was reduced in ∆parDE4 biofilms compared to WT, especially at longer time points when the biofilm reached maturation (Figure 2A, B). In addition, less eDNA was released in these mutant cultures (Figure 2C). We also observed an increase in attached biomass in the ∆parDE4 mutant (Figure 2D). These results support our previous findings that biofilm inhibition, eDNA release, and cell death are correlated (Berne et al., 2010). Furthermore, these results indicate that ParDE4 is involved in stimulating cell death and eDNA release, yielding a change in biofilm formation. Since eDNA stimulates dispersal from the biofilm (Berne et al., 2010), both the reduced cell death and eDNA release in the ∆parDE4 mutant might contribute to the increased biofilm formation. Figure 2 with 1 supplement see all Download asset Open asset Involvement of the ParDE4 TAS in cell death, extracellular DNA (eDNA) release, and biofilm regulation. (A) Biofilm formed on polyvinyl chloride (PVC) strips stained with the BacLight Live/Dead reagent at different incubation times. represent of the (live and signals by bars (B) Percentage of dead cells over time in the biofilm, calculated from BacLight Live/Dead stained cells using (C) eDNA release in the planktonic phase over time, quantified using Biofilm formation over time, quantified by the attached biomass with crystal C. crescentus WT and ∆parDE4 are by solid and were grown in M2G The results are given as the average of two independent experiments, each run in and the error bars represent the standard error of the mean (SEM). The antitoxin against cell death in the biofilm In TAS, cell death usually when is an in levels of toxins and produced in the cell (Harms et al., 2018). To assess the role of antitoxin we expressed it using the low plasmid (Fiebig et al., 2010; et al., and monitored biofilm formation and eDNA release when the antitoxin is When we expressed the antitoxin in WT, biofilm formation was increased and eDNA concentration was decreased (Figure However, in a mutant lacking the was of parD4 expression on biofilm formation and eDNA concentration (Figure that (1) has a against cell lysis and eDNA release, thereby biofilm formation, and (2) this depends on the of the toxin Figure with 1 supplement see all Download asset Open asset of parD4 antitoxin expression on biofilm formation and extracellular DNA (eDNA) release. The parD4 was into the low plasmid and expressed using the Biofilm formation and eDNA release for strains the parD4 antitoxin gene or the plasmid in WT (A) and were grown in M2G medium + 1 The results are given as the average of three independent experiments and the error bars represent the standard error of the mean (SEM). we wanted to determine if the of the ∆parDE4 mutant was due to of cell death and eDNA release, or also to an response to eDNA biofilm inhibition in the ∆parDE4 We tested how WT and ∆parDE4 behaved in the of eDNA by the amount of biofilm formed in the of from cultures of different We showed previously that eDNA present in medium from cultures inhibits biofilm formation (Berne et al., 2010). various of eDNA from cultures of WT or ∆parDE4 grown to phase were in biofilm assays, as previously (Berne et al., 2010). Because ∆parDE4 less eDNA than WT compared to of eDNA present in the medium of a for ∆parDE4 and WT, respectively, see Figure supplement we first the amount of eDNA present and an amount of medium to the same concentration of The amount of biofilm formed by WT and ∆parDE4 was for the same total amount of eDNA (Figure supplement that, when exposed to the same amount and of both WT and ∆parDE4 form of biofilm and that eDNA from WT or ∆parDE4 have the same biofilm Furthermore, the inhibition response is positively correlated with the amount of eDNA present in the medium in a for both strains (Figure supplement in with our previous results (Berne et al., 2010). Therefore, the increase in biofilm formation by the ∆parDE4 mutant is not due to an response to eDNA but is due to less cell death a more increase to less cell dispersal by ParDE4 promotes dispersal in the biofilm To the of biofilm formation in the WT and ∆parDE4 strains, we grew a of differently WT and ∆parDE4 at a in The of each in the biofilm was monitored over time (Figure in the of the biofilm could be observed at of biofilm maturation (Figure with formation of due to This is in with previous of C. crescentus biofilm growth in cells (Entcheva-Dimitrov and Spormann, et al., 2019). While in the of biofilm formation the of WT and ∆parDE4 was the mutant rapidly the WT at later After hr, around of the attached bacteria were ∆parDE4 (Figure Figure Download asset Open asset Biofilm formation and dispersion in of WT and ∆parDE4 were to a and grown in cells over (A) of biofilms grown in is in and the other one in (B) of each over Results are given as a percentage of total + both calculated from of of time point, independent experiments where were (C) Cell dispersal as released in the of the Results are to the of colonies for WT at hr of the were using M2G Results are expressed as an average of of samples time cells were run in independent experiments where were bars represent the standard error of the mean (SEM). To test the dispersal rate of both strains, we dispersal from the biofilm by the of cells released from the biofilm over time in the This was by samples of the cell and the of single cells released from each (Figure was more biomass of ∆parDE4 cells than WT in the biofilm were more WT cells released over time compared to that the dispersal of WT is more (Figure In these results with of previous suggest that the observed increased biofilm formation in the ∆parDE4 is due to a of increased attached biomass of reduced cell death and decreased dispersion The ParDE4 response is correlated with O2 availability Previous work the regulation of the parDE4 operon as a function of O2 availability on growing cells (Fiebig et al., 2010). by under stress O2 showed a in parD4 not statistically (Fiebig et al., 2010). In our PCD triggered by the ParDE4 TAS is more when the biofilm maturation (Figure 2). of environmental changes occur as the biofilm of O2 availability. Since C. crescentus is an O2 could be a detrimental triggering cell death and dispersal, as is the case in other species et al., In to test the of O2 on cell death, we grew cells with as compared to the ‘moderate (Figure a in eDNA release not occur in cells grown under aeration compared to the moderate aeration conditions (Figure and Figure 1—figure supplement that ParDE4 is not active under conditions. To determine if ParDE4 expression is regulated by aeration conditions, we monitored its using a under aeration growth compared to growth under moderate aeration conditions. To growth conditions as different O2 availability, we a to the of encoding the This gene is expressed when C. crescentus cells experience O2 levels et al., and its expression can be as a to O2 availability. expression was more active under moderate aeration conditions (Figure that O2 availability is limited under growth conditions. We that was two to three under aeration growth (Figure that ParDE4 expression is regulated by O2 availability. Figure Download asset Open asset expression is under aeration growth conditions. (A) of cultures grown in M2G under conditions different of and moderate (B) Quantification of extracellular DNA (eDNA) released in the planktonic phase of WT and ∆parDE4 quantified using Results are given as the average of independent experiments and the error bars represent the standard error of the mean (SEM). (C) activity of and in WT grown under and moderate aeration conditions in The results represent the average of independent cultures on three different and the error bars represent the Since the results suggested that O2 availability is important for biofilm we tested two growth conditions to an of O2 and a (Figure as by the levels in each (Figure We first that the expression of is correlated to the O2 in the cultures (Figure We then compared the of eDNA released in ∆parDE4 to WT in the different aeration conditions. We that was as eDNA released by WT compared to ∆parDE4 under conditions where O2 levels are the most reduced the levels were under aeration (Figure with the in eDNA release, we that biofilm formation by the ∆parDE4 increased

Genetic and microscopy analyses identify a programmed cell death mechanism that kills a cell subpopulation in a bacterial biofilm where oxygen is limiting, thereby promoting dispersion of newborn motile cells through the action of DNA released by dead cells.

In bacteria, many small regulatory RNAs (sRNAs) are induced in response to specific environmental signals or stresses and act by base-pairing with mRNA targets to affect protein translation or mRNA stability. In Escherichia coli, the gene for the sRNA IS061/IsrA, here renamed McaS, was predicted to reside in an intergenic region between abgR, encoding a transcription regulator and ydaL, encoding a small MutS-related protein. We show that McaS is a ∼95nt transcript whose expression increases over growth, peaking in early-to-mid stationary phase, or when glucose is limiting. McaS uses three discrete single-stranded regions to regulate mRNA targets involved in various aspects of biofilm formation. McaS represses csgD, the transcription regulator of curli biogenesis and activates flhD, the master transcription regulator of flagella synthesis leading to increased motility, a process not previously reported to be regulated by sRNAs. McaS also regulates pgaA, a porin required for the export of the polysaccharide poly β-1,6-N-acetyl-d-glucosamine. Consequently, high levels of McaS result in increased biofilm formation while a strain lacking mcaS shows reduced biofilm formation. Based on our observations, we propose that, in response to limited nutrient availability, increasing levels of McaS modulate steps in the progression to a sessile lifestyle.

The foodborne pathogen Staphylococcus aureus continues to challenge the food industry due to the pathogenicity and tolerance of the bacterium. As a common storage condition for frozen food during transportation, distribution, and storage, freezing does not seem to be entirely safe due to the cold tolerance of S. aureus. In addition, our study indicated that the biofilm formation ability of S. aureus was significantly increased in response to freezing stress. To explore the molecular mechanism regulating the response to freezing stress, the proteomics signature of S. aureus after freezing stress based on tandem mass tag (TMT) labeling and liquid chromatography tandem mass spectrometry (LC-MS/MS) was analyzed. Gene Ontology and pathway analysis revealed that ribosome function, metabolism, RNA repair, and stress response proteins were differentially regulated (P < 0.05). Furthermore, transpeptidase sortase A, biofilm operon icaADBC HTH-type negative transcriptional regulator IcaR, and HTH-type transcriptional regulator MgrA were involved in the modulation of increased biofilm formation in response to freezing stress (P < 0.05). Moreover, significant lysine acetylation and malonylation signals in the S. aureus response to freezing stress were observed. Collectively, the current work provides additional insight for comprehending the molecular mechanism of S. aureus in response to freezing stress and presents potential targets for developing strategies to control S. aureus. KEY POINTS: • TMT proteomic analysis was first used on S. aureus in response to freezing stress. • Ribosome-, metabolism-, and biofilm-related proteins change after freezing stress. • Increased biofilm formation in S. aureus responded to freezing stress.

An investigation of Pseudomonas aeruginosa attachment to a diverse range of polymers in a high-throughput microarray format resulted in the discovery of novel biofilm-resistant and biofilm-stimulating materials. These findings raised questions about the nature of the surface interactions involved and in particular the sensory mechanisms use by P. aeruginosa to distinguish between different surface chemistries. To further investigate this, Tn5 mutants of the P. aeruginosa PAO1 Washington sub-line were tested for biofilm formation on the polymer microarrays. This revealed that a Tn5::cpxR mutant had a significant difference in biofilm formation when compared with PAO1-W. In Escherichia coli cpxR forms an operon with cpxA and cpxP. Together they form the CPX two-component signalling system controlling biofilm formation in response to cell envelope stress. To further characterise the P. aeruginosa cpxsystem, ΔcpxR,ΔcpxP and ΔcpxA deletion mutants were constructed. No significant differences were observed in their growth or in the swimming, swarming or twitch motility phenotypes normally required for surface colonization. Unlike E. coli the PAO1-W ΔcpxAmutant was able to invade lung epithelial cells and did not display increased sensitivity to antibiotics. However, the ΔcpxRmutant showed increased biofilm formation on glass and eDNA secretion in both biofilm and liquid modes of growth. This work highlights the relationship between biofilm formation and the CPX system in P. aeruginosa. However, further assays need to be conducted in order to understand the sensory mechanism(s) involved in surface sensing via the CPX system.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that mainly causes otitis media in children and community-acquired pneumonia or exacerbations of chronic obstructive pulmonary disease in adults. A large variety of studies suggest that biofilm formation by NTHi may be an important step in the pathogenesis of this bacterium. However, the underlying mechanisms involved in this process are poorly elucidated. In this study, we used a transposon mutant library to identify bacterial genes involved in biofilm formation. The growth and biofilm formation of 4,172 transposon mutants were determined, and the involvement of the identified genes in biofilm formation was validated in in vitro experiments. Here, we present experimental data showing that increased bacterial lysis, through interference with peptidoglycan synthesis, results in elevated levels of extracellular DNA, which increased biofilm formation. Interestingly, similar results were obtained with subinhibitory concentrations of β-lactam antibiotics, known to interfere with peptidoglycan synthesis, but such an effect does not appear with other classes of antibiotics. These results indicate that treatment with β-lactam antibiotics, especially for β-lactam-resistant NTHi isolates, might increase resistance to antibiotics by increasing biofilm formation. IMPORTANCE Most, if not all, bacteria form a biofilm, a multicellular structure that protects them from antimicrobial actions of the host immune system and affords resistance to antibiotics. The latter is especially disturbing with the increase in multiresistant bacterial clones worldwide. Bacterial biofilm formation is a multistep process that starts with surface adhesion, after which attached bacteria divide and give rise to biomass. The actual steps required for Haemophilus influenzae biofilm formation are largely not known. We show that interference with peptidoglycan biosynthesis increases biofilm formation because of the release of bacterial genomic DNA. Subinhibitory concentrations of β-lactam antibiotics, which are often prescribed to treat H.influenzae infections, increase biofilm formation through a similar mechanism. Therefore, when β-lactam antibiotics do not reach their MIC in vivo, they might not only drive selection for β-lactam-resistant clones but also increase biofilm formation and resistance to other antimicrobial compounds.

Staphylococcus aureus glucose-induced biofilm accessory proteins, GbaAB, influence biofilm formation in a PIA-dependent manner

Campylobacter jejuni is a microaerophilic bacterium and is believed to persist in a biofilm to antagonize environmental stress. This study investigated the influence of environmental conditions on the formation of C. jejuni biofilm. We report an extracellular DNA (eDNA)-mediated mechanism of biofilm formation in response to aerobic and starvation stress. The eDNA was determined to represent a major form of constitutional material of C. jejuni biofilms and to be closely associated with bacterial lysis. Deletion mutation of the stress response genes spoT and recA enhanced the aerobic influence by stimulating lysis and increasing eDNA release. Flagella were also involved in biofilm formation but mainly contributed to attachment rather than induction of lysis. The addition of genomic DNA from either Campylobacter or Salmonella resulted in a concentration-dependent stimulation effect on biofilm formation, but the effect was not due to forming a precoating DNA layer. Enzymatic degradation of DNA by DNase I disrupted C. jejuni biofilm. In a dual-species biofilm, eDNA allocated Campylobacter and Salmonella at distinct spatial locations that protect Campylobacter from oxygen stress. Our findings demonstrated an essential role and multiple functions of eDNA in biofilm formation of C. jejuni, including facilitating initial attachment, establishing and maintaining biofilm, and allocating bacterial cells.IMPORTANCECampylobacter jejuni is a major cause of foodborne illness worldwide. In the natural environment, the growth of C. jejuni is greatly inhibited by various forms of environmental stress, such as aerobic stress and starvation stress. Biofilm formation can facilitate the distribution of C. jejuni by enabling the survival of this fragile microorganism under unfavorable conditions. However, the mechanism of C. jejuni biofilm formation in response to environmental stress has been investigated only partially. The significance of our research is in identifying extracellular DNA released by bacterial lysis as a major form of constitution material that mediates the formation of C. jejuni biofilm in response to environmental stress, which enhances our understanding of the formation mechanism of C. jejuni biofilm. This knowledge can aid the development of intervention strategies to limit the distribution of C. jejuni.

Biofilms are multicellular communities of microorganisms living as a quorum rather than as individual cells. The bacterial human pathogen Staphylococcus aureus uses oxygen as a terminal electron acceptor during respiration. Infected human tissues are hypoxic or anoxic. We recently reported that impaired respiration elicits a programmed cell lysis (PCL) phenomenon in S. aureus leading to the release of cellular polymers that are utilized to form biofilms. PCL is dependent upon the AtlA murein hydrolase and is regulated, in part, by the SrrAB two-component regulatory system (TCRS). In the current study, we report that the SaeRS TCRS also governs fermentative biofilm formation by positively influencing AtlA activity. The SaeRS-modulated factor fibronectin-binding protein A (FnBPA) also contributed to the fermentative biofilm formation phenotype. SaeRS-dependent biofilm formation occurred in response to changes in cellular respiratory status. Genetic evidence presented suggests that a high cellular titer of phosphorylated SaeR is required for biofilm formation. Epistasis analyses found that SaeRS and SrrAB influence biofilm formation independently of one another. Analyses using a mouse model of orthopedic implant-associated biofilm formation found that both SaeRS and SrrAB govern host colonization. Of these two TCRSs, SrrAB was the dominant system driving biofilm formation in vivo We propose a model wherein impaired cellular respiration stimulates SaeRS via an as yet undefined signal molecule(s), resulting in increasing expression of AtlA and FnBPA and biofilm formation.

Biofilm formation is commonly described as a developmental process regulated by environmental cues. In the current study we present a mechanistic model to explain regulation of Pseudomonas fluorescens biofilm formation by the environmentally relevant signal inorganic phosphate (P(i)). We show that activation of the Pho regulon, the major pathway for adaptation to phosphate limitation, results in conditional expression of a c-di-GMP phosphodiesterase referred to as RapA. Genetic analysis indicated that RapA is an inhibitor of biofilm formation and required for loss of biofilm formation in response to limiting P(i). Our results suggest that RapA lowers the level of c-di-GMP, which in turn inhibits the secretion of LapA, a large adhesion required for biofilm formation by P. fluorescens. The ability of c-di-GMP to modulate protein secretion is a novel finding and further extends the biological influence of c-di-GMP beyond that of regulating exopolysaccharide synthesis and motility. Interestingly, Pho regulon expression does not impinge on the rate of attachment to a surface but rather inhibits the transition of cells to a more stable interaction with the surface. We hypothesize that Pho regulon expression confers a surface-sensing mode on P. fluorescens and suggest this strategy may be broadly applicable to other bacteria.